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23354943
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pes2o/s2orc
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v3-fos-license
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Expression patterns of PRDM 10 during mouse embryonic development
It is well known that PR/SET family members participate in transcriptional regulation via chromatin remodeling. PRDM10 might play an essential role in gene expression, but no such evidence has been observed so far. To assess PRDM10 expression at various stages of mouse development, we performed immunohistochemistry using available PRDM10 antibody. Embryos were obtained from three distinct developmental stages. At E8.5, PRDM10 expression was concentrated in the mesodermal and neural crest populations. As embryogenesis proceeded further to E13.5, PRMD10 expression was mainly in mesoderm-derived tissues such as somites and neural crest-derived populations such as the facial skeleton. This expression pattern was consistently maintained to the fetal growth period E16.5 and adult mouse, suggesting that PRDM10 may function in tissue differentiation. Our study revealed that PRDM10 might be a transcriptional regulator for normal tissue differentiation during mouse embryonic development. [BMB reports 2010; 43(1): 29-33]
INTRODUCTION
PRDM contains a PR (PRDI-BF1 and RIZ homology) domain and is a subgroup of PR/SET transcription factor family, because its PR domain shows similarity to the catalytic motif of the SET domain of histone methyltransferase (HMTase) (1,2).There are approximately 20 PR and 30 SET domain-containing genes in the human genome, a growing number of which have been found to possess intrinsic HMT activity towards specific lysine residues along the N-terminal tails of histones (2)(3)(4)(5).These histone-modifying proteins participate in the regulation of genome expression and chromatin remodeling.
Most of the PRDM subfamily has been implicated in cell proliferation and gene repression in cancer and normal mammalian development (6)(7)(8).Human PRDM2 (also known as RIZ1) is a strong tumor suppressor that induces G2/M cell cycle arrest and/or apoptosis in various tumor cells (6,9,10).Its PR domain can methylate histone H3 lysine 9 (H3K9) and is necessary in the repression of estrogen signaling genes (3,11).Deletion of mouse PRDM2 was closely associated with B cell lymphoma in a knockout mouse experiment (10).Mouse PRDM1 (also known as Blimp1) has been identified as a key factor for PGC (primordial germ cell) specification, which is the major source for oocytes and spermatozoa (12).Interestingly, PGC specification requires the concerted orchestration of two PR domain-containing transcriptional regulators, PRDM1 and PRDM14 (13).PRDM14 is a key protein in the launching of the germ cell lineage in mice, suggesting that this event is accomplished by the sequential expression of multiple genes (13).PRDM14 also maintains the self-renewal of human ES cells by suppressing gene expression in embryoid bodies (14).The expression of multiple PRDM family genes (PRDM6, 8, 12, 13 and 16) in the developing mouse central nerve system occurs in a spatially and temporally restricted manner, thereby generating a great deal of interest.PRDM8 and 12 are found in the neuronal progenitor zones of developing spinal cord, where as PRDM12 and 16 occur in the ventricular zone of developing telencephalon in a lateral to medial graded manner; PRDM8 also is present in postmitotic neurons.This suggests that dynamic changes in PRDM family genes occur in the developing embryo (15).
In this study, we examined the expression of PRDM10 during mouse embryogenesis.Immunohistochemical analysis was performed on sections obtained from distinct developmental stages of the embryo during embryo to fetus or tissue differentiation.
Expression of PRMD10 at the embryo to fetus differentiation (E8.5)
Immunohistochemistry (IHC) with antibodies specific to PRDM10 was used to examine.PRDM10 expression during various stages of mouse embryogenesis 8.5 to 16.5 days post-coitus.Hematoxylin counterstaining was used as a control.During early stages (E8.5),PRDM10 was strongly expressed in the middle embryo region, suggesting a cell population in the paraxial mesodermal or migrating neural crest regions as shown in Fig. 1.All mesenchymal cell populations can differentiate after neural tube closure according to prohttp://bmbreports.org ).Sagittal sections of mouse embryos were prepared 13 days after fertilization (E13.5).PRDM10 proteins were detected mainly in the craniofacial, somital and notochord regions.Immunoreactivity with brown color was determined by incubation with PRDM10 antibody (B), whereas there was no reaction in the negative control (A).NC: notochord, TG: tongue, VT: ventricle, S: somite, L: liver.gramming (16,17).Therefore, PRDM10 is likely expressed in robust mesenchymal cells derived from the mesoderm and neural crest during early mouse development.
PRMD10 expression in somital and craniofacial tissues during organogenesis
To assess PRDM10 expression during late mouse development, sagittal sections from E13.5 were analyzed.Compared to the IgG negative control, PRDM10 was mainly expressed in the cra-niofacial, somital and notochord regions (Fig. 2).PRDM10 expression was also apparent in the ventricles at this stage, but not in the intestines or liver.Although experiments were not quantitative, the relative levels of PRDM10 expression are apparent.A more detailed examination was performed using sagittal sections of the embryo obtained from E16.5.As shown in Fig. 3, IHC demonstrated that PRDM10 is ubiquitously expressed patterns in various tissues.Specifically, PRDM10 was mainly visible in the developing reticular dermis layer of differentiated head tissues (Fig. 3A), mandible regions (Fig. 3B), and the adrenal medulla (Fig. 3C), but was concentrated in the skeletal cartilage (Fig. 3D).Since these PRDM10-expressing tissues were mainly derived from mesodermal or neural crest cell populations, the results suggest that PRDM10 is consistently expressed for mesodermal differentiation during mouse development.
PRMD10 expression in adult mouse tissues
Adult mice were sacrificed in order to examine PRDM10 expression in various tissues by IHC and RT-PCR analysis.IHC detected PRDM10 expression in cartilage and kidney, in agreement with PRDM10 expression during embryonic development (Fig. 4A).However, RT-PCR analysis found that the patterns of PRDM10 expression in certain tissues, such as liver, spleen and rectum, were slightly different compared to embryonic stages, suggesting that PRDM10 is distinctly expressed in adult mice (Fig. 4B).This discrepancy could be explained by the fact that the embryonic tissues were too small.Therefore, these results suggest that PRDM10 plays a role in tissue differentiation during mouse embryogenesis and adult development.
DISCUSSION
Although PRDM10 shares characteristics with the PR/SET fam- ily with the PR/SET domain and clusters of krüppel-like zinc fingers DNA binding motifs, the functions of PRDM10 are only beginning to be determined (18).It is known that most of the PR/SET gene family modulates gene expression, specifically the spatial and temporal regulation of tissue differentiation during mouse embryonic development (5,15,19,20).Functionally, PR/SET gene family members directly or indirectly interact with histone methylation proteins that possess HMTase activity as well as components of the transcription machinery (1,8,11).
Our present study suggests that PRDM10 expression stimulates the sophisticated morphogenesis of tissue differentiation, including somites and craniofacial tissue formation.Therefore, we postulate that PRDM10 has an essential role in regulating gene expression by binding target gene promoters and regulating tissue differentiation genes.PRDM10 could be recruited to its target promoters, depending on the activity derived from its conserved PR/SET domain.Even though the intrinsic activity of PRDM10 remains unknown, we have consistently obtained data showing that PRDM10 is a tri-H3K9 HMTase-related protein (unpublished data).In general, silenced genes are associated with tri-methylated H3K9, which represses transcription (11).This suggests that PRDM10 expression during mouse embryonic development may function as a gene repressor for normal somite and craniofacial formation.
Previous reports have suggested that PRDM10 may be involved in the pathogenesis of gangliosidosis (GM2), a neuronal storage disease (18).Based on its high expression in the mesodermal region, PRDM10 might be associated with the pathogenesis of the other mesoderm-derived diseases as well.Therefore, further studies are needed to explore the relationship between PRDM10 expression and diseases associated with cartilage defects such as chondroma or inflammatory arthritis.Although there is no expression data for craniofacial syndromes, these diseases have genetic backgrounds containing mutated candidate target genes (21).PRDM10 might also be an important target protein for the growth and development of the craniofacial complex based on its expression in craniofacial tissues.
In summary, our study suggests that PRDM10 was expressed in somites and craniofacial tissues during mouse embryonic development.PRDM10 may be a target gene for tissue differentiation and bone formation defects.
Antibody and reagents
Polyclonal PRDM10 antibody was kindly provided from Abgents Inc. (CA, USA).Secondary antibody was purchased from Santa Cruz Inc. (CA, USA).Reagents for IHC analysis were obtained from Vector Laboratories, Inc. (MI, USA).3,3'diaminobenzidine (DAB) was used as a chromogenic substrate and was counterstained with hematoxylin.
Animals and mating
Eight week-old ICR mice were obtained from Orient Bio Inc. (Korea) and either used for mating or sacrificed for further analysis.Mating was performed according to a 3 : 1 female to male ratio and was verified by vaginal plug formation.The embryonic stage was designated embryonic day 0.5 (E0.5) on the day of vaginal plug formation.Embryos or fetuses were prepared on E8.5, E13.5 and E16.5.Animals were handled according to the Guide for the Care and Use of Laboratory Animals at Kangwon National University, Chuncheon, S. Korea.
Immunohistochemistry (IHC)
Following Forane anaesthesia, embryos were removed from the maternal uterus and fixed in neutral formalin for 2 days, after which they were cut in half and fixed in neutral formalin for an additional 3 days.The fixed samples were processed in gradient alcohols, xylene and embedded in paraffin prior to being sectioned at 4 μm for IHC analysis.Paraffin was removed from sections using a citrus clearing solvent for 20 min.To block endogenous peroxidase activity, sections were incubated in a 3% hydrogen peroxidase-methanol mixture for 15 min.Primary PRDM10 antibody was diluted 1 : 100, incubated for 2 hours, and treated with secondary antibody.Chromogenic DAB was used as a substrate and was couterstained with hematoxylin.Finally, the slides were hydrated through alcohol, cleared with citrus clearing solvent and mounted with Permount mounting medium (Fisher Scientific Inc., NJ, USA).
Fig. 1 .
Fig.1.Expression of PRDM10 protein in the embryonic head region 8.5 days after fertilization (E8.5).Transverse sections of the embryonic head were prepared and stained by IHC using PRDM10 antibody.Immunoreactivity was found in the mesodermal and neural crest regions.Arrowhead: neural tube, Brown: PRDM10, Purple: hematoxylin counterstaining.
Fig. 2 .
Fig. 2. PRDM10 expression in the developmental stage (E13.5).Sagittal sections of mouse embryos were prepared 13 days after fertilization (E13.5).PRDM10 proteins were detected mainly in the craniofacial, somital and notochord regions.Immunoreactivity with brown color was determined by incubation with PRDM10 antibody (B), whereas there was no reaction in the negative control (A).NC: notochord, TG: tongue, VT: ventricle, S: somite, L: liver.
Fig. 4 .
Fig. 4. PRDM10 expression in adult mouse tissues.(A) IHC analysis of PRDM10 expression in adult mouse.Left, cartilage; Right, kidney; Brown: PRDM10, Purple: hematoxylin counterstaining.(B) RT-PCR analysis of PRDM10 expression in various mouse tissues.cDNA was prepared from various tissues and used as a DNA template for PCR amplification using PRDM10 specific primers.A single band of PRDM10 was detected at 401 bp.GAPDH was used as a loading control to confirm correct cDNA synthesis.
5'-CTG TTG CTG TAG CCG TAT TCA TT-3'.GAPDH was used as a loading control.Samples were incubated at products were resolved on a 1% agarose gel.
|
2018-04-03T03:58:13.643Z
|
2010-01-01T00:00:00.000
|
{
"year": 2010,
"sha1": "78886393b46c445adf6359730147ba0b36d16667",
"oa_license": "CCBYNC",
"oa_url": "http://society.kisti.re.kr/sv/SV_svpsbs03V.do?cn1=JAKO201010103426014&method=download",
"oa_status": "GOLD",
"pdf_src": "ScienceParseMerged",
"pdf_hash": "78886393b46c445adf6359730147ba0b36d16667",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
}
|
1513117
|
pes2o/s2orc
|
v3-fos-license
|
The use of auxiliary devices during irrigation to increase the cleaning ability of a chelating agent
Objectives This study investigated the cleaning ability of ultrasonically activated irrigation (UAI) and a novel activation system with reciprocating motion (EC, EasyClean, Easy Equipamentos Odontológicos) when used with a relatively new chelating agent (QMix, Dentsply). In addition, the effect of QMix solution when used for a shorter (1 minute) and a longer application time (3 minutes) was investigated. Materials and Methods Fifty permanent human teeth were prepared with K3 rotary system and 6% sodium hypochlorite. Samples were randomly assigned to five groups (n = 10) according to the final irrigation protocol: G1, negative control (distilled water); G2, positive control (QMix 1 minute); G3, QMix 1 minute/UAI; G4, QMix 1 minute/EC; G5, QMix 3 minutes. Subsequently the teeth were prepared and three photomicrographs were obtained in each root third of root walls, by scanning electron microscopy. Two blinded and pre-calibrated examiners evaluated the images using a four-category scoring system. Data were statistically analyzed using Kruskal-Wallis and Dunn tests (p < 0.05). Results There were differences among groups (p < 0.05). UAI showed better cleaning ability than EC (p < 0.05). There were improvements when QMix was used with auxiliary devices in comparison with conventional irrigation (p < 0.05). Conventional irrigation for 3 minutes presented significantly better results than its use for 1 minute (p < 0.05). Conclusions QMix should be used for 1 minute when it is used with UAI, since this final irrigation protocol showed the best performance and also allowed clinical optimization of this procedure.
Introduction
Irrigation of root canal system (RCS) is a crucial stage during endodontic therapy, as it allows cleaning and disinfection beyond the reach of mechanical activity of root canal instruments. 1 Effectiveness in the conventional irrigation technique by using syringe and needle depends on RCS anatomy and the depth of needle penetration according to root diameter and curvature. 2 This method is capable of inserting root canal irrigants to only 0 -1.1 mm beyond the needle tip, and it is generally inefficient for reaching mechanically inaccessible regions and the apical root third. 1,3 Effective action of irrigating solutions is achieved by direct contact with dentin walls. 2 This contact may be restricted by the presence of smear layer (SL) and debris, irrigation method, and/or root canal anatomy, mainly in the apical region. 1,[4][5][6][7] Therefore, different auxiliary devices and irrigation techniques have been proposed, not only to improve the flux distribution of irrigants, but also to increase their action in RCS. [8][9][10][11] The use of passive ultrasonic irrigation produces and transmits acoustic energy by ultrasonic waves from an oscillating file or smooth wire, creating cavitation, and acoustic streaming that increases the effect of irrigants on root canal walls and in mechanically inaccessible areas. 2,12 As this process is in fact active, the term 'passive' does not correctly describe the technique; 12 thus, the term 'ultrasonically activated irrigation (UAI)' has been used. An acrylonitrile butadiene styrene (ABS) plastic endodontic finishing instrument, with a size of 25/0.04, named EasyClean (EC, Easy Equipamentos Odontológicos, Belo Horizonte, Minas Gerais, Brazil) was recently designed to increase the activity of root canal irrigants. As EC operates in reciprocating motion (180º clockwise turn, followed by a 90º counterclockwise turn), this kinematic model may avoid file fracturing or locking within the canals. 13 A recent study concluded that the reciprocating activation of EC with 17% ethylenediaminetetraacetic acid (EDTA) was effective for debris removal from the more apical regions of root canals. 13 Therefore, this reciprocating system may yield a further advantageous option among the available auxiliary irrigation devices.
The aim of this study was to evaluate the cleaning ability of UAI and a novel activation system with reciprocating motion (EC) in all root canal regions, when used with a relatively new chelating agent, QMix 2in1 (Dentsply, Tulsa Dental Specialties, Tulsa, OK, USA), as the final irrigation protocol of endodontic therapy. In addition, the effect of QMix solution when used for a shorter (1 minute) and a longer application time (3 minutes) was investigated. The null hypotheses tested were: (1) There were no differences in the cleaning ability among all irrigation protocols; (2) There were no differences in the cleaning ability between UAI and EC devices; (3) There was no difference in the cleaning ability of QMix when used with conventional irrigation in comparison with using it with auxiliary devices.
Sample selection and preparation
This study was approved by the University Ethics Committee (Protocol: 27745514.0.0000.5259). Fifty singlerooted anterior teeth with single canal and straight roots were selected for this study. The presence of immature root, root damage, calcifications, atresic root canals, or root length shorter than 13 mm were established as the exclusion criteria. Using a diamond disc, the selected teeth were sectioned at 12.5 mm from the anatomic apex, and two longitudinal grooves were prepared in the mesial and distal regions of roots to facilitate later splitting of the samples. After this, patency of all specimens was confirmed with a size 10 K file (Dentsply Maillefer, Rio de Janeiro, RJ, Brazil) and the apical foramen was standardized with a size 25 K file (Dentsply Maillefer).
Chemomechanical preparation
The samples were chemomechanically prepared with the K3 rotary system (SybronEndo, Orange, CA, USA) and 6% sodium hypochlorite (NaOCl) solution as chemical auxiliary substance. The working length was established at the total root length, in the apical foramen. To ensure that instrumentation occurred in a closed system, the apex was sealed with utility wax. A closed system enabled the authors to simulate clinical conditions, avoiding irrigant extrusion through the apical foramen and inducing production of the vapor lock phenomenon; thus allowing more reliable results. The K3 NiTi instruments were used in the following order (size/taper): 25/0.06 in the cervical and middle thirds; and 15/0.04, 20/0.02, 20/0.04, 25/0.04, 20/0.06, and 25/0.06 in the apical region. Between each file change, 1 mL of NaOCl was inserted with a syringe and needle (10 mL syringe with 25 x 0.7 mm needle) and patency was confirmed with a size 10 K file (Dentsply Maillefer).
After preparation, 10 mL of distilled water (DW) was used to neutralize any traces of NaOCl, and samples were randomly assigned to five groups (n = 10) according to the final irrigation protocol (Table 1): G1, negative control (DW); G2, positive control (QMix 1 minute); G3, QMix 1 minute/UAI; G4, QMix 1 minute/EC; G5, QMix 3 minutes. In G1, the negative control group, DW was used alone at the final rinse; thus, SL was not removed. In G2, G3, and G4, QMix was used for 1 minute, as this is the minimum time required for this solution to act, according to manufacturer, allowing clinical optimization of final rinsing. However, in contrast to G3 and G4, no auxiliary irrigation device was used in G2, denominated the positive control group. In G5, QMix was used for a longer time (3 minutes) with no auxiliary irrigation device, allowing the authors to analyze whether an increase in application time of this irrigant could compensate the non-use of UAI or EC and yield satisfactory results.
In the groups in which conventional irrigation was used at the final rinse (G1, G2, and G5), the needle was introduced in the middle third of specimens. In the groups in which auxiliary irrigation device were used during the final irrigation protocol (G3 and G4), the needle was introduced in the middle third of specimens and EC or UAI were inserted into the apical third of specimens. The UAI was performed with a piezoelectric ultrasound machine (ENAC, Osada Inc., Los Angeles, CA, USA) set at 30 Hz, which provided an ultrasonic activation power of irrigants.
As regards the use of EC, an electric motor (VDW.Silver Reciproc, VDW GmbH, Munich, Germany), in reciprocation motion, was used, providing agitation of QMix with this kinematic model. Afterwards samples received a final rinse with 10 mL of DW and were dried with absorbent paper points.
Scanning electron microscopy analysis
Samples were longitudinally split into two root halves through the previously prepared grooves, and the most preserved portion was sputter coated with gold for analysis. Then, each root was figuratively divided into three parts measuring 4 mm, equivalent to the cervical, middle and apical root thirds. According to a previous study, three representative images (magnification ×1,000) were prepared in each root third of samples by scanning electron microscopy (SEM, JSM 6460 LV, JEOL, Tokyo, Japan) at 20 KV. 14 Thus, nine images were prepared for each sample, totaling ninety images per group for evaluation.
The SEM images obtained were evaluated by two blinded and previously calibrated examiners. The cleaning ability of irrigation protocols was based on a four categorical scoring system (Figure 1), through qualitative analysis of the existing amounts of SL and debris on the root canal walls and dentinal tubules: Score 1, complete absence of SL or debris; Score 2, the largest part of the root surface without SL and debris; Score 3, the largest part of the root surface covered with SL and debris; Score 4, root surface completely covered with SL and debris without any visible dentinal tubules. 14 The use of auxiliary devices with QMix
Statistical analysis
Kappa test was carried out to analyze the interexaminer reliability regarding SEM images. The non-parametric Kruskal-Wallis and Dunn post hoc tests were applied, at a 5% significance level (p < 0.05), to detect statistical difference between the groups.
Results
The Kappa test indicated an excellent agreement between examiners (value of 0.93). All groups presented residual SL and debris on root canal walls and dentinal tubules. No significant erosion was observed in the samples. Table 2 summarizes the values of SL scores presented by each group and root thirds, with the statistical difference between them. There were differences in the cleaning ability among the irrigation protocols (Kruskal-Wallis test; p < 0.05). The UAI group presented the lowest SL scores and superior results in comparison with those of the other groups evaluated (p < 0.05).
The cleaning ability of UAI was statistically superior to that of the EC device (Dunn test; p < 0.05). Furthermore, there was an improvement in the cleaning ability of QMix when used with auxiliary devices in comparison with conventional irrigation in the same period of time (Dunn test; p < 0.05). Finally, EC group and QMix 3 minutes showed similar results (Dunn test; p > 0.05) and the conventional irrigation with QMix for 3 minutes presented significantly better results than its use for 1 minute (Dunn test; p < 0.05).
Discussion
All groups presented residual SL and debris on root canal walls and in dentinal tubules, in agreement with the overall findings in endodontic literature. 8,10,11,15,16 Although the goal of the final rinse was complete removal of the SL, it seemed to be virtually impossible to achieve the objective by means of all the available techniques. Nevertheless, maximum cleaning of the root canals should be reached without causing the erosion of dentinal tubules, since the presence of SL and debris reduced the effectiveness of disinfecting agents and interfered in the adhesion of filling materials. 4,6,17 Statistically significant differences were observed among groups (p < 0.05). Consequently, the first null hypothesis that 'there was no difference in the cleaning ability among all irrigation protocols' was clearly rejected. The use of UAI demonstrated the best results, followed by EC and QMix 3 minutes, which presented similar scores. Indeed, the excellence of UAI could not be regarded as a novelty, because it has been well documented in the literature. 12,18 QMix agitated with the EC system led to less effective results, probably because this device may reduce the phenomena of acoustic streaming (fast vortex kinematics) and cavitation (creation of steam bubbles or the expansion, contraction and/or distortion of pre-existing bubbles in a liquid) compared with ultrasonic irrigant activation. 12 Therefore, the second null hypothesis that 'there were no differences in cleaning ability between the UAI and EC devices' was also rejected.
The increase in the application time of QMix up to 3 minutes, without auxiliary devices, yielded similar results to those of the EC group, in which QMix was used for 1 minute. Chelation action of QMix must have been enhanced with time, so that it achieved its optimal effect after 1 minute. This finding may suggest that the absence of an auxiliary device could be compensated by increasing the time of action of this solution. However, the toxic effect of an agent may increase with time and thus the application time of irrigants must be selected with caution.
An increase in the application time of UAI and EC devices may yield better cleaning ability. However, as auxiliary devices increase the flux and effectiveness of irrigants, the use of these devices in association with longer application time of solutions could significantly raise further risks of dentin erosion and toxic effects. [8][9][10][11] Therefore, the time used during irrigant activation should be the minimum required for effective results. As an adequate performance was obtained by using UAI for 1 minute, it is unnecessary to increase the application time of QMix with this device. Nevertheless, when associated with EC, a slight increase in the application time of QMix, such as adding 30 seconds, could be interesting to obtain better results. Further researches are required to confirm this assumption. As regards the cleaning potential of irrigation protocols in the critical apical area, G1 (negative control -DW) and G2 (positive control -QMix 1 minute) presented the worst results, whereas G3 (QMix 1 minute/UAI), G4 (QMix 1 minute/EC) and G5 (QMix 3 minutes) groups showed similarly improved results. The use of QMix for 1 minute was not effective for SL removal in the apical area. As previously stated, QMix probably achieved its optimal effect after 1 minute; however, this application time was insufficient for obtaining effective SL removal in the apical third. Moreover, specifically in this region, G3 (QMix 1 minute/UAI) and G4 (QMix 1 minute/EC) showed similar results. This can be explained because both systems allowed agitation of the solution associated with elimination of the vapor lock phenomenon, improving the irrigant effectiveness. In this aspect, Kato et al. 13 observed superior cleaning ability results in the apical region for EC in comparison with UAI. The differences in results can be explained by the methodology. The authors of the cited study used the mesiobuccal root canals of mandibular molars and previously prepared cleaved specimens, while in the present study we used unirradicular teeth and the teeth were cleaved after preparation.
EC is composed of ABS plastic, which reduces or even prevents damage when it comes into contact with the root canal walls. This material also provides the instruments with flexibility, allowing it to be used right up to the working length, possibly even in the presence of curvatures. 13 Moreover, the EC file presents a particular 'aircraft wing' design, characterized by a central matrix associated with two delicate edges throughout the instrument (Figure 2). These combined design features generate two mechanisms of action, that is, the well-known agitation of solutions, which disperses forces within the canal, and direct contact with the root walls that allows mechanical drag of the adhered debris and biofilms. 13 Sodium hypochlorite (0.5 -6%) is the most widely used chemical auxiliary substance during root canal cleaning and shaping, because it presents a considerable body of supporting evidence. 5,19 At the final rinse, EDTA (15 -17%) is usually associated with NaOCl for SL removal, leading to an undesirable formation of chlorine gas, a toxic byproduct. 19,20 Therefore, intermediate flushes with distilled water should be performed to prevent or at least reduce the formation of this by-product. 20 QMix 2in1, which is a one-step, ready-to-use solution composed of EDTA and chlorhexidine, 17 effectively removes the inorganic portion of SL and kills bacteria, 4,21 thereby optimizing the final irrigation protocol and avoiding the formation of chlorine gas during root canal therapy.
QMix solution effectively removed SL in the total area of root canals since all experimental groups presented results that were superior to those of the Negative Control group, in which only DW was used in the final irrigation protocol. However, the method of application of this irrigant must be reviewed. Despite the manufacturer's indication to use QMix for 60 -90 seconds, the ability of this solution was only effective for cleaning the apical region in this minimum time interval when associated with auxiliary irrigation devices. Hence, the third null hypothesis tested that 'there were no differences in the cleaning ability of QMix when used with conventional irrigation in comparison with its The use of auxiliary devices with QMix use with auxiliary devices' was rejected. The agitation of QMix increased the flux and distribution of this solution, reflecting in cleaner root canal walls and dentinal tubules in apical region.
Conclusions
The use of UAI presented cleaning ability that were superior to those of the EC reciprocating system when these devices were used with QMix for 1 minute. QMix application for 3 minutes presented better results than its use for 1 minute when no auxiliary irrigation device was used. QMix should be used for 1 minute when it is used with UAI, since this final irrigation protocol showed the best performance in cleaning ability and also allowed clinical optimization of this procedure.
|
2018-04-03T04:50:27.941Z
|
2017-02-03T00:00:00.000
|
{
"year": 2017,
"sha1": "d1a1d4298906bef50b865a95802f9a0a08b6312a",
"oa_license": "CCBYNC",
"oa_url": "https://doi.org/10.5395/rde.2017.42.2.105",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
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"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Mathematics",
"Medicine"
]
}
|
12852732
|
pes2o/s2orc
|
v3-fos-license
|
Increase methylmercury accumulation in Arabidopsis thaliana expressing bacterial broad-spectrum mercury transporter MerE
The bacterial merE gene derived from the Tn21 mer operon encodes a broad-spectrum mercury transporter that governs the transport of methylmercury and mercuric ions across bacterial cytoplasmic membranes, and this gene is a potential molecular tool for improving the efficiency of methylmercury phytoremediation. A transgenic Arabidopsis engineered to express MerE was constructed and the impact of expression of MerE on methylmercury accumulation was evaluated. The subcellular localization of transiently expressed GFP-tagged MerE was examined in Arabidopsis suspension-cultured cells. The GFP-MerE was found to localize to the plasma membrane and cytosol. The transgenic Arabidopsis expressing MerE accumulated significantly more methymercury and mercuric ions into plants than the wild-type Arabidopsis did. The transgenic plants expressing MerE was significantly more resistant to mercuric ions, but only showed more resistant to methylmercury compared with the wild type Arabidopsis. These results demonstrated that expression of the bacterial mercury transporter MerE promoted the transport and accumulation of methylmercury in transgenic Arabidopsis, which may be a useful method for improving plants to facilitate the phytoremediation of methylmercury pollution.
Introduction
Mercury pollution is still a worldwide problem in environments because of natural events and human activities such as coal burning, industrial use and gold-mining activities (Harada 1995). Metallic and ionic form of mercury can accumulate in sediments, where they are readily converted to highly toxic methylmercury by microbes (Barkay et al. 2003). Clinical investigations have shown that methylmercury is the principal form of mercury that accumulated in fish and biomagnifies in their consumers, causing severe neurodegenerative symptoms (Harada 1995). The severe adverse effects of this contaminant mean there is an urgent need to develop an effective and affordable technology to facilitate its removal from the environment.
Phytoremediation refers to the use of green plants in the removal of environmental pollutants, which is recognized as a cost effective, sustainable, and environmentally friendly approach that has many advantages during the large-scale clean-up of contaminated sites (Clemens et al. 2002;Kramer 2005;Malik 2004; Ruiz and Daniell 2009;Salt 1998). In recent studies, Meager et al. engineered bacterial mer operons such as MerA (mercuric reductase) to reduce reactive mercuric ions to volatile and relatively inert monoatomic Hg(0) vapor, and MerB (organomercurial lyase) to degrade methylmercury to mercuric ions into plants, thereby remediating methylmercury contamination (Meagher and Heaton 2005). Plants such as cottonwood trees (Lyyra et al. 2007) and tobacco (Heaton et al. 2003) have been modified to express either MerB or both MerB and MerA, which convert methylmercury to mercuric ions or mercury vapor, respectively. The disadvantage of this approach is that elemental mercury Hg(0) is released into the environment, where it accumulates and can eventually be converted into highly toxic methylmercury. To help address this environmental problem, a new methylmercury remediation method is required to replace the merA-mediated mercury reduction mechanism so plant cells can accumulate methylmercury from contaminated sites without releasing mercury vapor into the ambient air.
In general, rehabilitation of metal-contaminated soils by plants requires a long time for the purification process to be completed. McGrath and Zhao reported that several months were required to reduce the mercury content by half in contaminated soils (McGrath and Zhao 2003). The expression of mercury transporter in the plant may provide a means of improving mercury uptake, thereby shortening the purification completion time. In a previous study, we demonstrated for the first time that the MerE protein encoded by pE4 is localized in the membrane cell fraction and that MerE is a novel, broad-spectrum mercury transporter, which governs the transport of CH 3 Hg(I) and Hg(II) across bacterial cytoplasmic membranes (Kiyono et al. 2009;Sone et al. 2010).
The current study evaluated the feasibility of engineering transgenic Arabidopsis plants to express bacterial broad-spectrum mercury transporter, MerE and its potential use in the phytoremediation of methylmercury pollution. This study showed that expression of MerE promoted the transport and accumulation of methylmercury in transgenic Arabidopsis. Enhanced methylmercury accumulation mediated by MerE represents one way for improving plants to be more suitable for use in phytoremediation of methylmercury pollution. Table 1 shows the Escherichia coli (E. coli), Arabidopsis thaliana (A. thaliana) -cultured cells and plasmids used in this study. E. coli XL1-Blue was grown at 37°C in Luria-Bertani (LB) medium and used for routine plasmid propagation. When necessary, the medium was supplemented with 25 μg/mL kanamycin. Suspension-cultured A. thaliana cells were maintained in Murashige and Skoog (MS) medium on a rotary shaker at 22°C with continuous white light and sub-cultured once a week. A. thaliana ecotype Columbia was used for the plant transformations. The seeds produced were germinated and grown on jiffy-7 peat pellets, whereas the surfacesterilized seeds were sown onto MS agar plates. The plants were grown in an environmental growth chamber (Sanyo, Tokyo, Japan) at 22°C in long-day (16 h light/8 h dark) conditions.
Enzymes and reagents
The restriction enzymes, the DNA ligation kit and Taq polymerase were obtained from Takara Shuzo Corp. (Kyoto, Japan). Analytical reagent grade mercury was purchased from Wako Chemicals (Tokyo, Japan).
Plasmid construction
The recombinant plasmids and oligonucleotide primers used in this study are described in Table 1 and Table 2, respectively. The plasmid pE18 that carried the gfp-merE fusion gene was constructed in pUC18 as follows. A 0.23 kb fragment containing merE was PCR-amplified using the primers U-Bgl-merE and L-Kpn-merE, which contained BglII and KpnI sites. The pE4 plasmid (Kiyono et al. 2009) was used as a template. After digestion the PCR product with BglII and KpnI, the fragment was cloned into the corresponding sites in CaMV35S-sGFP(S65T)-NOS3′ (Uemura et al. 2004) and sequenced, and then designated as pE18.
The plasmid pMAE2 that carried the merE gene was constructed in pMAT137 (Matsuoka and Nakamura 1991) as follows. The primers U-Not-merE and L-Xba-merE were used to amplify the merE region (0.23 kb) with pE18 as a template. After digestion the PCR products with NotI-XbaI, the fragment was cloned into the NotI-XbaI sites of pMAT137. The cloned fragment was sequenced and the resulting plasmid was designated as pMAE2. Confocal laser scanning microscopy GFP-fused proteins were transiently expressed in A. thaliana suspension-cultured cells using a published method (Uemura et al. 2004). Cells transformed with pE18 were viewed without fixation under an Olympus BX60 fluorescence microscope, which was equipped with a Model CSU10 confocal scanner (Yokogawa Electric) (Nakano 2002) and a Zeiss LSM510 META or LSM5 PASCAL microscope, which were equipped with green HeNe and argon lasers.
Transformation of plants and confirmation of transgenic plants
The pMAE2 plasmid was introduced into Agrobacterium tumefaciens (A. tumefaciens) via electroporation (Mozo and Hooykaas 1991). A. tumefaciens was grown at 25-28°C in LB medium added with 25 μg/mL kanamycin and employed for the transformation of A. thaliana. A. thaliana ecotype Columbia plants were transformed using the floral dip method (Clough and Bent 1998) by Inplanta Innovations Inc. (Kanagawa, Japan). The progeny seedlings were selected on MS medium containing 50 mg/L kanamycin. The third generations of merE transgenic plants (T 3 ) were used for all experiments described in this paper. Plant genomic DNA was isolated from transformed and untransformed leaves using the FTA Kit (GE Healthcare, Buckinghamshire, England) in accordance with the manufacturer's instructions. The target genome DNA was amplified using specific primers, i.e., U-Not-merE and L-Xba -merE for merE, according to the manufacturer's instructions ( Table 2). The primers U-321NPTII and L-1109NPTII were used to amplify NOS-NPTII in each PCR reaction, as control ( Table 2). The PCR products were separated on 2% agarose gels and visualized by ethidium bromide staining.
Quantitation of mRNA levels by reverse transcription PCR
Total RNA was extracted from cells using an RNeasy Plant Mini Kit (Qiagen, CA, USA), according to the manufacturer's instructions. The Superscript First-strand Synthesis System for reverse transcription PCR (Life Technologies, CA, USA) was used to prepare singlestranded cDNA. The target cDNAs were amplified using specific primers, i.e., U-Not-merE and L-Xba-merE to merE, according to the manufacturer's instructions. The primers β-ACT-Fd and β-ACT-Rv were used to amplify ACT1 in each PCR reaction as controls ( Table 2). The PCR products were separated on 2% agarose gels and visualized by ethidium bromide staining.
Subcellular fractionation of Arabidopsis tissues
To generate roots, surface-sterilized A. thaliana seeds were germinated in sterile MS liquid medium with 100 rpm shaking using a rotary shaker in dark conditions. The roots of 14-day-old plants were homogenized in a grinding buffer, which contained 50 mM Tris-HCl, pH 7.5, 250 mM sorbitol, 5 mM EDTA, 5 mM EGTA, 1 mM dithiothreitol, and 100 μM p-(amidinophenyl) methanesulfonyl fluoride hydrochloride (APMSF). The homogenate was filtered through four layers of Miracloth (EMD Biosciences, Darmstadt, Germany), and centrifuged at 10,000 × g for 10 min. The supernatant was centrifuged at 100,000 × g for 30 min and the precipitate was then suspended in the grinding buffer.
SDS-PAGE and immunoblotting
Proteins were separated by SDS-PAGE and transferred to an Immobilon-P membrane (Millipore, Billerica, USA). After blocking with de-fatted milk, the membrane filter was incubated with rabbit anti-MerE polyclonal antibody (Kiyono et al. 2009). The membranes were washed and reacted with peroxidase-conjugated anti-rabbit IgG antibody (Sigma Aldrich, MO, USA). Anti-MerE polyclonal and peroxidase-conjugated anti-rabbit IgG antibodies were used at a dilution of 1:3,000. Chemiluminescent reagents ECL (GE Healthcare, Chalfont St Giles, UK) were used to detect antigens.
Mercury accumulation
T 3 transgenic plants were cultured in MS gellan gum plates with different concentrations of HgCl 2 or CH 3 Hg Cl for 2 weeks at 22°C. After treatment with 10 μM HgCl 2 or 0.3 μM CH 3 HgCl, the total amount of mercury accumulated by an entire plant was determined as follows, using 60 plants in total. Entire plants samples were digested with a concentrated acid mixture (nitric acid: perchlonic acid = 4: 1) for 4 h at 90°C and their total cellular mercury contents were measured using an atomic absorption spectrometry analyzer HG-310 (Hiranuma, Japan). The standard deviation of the measurements was less than 10%.
Mercury resistance
The sensitivities of T 3 transgenic plants to mercury were tested using the following method. The sterilized seeds from wild-type and transgenic plants were aligned in a horizontal array of MS gellan gum plates, which contained 5 μM HgCl 2 or 0.3 μM CH 3 HgCl, where they the seeds germinated and grew vertically. The root lengths of the seedlings were measured after 2 weeks' cultivation at 22°C.
Statistical analysis
Data analysis was performed using the statistical tools (Student's t-test) of Microsoft Excel software.
Cellular localizations of GFP-MerE in suspension-cultured plant cells
To determine the subcellular localization of GFP-fusion proteins in suspension-cultured plant cells, GFP-tagged MerE was expressed transiently ( Figure 1A) and its fluorescence was visualized by confocal laser scanning microscopy ( Figure 1B). GFP-MerE was detected primarily in the endoplasmic reticulum (Closed arrowheads in Figure 1B), but some fusion proteins were detected in the plasma membrane (Open arrowheads in Figure 1B).
Expression of MerE in transgenic plants
The PCR-amplified merE DNA fragment was subcloned into a binary vector, pMAT137, to generate the plasmid pMAE2 ( Figure 2A). The plasmid was transformed into A. thaliana ecotype Columbia via Agrobacterium-mediated gene transfer (Clough and Bent 1998). Eleven merE independent transgenic lines were obtained by selection using MS medium containing 50 mg/L kanamycin. The MerE transformants exhibited no distinctive phenotypes.
For instance, the MerE transgenic plants had an equally normal growth as wild-type Arabidopsis. Six of eleven merE transgenic lines (lines E2, E3, E4, E5, E6, and E7) were selected for further studies. To confirm the presence of the merE transgene in the transgenic plants, total genomic DNA was extracted from the mature leaves of T 3 progeny, and the regions of the introduced merE fragments were amplified using the genomic DNA as a template. As expected, 0.23 kb PCR fragment was detected in the merE (lines E2, E3, E4, E5, E6 and E7) when U-Not-merE/L-Xba-merE were used as PCR primers ( Figure 2B). The expression levels of merE in plants were determined by reverse transcription-PCR (RT-PCR). The total RNA was isolated from leaves and specific primers were used to detect merE and Actin mRNA.
The merE mRNA was detected in all transformants tested ( Figure 2C). There was no significant difference in the mRNA levels of the individual transgenic lines, so the E2 transgenic plant was selected for further studies. The expression levels of MerE protein were measured in transgenic plants (lines E2) by immunoblot analysis using a polyclonal anti-MerE antibody, which was prepared in a previous study (Kiyono et al. 2009). A novel protein band of 8 kDa, which reacted specifically with the anti-MerE antibody, was identified in the crude cell extract and crude membrane fraction from transgenic plants (Figure 3, lane 1&3), whereas no protein band reacted with anti-MerE antibody was detected in the soluble fraction (Figure 3, lane 2). The protein size was consistent with the size predicted from the translation of the DNA sequence of the merE gene. These results demonstrated that the merE gene in transgenic plants was transcribed and translated into proteins with molecular mass of 8 kDa, which were probably located in the membrane fraction.
Mercury accumulation was determined in transgenic
Arabidopsis plants after exposure to HgCl 2 or CH 3 HgCl in MS gellan gum plates for 2 weeks. After treatment with 10 μM HgCl 2 , the amount of mercury accumulated in the entire merE transgenic plants were higher than that in wild-type plants ( Figure 4A). After treatment with 0.3 μM CH 3 HgCl, the amount of mercury accumulated in the entire merE transgenic plants were about 2-fold higher than that in the wild-type plants ( Figure 4B).
The effect of MerE expression on mercury resistance was evaluated in transgenic Arabidopsis plants (lines E2) by monitoring the root and shoot growth of seedlings after exposure to HgCl 2 or CH 3 HgCl in MS gellan gum plates for 2 weeks. In the absence of mercury, merE transgenic plants exhibited the same normal growth as wild-type Arabidopsis ( Figure 5A). In the presence of 5 μM HgCl 2 or 0.3 μM CH 3 HgCl, root and shoot growth were inhibited in transgenic seedlings and wild-type Arabidopsis ( Figure 5A). However, the shoot and root growth of the merE transgenic seedlings indicated significant tolerance compared with wild-type Arabidopsis in the presence of 5 μM HgCl 2 ( Figure 5A-C). The shoot and root growth of merE transgenic seedlings also appeared to be better than that of wild-type Arabidopsis in the presence of 0.3 μM CH 3 HgCl ( Figure 5A-C).
Discussion
Phytoremediation, using green plants to remove environmental pollutants including hazardous toxic metals removal from a large volume of contaminated sites is recognized as a cost-effective, sustainable and aesthetically pleasing technology (Tong et al. 2004). However, the use of plants, like all biological methods, does not allow 100% removal of contaminants because the remediation rates decrease as the concentrations of contaminant decrease (Clemens et al. 2002). In addition, phytoremediation is a slow process that requires a long time to complete the purification (McGrath and Zhao 2003). These potential faults may predominantly result from the low metaluptake activity and thereby limit its usefulness for practical application.
Among the strategies being used to overcome these disadvantages is the use of metal transporter to boost the uptake and transport of metal from soil into transgenic plants (Song et al. 2003). Expression of heavy metal transporter or periplasmic Hg(II)-binding protein genes under the control of a constitutive or inducible promoter may provide a means of improving metal uptake, thereby shortening the phytoremediation completion time (Kiyono et al. 2013;Kiyono et al. 2012;Nagata et al. 2009 Hsieh et al. 2006). In the present study, a transgenic Arabidopsis plants engineered to express mercury transporter, MerE (Kiyono et al. 2009;Sone et al. 2010) was constructed and the impact of expression of MerE on methylmercury accumulation was evaluated. By using the Agrobacterium-floral dip method, many independent transgenic Arabidopsis plants were obtained. The results obtained by genomic PCR (Figure 2B), RT-PCR ( Figure 2C) and immunoblot (Figure 3) analysis demonstrated that the gfp tagged merE was successfully integrated into the genome of Arabidopsis plants and substantially transcribed into mRNA and then translated into the expected fusion proteins in the transgenic plants. Transgenic Arabidopsis plants expressing merE grew vigorously at rates similar to those of wild-type plants, without exhibiting notable symptoms of stress ( Figure 5A).
These results suggest that the integration of merE gene had no deleterious effects on the plant growth. The transgenic Arabidopsis expressing MerE accumulated significantly more Hg(II) and CH 3 Hg(I) than the wild-type Arabidopsis from the mercurial-containing medium (Figure 4). These results reveal that MerE is indeed functional as a broad-spectrum mercury transporter in transgenic Arabidopsis, and suggest that accelerated mercurials uptake into the plants mediated by MerE would provide one possible way for shortening the completion time of phytoremediation of mercury pollution. Growth in a relatively higher concentration of mercurials and constitutive expression of mercury transport activity seem to be necessary for the plants applied in mercury remediation. The transgenic Arabidopsis displayed a relatively high level of Hg(II) and CH 3 Hg(I) resistance compared with the wild type ( Figure 5). These results demonstrated that the toxic Hg(II) and CH 3 Hg(I) in the culture medium may have been transported into plant cells by the integrated merE and substantially inactivated in the cells by the physiological activity of the plant.
Phytoremediation is an effective and aesthetically pleasing technique for cleaning up soils contaminated with mercurials where excavation or bioremediation is not practical or possible (Heaton et al. 2003;Lyyra et al. 2007;Meagher and Heaton 2005). However, the technique is still in its infancy stage. This study showed that the expression of the bacterial mercury transporter MerE promoted the transport and accumulation of mercuric ions and methylmercury in transgenic Arabidopsis, which may be a useful method to facilitate the improvement of plants that could be applied to the phytoremediation of mercuric ions and methylmercury pollution. It is hoped that the efficiency of these newlydesigned transgenic Arabidopsis plants will be validated in field experiments in the near future.
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2016-05-12T22:15:10.714Z
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2013-09-03T00:00:00.000
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220048226
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pes2o/s2orc
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v3-fos-license
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Integrin-Targeting Dye-Doped PEG-Shell/Silica-Core Nanoparticles Mimicking the Proapoptotic Smac/DIABLO Protein
Cancer cells demonstrate elevated expression levels of the inhibitor of apoptosis proteins (IAPs), contributing to tumor cell survival, disease progression, chemo-resistance, and poor prognosis. Smac/DIABLO is a mitochondrial protein that promotes apoptosis by neutralizing members of the IAP family. Herein, we describe the preparation and in vitro validation of a synthetic mimic of Smac/DIABLO, based on fluorescent polyethylene glycol (PEG)-coated silica-core nanoparticles (NPs) carrying a Smac/DIABLO-derived pro-apoptotic peptide and a tumor-homing integrin peptide ligand. At low μM concentration, the NPs showed significant toxicity towards A549, U373, and HeLa cancer cells and modest toxicity towards other integrin-expressing cells, correlated with integrin-mediated cell uptake and consequent highly increased levels of apoptotic activity, without perturbing cells not expressing the α5 integrin subunit.
Introduction
Apoptosis, or programmed cell death, is an essential process in the homeostasis of multicellular organisms. Apoptosis initiates through either the extrinsic death receptor pathway or the intrinsic mitochondrial signaling pathway, which both culminate with the activation of Cysteine ASPartic acid-specific proteASES (CASPASES), enzymes that degrade specific substrates implied in fundamental cellular processes. In mammals, caspase-3, -7 and -9 activity is regulated by the inhibitor of apoptosis proteins (IAPs) [1,2]. The mammalian IAP family includes eight members, all of which share the family-defining baculovirus IAP repeat (BIR) domain at the N-terminal end of the protein [3]. BIRs are protein-interacting modules with distinct binding properties, necessary for the anti-apoptotic activity [4][5][6].
A strict regulation of apoptosis is involved in many human diseases [7]. Tumorigenic cells exhibit significantly elevated expression levels of IAPs, resulting in the elusion of apoptosis, one of the defining hallmarks of cancer and an underlying cause of therapeutic resistance [8]. IAP-mediated caspase inhibition is depressed by the second mitochondria-derived activator (Smac)/direct inhibitor of apoptosis-binding protein with low pI (DIABLO), a mitochondrial protein that is translocated to the cytoplasm in apoptotic conditions [9]. Structural analysis proved that the N-terminal sequence of Smac/DIABLO is essential for its function in the interaction with the BIR domain of IAP [10]. As a consequence, peptides derived from the N-terminal sequence of Smac/DIABLO may represent attractive anticancer molecules.
In general, native peptides show too scarce stability and bioavailability to consent therapeutic or diagnostic applications [11,12]. To increase cellular uptake, the native seven-residue N-terminus of Smac/DIABLO (SmacN7) was bonded to a cell membrane-permeable octaArg peptide (R8). The resulting SmacN7-R8 was able to induce the apoptosis of human non-small lung cancer (NSCLC) cells H460 [13]. More recently, peptidomimetics [12] of the N-terminus of Smac/DIABLO with potentially higher stability and bioavailability, including retro-inverso [14], C-naphthyl substituted [15], and aza-peptides [16] have been designed and tested.
Nanoparticles (NPs) provide extraordinary opportunities as drug nanocarriers [17][18][19], due to their prolonged circulation time and both passive and active targeting abilities towards cancerous tissues/cells. The conjugation of peptides to NPs represents an effective approach to addressing the intrinsic drawbacks of the peptides, allowing the access to a variety of biomedical uses [20,21]. Specifically, this conjugation increases the circulating half-lives of the peptides in vivo, reducing the need for frequent administrations to sustain their efficacy [22]. As concerns the transport of Smac/DIABLO, Seneci et al. reported non-covalent and covalent superparamagnetic iron oxide NPs (SPIONs)-Smac/DIABLO mimetic nanoconjugates. Unfortunately, the nanoconjugates were almost inactive in assays against breast cancer MDA-MB-231 cells, ovarian carcinoma IGROV-1 cells, and cervical cancer HeLa cells [23]. Li et al. prepared a SmacN7-conjugated polymer containing the cell-penetrating R8 peptide and four hydrophobic tails. The Smac-conjugated polymer could self-assemble, giving NPs in the aqueous environment. At high concentrations (>10 µM), Smac-NPs elicited a measurable effect in MDA-MB-231 and H460 cells. The same NPs have been also used as a drug delivery system for doxorubicin (DOX) in combination therapy; DOX-loaded NPs exhibited higher cellular uptake and antitumor effect [24].
In this context, we designed nanosystems mimicking the Smac/DIABLO protein, based on inorganic fluorescent NPs coated with a biocompatible organic shell, functionalized with a Smac/DIABLO-derived peptide and/or a tumor-homing RGD integrin ligand peptide. We opted for micellar NPs composed of the tri-block surfactant copolymer Pluronic ® F127 (PF127) (polyethylene glycol-polypropyleneoxide-polyethylene glycol, PEG 100 -PPO 65 -PEG 100 ) and a dye-doped silica core. The PEG-PPO-PEG block copolymers alone can form micelles in aqueous media with a hydrophilic core, which can be used to non-covalently encapsulate hydrophobic dyes with minimal leakage [37,38]. Micelles are valuable systems for application in the field of imaging and drug delivery, since they are self-organized systems, and the encapsulation of dyes and drugs in these systems is straightforward from the preparation point of view. However, they are dynamic systems, and their stability is strongly influenced by their local concentration, pH, and eventually by the presence of other species like apolar molecules or proteins [21,38].
On the other hand, silica NPs as tools to develop targeting probes have several advantages over other nanomaterial and self-organized systems [39]. Indeed, silica is photophysically inert, is an intrinsically non-toxic material, and there are many synthetic approaches available to tune these nanosystems in terms of size and functionalization. The luminescence emission of these systems depends on the doping dye, so that a large variety of emission properties can be achieved by just choosing the right doping dye(s). The inclusion of dye molecules in a rigid matrix like silica often increases the quantum yield of the dyes and also their photostability, because of the rigidification of dye structure and the protection towards quenching molecules present in the environment [39]. These last two features are of prominent importance to univocally assign the recorded fluorescent signal to the presence of the NPs and to control the local concentration of the cytotoxic compound during the recognition event toward the targeted receptor.
For the purpose of peptide conjugation, NPs were prepared from a mixture of PF127 and its diazide derivative PF127-(N 3 ) 2 . After synthesis of the NPs and characterization, the azide terminations of the outer shell were exploited to covalently bind a Smac/DIABLO-derived peptide and/or a tumor-homing integrin ligand peptide. Then, the cytotoxicity, pro-apoptotic efficacy, and cellular uptake were determined for these peptide-NPs in diverse cells. Particularly, the role of integrin-mediated cell uptake was investigated by confocal microscopy.
General Methods
Standard chemicals, including protected amino acids, were purchased from commercial sources and used without further purification. Peptide purity was assessed by analytical RP HPLC performed on an 1100 series apparatus Agilent Technologies, Milan, Italy, using an XSelect Peptide CSH C18 column (Waters, Milford, MA, USA), 4.6 mm × 100 mm, 130 Å, 3.5 µm. MS (ESI) analysis was performed using an MS single quadrupole HP 1100 MSD detector (Agilent Technologies, Milan, Italy). The synthetic procedures by MW irradiation were performed with a Microwave Labstation for Synthesis (Micro-SYNTH, Bergamo, BG, IT) equipped with a built-in ATC-FO advanced fiber-optic automatic temperature control. Peptides isolation was performed by preparative RP HPLC performed on an 1100 series apparatus (Agilent), using an XSelect Peptide CSH C18 OBD column (Waters) 19 mm × 150 mm, 130 Å, 5 µm. The molecular weights of the purified peptides were verified by electrospray ionization (ESI)-mass spectrometry (MS) using an MS single quadrupole HP 1100 MSD detector (Agilent). Fluorescence measurements were performed with an LS-55 Fluorescence Spectrometer (Perkin Elmer, Milan, Italy). DLS measurements were performed with a Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK). For full details, please see the Supporting Information.
The precursor Boc-Ala-Val-Pro-Ile-Gly-OH was prepared by SPPS and cleavage using the same protocol as described above; all details are given in the Supporting Information. Briefly, the crude peptide was coupled to 4-pentyn-1-amine with HOBt/TBTU/DIPEA in DMF/DCM, under MW irradiation, and the tert-butyloxycarbonyl (Boc) was removed with TFA.
The dimesylate derivative of BASF Pluronic ® F127 (PF127), obtained in turn by the treatment of PF127 surfactant with trimethylamine/methanesulfonyl chloride, was reacted with NaN 3 . The resulting PF127-(N 3 ) 2 (20 mg) was mixed with PF127 (200 mg) and RhB-TES (4.0 mg) in DCM, followed by tetraethyl orthosilicate (TEOS, 350 µL) and trimethylchlorosilane (40 µL) in the presence of AcOH/NaCl. The obtained NPs were purified by dialysis and diluted with water to the final concentration of 29 µM [43]. For full details, please see the Supporting Information.
Cells and Culture Conditions
Human umbilical vein endothelial cells (Huvec), adenocarcinoma human alveolar basal epithelial cells (A549), human glioblastoma (U373), and human fibroblasts were obtained from Thermofisher Scientific, Waltham, MA, USA. The human cervical carcinoma (HeLa) cells and human colon cancer (HT29) cells were obtained from ATCC. DMEM, trypsin, PBS, Gly, and BSA 1% were purchased from Merck Co Ltd., Serono, Italy. Mouse anti-α-tubulin primary antibody was purchased from BioLegend, San Diego, CA. Anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody and Hoechst33342 were purchased from ThermoFisher. The MTS assay CellTiter 96 ® AQueous One Solution Cell Proliferation Assay and Caspase-Glo ® 9 assay were purchased from Promega, Italy. A Synergy HT microplate reader Biotek, Milan, Italy, was used.
Apoptosis
We incubated 1.0 × 10 4 cells/well with the peptides-NPs at the concentration of 1 µM for 6 h. The apoptotic process onset was evaluated by the Caspase-Glo ® 9 assay (Promega), according to the manufacturer's instructions. After 30 min, the luminescence was measured using a Synergy HT microplate reader (Biotek).
Cell Internalization
HT29 and HeLa cells were grown on sterile glass coverslips for 48 h and then treated with 1 µM peptide-NPs for 1 h. The cells were washed (3×) with PBS and fixed in 500 µL of 3% paraformaldehyde. The glass slides were washed twice with 1 mL of PBS-Gly 0.1 M and washed twice again with 1 mL of PBS-BSA 1%. The samples were first incubated with mouse anti-α-tubulin primary antibody for 1 h in agitation at rt. The samples were washed again twice with 1 mL of PBS-BSA 1% and then incubated with anti-mouse FITC-conjugated secondary antibody for 1 h at rt. Finally, the specimens were embedded in Mowiol and analyzed by confocal microscopy. Confocal images were obtained with a C1s confocal laser-scanning microscope equipped with a PlanApo, 60X or 40X, oil immersion lens (Nikon, Tokyo, Japan). The visualization and quantification of cells that internalized the rhodamine B (RhB)-NPs were performed using ImageJ (NIH, Bethesda, MD, USA).
Competition Experiments
HeLa cells were seeded on sterile glass coverslips for 48 h. Then, the cells were first pre-exposed to IgG isotype or anti-CD49e antibody for 1 h and then incubated with 1 µM peptide-NP for 1 h. The cells were stained with Hoechst33342, and the specimens were analyzed by confocal microscopy as described above. The number of cells, counterstained with Hoechst 33342, showing intracellular red fluorescence was expressed as % of the total cells.
Chemistry
Monodispersed fluorescent silica-core/PEG-shell NPs functionalized with azide moieties and incorporating the dye rhodamine B triethoxysilane (RhB-TES) [44] ( Figure 1A) were expediently obtained using a direct micelle-assisted method [45]. These nanostructures were formed by the condensation of the silica precursor TEOS in an aqueous acid environment in the presence of co-aggregates composed by a 10:1 mixture of the tri-block surfactant copolymer PF127 and its diazide derivative PF127-(N 3 ) 2 [43]. The condensation of RhB-TES within the silica core of the NP conferred the desired fluorescent properties to this nanosystem, preventing also the leaking of the fluorophore in the external environment. Transmission electron microscopy (TEM) images showed a silica core diameter dc = (10 ± 2) nm, while the hydrodynamic diameter measured by dynamic light scattering (DLS) was dH = 22 ± 1 nm (PDI = 0.10) ( Figure 1B-D), confirming the core/shell type architecture of the resulting NPs-N 3 .
By adapting reported procedures [47], the number of NP-bonded AVPI molecules was estimated by fluorimetric quantitation with fluorescamine (an amine-reactive fluorogenic tracer), against a standard calibration curve obtained with PEG-amine/fluorescamine (λex 390 nm, λem 480 nm). An aliquot of the AVPI-NP suspension (25 μL, 29 mM) was treated with fluorescamine, and the relative fluorescence intensity measured allowed to estimate 7.8 ± 1 peptides/NP. Alternatively, NPs functionalization was appraised by the fluorimetric quantitation of the dansyl group after CuAAC reaction with dansyl-AVPI-alkyne (Supporting Information), against a calibration curve obtained with unconjugated dansyl-AVPI-alkyne (λex 340 nm, λem 477 nm). Consistent with the fluorescamine method, this test gave 9.3 ± 1 dansyl-AVPI/NP and gave us the possibility to measure the amount of dansyl-AVPI bounded to a sample obtained by CuAAC reaction with a 1:1 mixture of dansyl-AVPI-alkyne and cRGD-alkyne. For this sample, an average number of 4.8 ± 1 dansyl-AVPI/NP was determined, indicating by the difference that cRGD-alkyne reacted circa to the same extent.
Cytotoxicity of Peptide-NPs
The in vitro cell growth inhibitory efficacy was determined for the NPs and the unconjugated AVPI peptide, by incubating A549, U-373, HeLa, Huvec, and fibroblast cells with increasing concentrations of the compounds (0.1, 1.0, 3.0 μM) for 48 h. Cell viability is reported in Figure 2A; in By adapting reported procedures [47], the number of NP-bonded AVPI molecules was estimated by fluorimetric quantitation with fluorescamine (an amine-reactive fluorogenic tracer), against a standard calibration curve obtained with PEG-amine/fluorescamine (λex 390 nm, λem 480 nm). An aliquot of the AVPI-NP suspension (25 µL, 29 mM) was treated with fluorescamine, and the relative fluorescence intensity measured allowed to estimate 7.8 ± 1 peptides/NP. Alternatively, NPs functionalization was appraised by the fluorimetric quantitation of the dansyl group after CuAAC reaction with dansyl-AVPI-alkyne (Supporting Information), against a calibration curve obtained with unconjugated dansyl-AVPI-alkyne (λex 340 nm, λem 477 nm). Consistent with the fluorescamine method, this test gave 9.3 ± 1 dansyl-AVPI/NP and gave us the possibility to measure the amount of dansyl-AVPI bounded to a sample obtained by CuAAC reaction with a 1:1 mixture of dansyl-AVPI-alkyne and cRGD-alkyne. For this sample, an average number of 4.8 ± 1 dansyl-AVPI/NP was determined, indicating by the difference that cRGD-alkyne reacted circa to the same extent.
Cytotoxicity of Peptide-NPs
The in vitro cell growth inhibitory efficacy was determined for the NPs and the unconjugated AVPI peptide, by incubating A549, U-373, HeLa, Huvec, and fibroblast cells with increasing concentrations of the compounds (0.1, 1.0, 3.0 µM) for 48 h. Cell viability is reported in Figure 2A; in general, all the combinations tested were ineffective at the concentration of 0.1 µM; therefore, these data were omitted. As expected, the simple peptide AVPI did not show any toxicity (data not shown), plausibly due to poor-to-null intracellular uptake [24]. The NP-N 3 appeared well tolerated, since no detectable decrease in cell viability was observed after 48 h.
general, all the combinations tested were ineffective at the concentration of 0.1 μM; therefore, these data were omitted. As expected, the simple peptide AVPI did not show any toxicity (data not shown), plausibly due to poor-to-null intracellular uptake [24]. The NP-N3 appeared well tolerated, since no detectable decrease in cell viability was observed after 48 h. As for the peptide-NPs, 1 μM cRGD-NPs showed very little toxicity, and modest toxicity when the concentration was increased to 3 μM. At the concentration of 1 μM, AVPI-NPs induced a decrease of viability of about 25% in A549, U373, and HeLa cells, and of 33% in Huvec and fibroblast cells. At the concentration of 3 μM, AVPI-NPs showed much higher toxicity against Huvec and fibroblasts, reducing their viability by 60%, while the effect was lower in U373, A549, and HeLa cells, whose vitality was decrased by 37% and 30%, respectively. In contrast, 1 μM AVPI/cRGD-NPs significantly inhibited the proliferation of A549, U373, HeLa, and Huvec cells of about 60% and showed a comparatively lower effect towards fibroblasts. Increasing the concentration of AVPI/cRGD-NPs to 3 μM led in general to higher toxicity, whit the exclusion of A549 cells for which the toxicity remained the same.
Caspase-9 Activity
The activity of caspase-9 [9] was assayed by a fluorimetric method in A549, U373, HeLa, Huvec, and fibroblast cells treated with 1 µ M peptide-NPs for 6 h ( Figure 2B). The AVPI-NPs gave a moderate but well measurable increase of activity, about four-fold, as compared to untreated control cells. On the other hand, AVPI/cRGD-NPs showed a >40-fold increase towards A549 and HeLa cells, Huvec, U373, and a comparatively lower 10-fold increase in activity in fibroblasts.
Cellular Uptake of Peptide-NPs
The internalization of the fluorescent peptide-NPs was observed by confocal microscopy in HeLa (α5 subunit-positive) [48] and in HT29 (α5 subunit-negative) cells [49,50]. In control HT29 cells, the internalization of AVPI-NPs was modest, and that of cRGD-NPs and AVPI/cRGD-NPs was very poor, as shown by the low fluorescent signal in the cytoplasm ( Figure 3A,B). By contrast, HeLa cells exposed to cRGD-NPs and AVPI/cRGD-NPs, but not AVPI-NPs, gave a similar, very high number of fluorescence-positive cells ( Figure 3D,E). The scarce internalization of AVPI-NPs was consistent with the modest decrease of viability of HT29 (about 20%, Figure 3C) and HeLa (approx. 25%, Figure 2A) cells. As for the peptide-NPs, 1 µM cRGD-NPs showed very little toxicity, and modest toxicity when the concentration was increased to 3 µM. At the concentration of 1 µM, AVPI-NPs induced a decrease of viability of about 25% in A549, U373, and HeLa cells, and of 33% in Huvec and fibroblast cells. At the concentration of 3 µM, AVPI-NPs showed much higher toxicity against Huvec and fibroblasts, reducing their viability by 60%, while the effect was lower in U373, A549, and HeLa cells, whose vitality was decrased by 37% and 30%, respectively. In contrast, 1 µM AVPI/cRGD-NPs significantly inhibited the proliferation of A549, U373, HeLa, and Huvec cells of about 60% and showed a comparatively lower effect towards fibroblasts. Increasing the concentration of AVPI/cRGD-NPs to 3 µM led in general to higher toxicity, whit the exclusion of A549 cells for which the toxicity remained the same.
Caspase-9 Activity
The activity of caspase-9 [9] was assayed by a fluorimetric method in A549, U373, HeLa, Huvec, and fibroblast cells treated with 1 µM peptide-NPs for 6 h ( Figure 2B). The AVPI-NPs gave a moderate but well measurable increase of activity, about four-fold, as compared to untreated control cells. On the other hand, AVPI/cRGD-NPs showed a >40-fold increase towards A549 and HeLa cells, Huvec, U373, and a comparatively lower 10-fold increase in activity in fibroblasts.
Cellular Uptake of Peptide-NPs
The internalization of the fluorescent peptide-NPs was observed by confocal microscopy in HeLa (α5 subunit-positive) [48] and in HT29 (α5 subunit-negative) cells [49,50]. In control HT29 cells, the internalization of AVPI-NPs was modest, and that of cRGD-NPs and AVPI/cRGD-NPs was very poor, as shown by the low fluorescent signal in the cytoplasm ( Figure 3A,B). By contrast, HeLa cells exposed to cRGD-NPs and AVPI/cRGD-NPs, but not AVPI-NPs, gave a similar, very high number of fluorescence-positive cells ( Figure 3D,E). The scarce internalization of AVPI-NPs was consistent with the modest decrease of viability of HT29 (about 20%, Figure 3C) and HeLa (approx. 25%, Figure 2A The merge of the images of HeLa treated with cRGD-NPs or AVPI/cRGD-NPs showed some cells colored in yellow/orange, but this observation was not indicative of colocalization between RhB and α-tubulin. These interactions were quantified by analyzing the correlation and/or the overlap between images, using the Pearson's and Manders' coefficients, respectively. Finally, the cells were pre-incubated with anti-CD49e antibody or mouse immunoglobulin G (IgG) antibody before exposure to AVPI/cRGD-NPs ( Figure 3F). Microscopic observations showed that, in the presence of the antibody, the internalization was considerably reduced, as compared to cells incubated in the presence of control IgG ( Figure 3G). The merge of the images of HeLa treated with cRGD-NPs or AVPI/cRGD-NPs showed some cells colored in yellow/orange, but this observation was not indicative of colocalization between RhB and α-tubulin. These interactions were quantified by analyzing the correlation and/or the overlap between images, using the Pearson's and Manders' coefficients, respectively.
Finally, the cells were pre-incubated with anti-CD49e antibody or mouse immunoglobulin G (IgG) antibody before exposure to AVPI/cRGD-NPs ( Figure 3F). Microscopic observations showed that, in the presence of the antibody, the internalization was considerably reduced, as compared to cells incubated in the presence of control IgG ( Figure 3G).
Discussion
The aim of our study was to develop fluorescent probes with sufficient brightness, able to selectively sustain a long-term interaction with cellular receptors in dilute conditions. High brightness, low in vivo toxicity, and ease of functionalization with pharmacologically active biomolecules make fluorescent silica-based NPs attractive platforms for diagnostic and theranostic applications in cancer [39]. Hence, we prepared dye-doped silica NPs surrounded by an outer shell of the biocompatible polymer PEG, which is expected to increase NP dispersion in physiological conditions ( Figure 1A) and to oppose the uptake by the reticuloendothelial system [51,52]. The covalent inclusion of the RhB derivative was adopted to prevent dye leaking in the external environment, a behavior that can affect the signal-to-noise ratio during optical imaging experiments. The influence of "minimal leakage" of fluorescent dyes from NPs or nanosystems on the overall fluorescent signal recorded during real experiments with cells is often a very difficult variable to quantify. For this reason, we preferred to circumvent this problem by the covalent linking of the dye to the NP silica core.
To avoid the leakage of the cytotoxic and targeting compounds, the azide termini of PEG were exploited for the covalent functionalization with peptide-alkyne partners. We designed the sequence AVPI-alkyne: the AVPI N-terminal tetrapeptide of Smac/DIABLO maintained a binding affinity for IAP BIR of 0.5 µM [10], while Gly and the C5 amine served as spacers. The cRGD-alkyne sequence was designed on the basis of the well-known Kessler's α5β1 integrin ligand c[RGDfK] [46]. It is well known that simple peptides, such as the AVPI and RGD sequences, delivered to the body are subject to enzymatic degradation and are poorly permeable through biological membranes. Nevertheless, inorganic NP carriers can support the transport of peptides by protecting them from environmental conditions while maintaining their stability [20,22].
The stability of the NPs was previously tested under different pseudo physiological and in vivo experimental situations. Pluronic ® F127/silica-core/PEG-shell NPs, doped with RhB and/or polymethine cyanine dye, demonstrated outstanding stability in the presence of phosphate-buffered saline and bovine serum albumin (PBS/BSA) [53]. These NPs were tested in small animals for in vivo total-body imaging and intravital 3D imaging, giving well detectable signals for hours after injection. The same silica-core/PEG-shell NPs, doped with cyanine 7 dye, were subcutaneously injected in animals, and the in vivo fluorescence signal in the right axillary lymph node was detected for at least 8 h [54].
The cytotoxicity of the resulting peptide-NPs was evaluated in A549, U-373, HeLa, Huvec, and fibroblast cells. Compared to AVPI-NPs, the dual functionalized AVPI/cRGD-NPs showed much higher toxicity towards the cancer cells and reduced toxicity towards fibroblasts. Indeed, the AVPI/cRGD-NPs inhibited the proliferation of A549, U373, HeLa, and Huvec cells of about 60% already at the concentration of 1 µM, a significantly higher efficacy as compared to that of the previously reported Smac/DIABLO-NPs [23,24]. To understand whether the peptide-NPs restored apoptotic cancer cell death, the activity of caspase-9 was measured. While AVPI-NPs gave a moderate increase of caspase activity, AVPI/cRGD-NPs showed a >40-fold increase in A549, HeLa, Huvec, and U373 cells and a comparatively lower effect in fibroblasts.
The much higher apoptotic effect of AVPI/cRGD-NPs over AVPI-NPs towards cancer cells appeared clearly correlated to integrin-mediated cellular uptake. Internalization of peptide-NPs was observed in HeLa (α5 subunit-positive) and in control HT29 (α5 subunit-negative) cells. cRGD-NPs and AVPI/cRGD-NPs showed much higher internalization in HeLa cells ( Figure 3D,E) than AVPI-NPs, likely mediated by the interaction between the RGD peptides and the integrin receptors. This observation was supported by the almost negligible uptake of both RGD-NPs and AVPI/cRGD-NPs in the HT29 cell line not expressing the α5 subunit ( Figure 3A). To confirm that the uptake of AVPI/cRGD NPs was integrin-mediated, exclusion studies were carried out by incubating the cells with anti-CD49e antibody or mouse IgG antibody before exposure to AVPI/cRGD-NPs. Microscopic observations showed that, in the presence of anti-CD49e, the internalization was strongly reduced, as compared to cells incubated in the presence of the control IgG ( Figure 3F).
Conclusions
In this paper, we described synthetic NPs mimicking the proapoptotic protein Smac/DIABLO, constituted by a fluorescent silica core doped with RhB, coated with a PEG shell, and carrying the AVPI peptide and/or a tumor-homing cRGD peptide. The bifunctional AVPI/RGD-NPs showed superior toxicity towards cancer cells, correlated to increased levels of caspase activity, plausibly due to efficient integrin-mediated transport into the cells, as shown by confocal microscopy, and modest toxicity towards cells not expressing the α5 integrin subunit. In perspective, these Smac/DIABLO-mimetic nanosystems can find applications in the treatment of cancer and, thanks to the combination with the fluorescent dye, they can provide new insight into integrin-mediated internalization.
Conflicts of Interest:
The authors declare no conflict of interest.
|
2020-06-25T09:09:26.447Z
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2020-06-01T00:00:00.000
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249256580
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pes2o/s2orc
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v3-fos-license
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Low-Frequency Vibrational Spectroscopy Characteristic of Pharmaceutical Carbamazepine Co-Crystals with Nicotinamide and Saccharin
The pharmaceutical co-crystal has attracted increasing interest due to the improvement of physicochemical properties of active pharmaceutical ingredients. The characterization of pharmaceutical co-crystal is an integral part of the pharmaceutical field. In this paper, the low-frequency vibrational properties for carbamazepine co-crystals with nicotinamide and saccharin (CBZ-NIC and CBZ-SAC) have been characterized by combining the THz spectroscopy with low-wavenumber Raman spectroscopy. The experiment results show that, compared with the individual constituents, CBZ-NIC and CBZ-SAC co-crystals not only have different characteristic absorption peaks in the 0.3-2.5 THz region, but also have significant low-wavenumber Raman characteristic peaks in 0–100 cm−1. Density functional theory was performed to simulate the terahertz and low-wavenumber Raman spectra of the two co-crystals, where the calculation agreed well with the measured vibrational peak positions. The vibrational modes of CBZ-NIC and CBZ-SAC co-crystals were assigned through comparing theoretical results with the experimental spectra. Meanwhile, the low-frequency infrared and/or Raman active of characteristic peaks for such co-crystals were discussed. The results indicate the combination of THz spectroscopy and low-wavenumber Raman spectroscopy can provide more comprehensive low-frequency vibrational information for pharmaceutical co-crystals, such as collective vibration and skeleton vibration, which could play an important role in pharmaceutical science.
Introduction
Carbamazepine (CBZ) is a common psychotropic drug, which is preferred in the treatment of epilepsy and has had better therapeutic effects in treating trigeminal neuralgia and bipolar affective disorder. As a typical oral drug, however, CBZ shows low solubility and limited bioavailability [1,2]. The pharmaceutical co-crystal is an effective approach to improve CBZ solubility and bioavailability, which form the combination between active pharmaceutical ingredients (APIs) and co-crystal conformers (CCFs) by hydrogen bonding or intermolecular interactions [3][4][5][6]. CBZ co-crystals show unique physicochemical properties without altering the chemical nature and bioactivity of CBZ, such as high solubility and bioavailability [7][8][9][10]. In the past, a series of co-crystals between CBZ and various CCFs have been reported [11,12].
Currently, the basic physicochemical properties of pharmaceutical co-crystals are usually characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), infrared spectroscopy (IR) and Raman spectroscopy. PXRD, as a common method was collected by THz detector. The spectrum was measured from 0.3 to 2.5 THz with a frequency resolution of 7.6 GHz and dynamic range of 70 dB. All the experimental results were obtained using transmission mode at room temperatures. The relative humidity was always kept below 3% by purging the drying air during the measurements in order to prevent the effect of the absorption of atmospheric vapor. The final spectrum of each sample was the average of five measurements. The time domain signals of each sample and reference (without sample) were converted to frequency domain signals through the Fourier transform. Then, the THz spectrum was the result of the sample frequency signal divided by corresponded reference [24].
Theoretical Calculation
Quantum chemical computation of theoretical structures was carried out using the DFT. The initial structures of the CBZ-NIC and CBZ-SAC co-crystals were obtained from the Cambridge Crystallographic Data Centre (CCDC), as shown in Figure 2. The CCDC number of CBZ-NIC and CBZ-SAC are 1544195 and 1562073, respectively. The simulated low-wavenumber Raman and THz spectra of the CBZ-NIC and CBZ-SAC co-crystals were calculated with the B3LYP functional and 6-311++G (d, p) basis set. Lorentzian line shapes
Apparatus and Procedure
In order to confirm the formation of the co-crystal, CBZ-NIC and CBZ-SAC co-crystals were identified using PXRD. PXRD patterns of the two co-crystals were recorded with the Rigaku Smartlab 9 KW diffraction system using a Cu Kα source (λ = 0.15406 nm) over the 2θ range from 20 • to 70 • . The scanning speed was set to 3 • /s. Low-wavenumber Raman spectra were measured using a commercial THz-Raman microscope system, as shown in Figure 1c. This system mainly contains an integrated laser module compatible (Ondax, Monrovia, CA, USA), a microscope (Leica, Wetzlar, Germany) and a spectrometer (Horiba, Japan) equipped with a CCD detector cooled to −50°C (Syncerity, Horiba, Kyoto, Japan). The integrated laser module compatible comprises of a stable 785 nm laser as an excitation light source and a series of high-throughput volume holographic grating (VHG) filters. Briefly, two VHG amplified spontaneous emission (ASE) filters (NoiseBlockTM, Ondax, Inc., Monrovia, CA, USA) were used to remove ASE from the laser. The beam was focused on the samples by a 10× objective lens. Then the 180 • backscattered light from sample was collected and filtered through the VHG beam splitter towards the two ultra-narrowband VHG notch filters (SureLockTM, Ondax, Inc.), which can further remove the collected Ralyeigh scattered light [23]. The filtered signal was focused into a spectrometer within a 1200 grooves/mm grating via a fiber. The Raman scattered light was detected by a CCD detector. The measurement range was from −200 cm −1 to 2000 cm −1 with a spectral resolution of 2.5 cm −1 . Each Raman spectrum was recorded using 70 mW laser power with 10 s acquisition times. In order to compare the Raman spectrum with the THz spectrum, the analysis range of the Raman spectrum was chosen as 0-100 cm −1 . The final Raman spectrum of each sample was the averaged result of five measurements, where the spectrum of each measurement was acquired at different positions. The experimental temperature was maintained at room temperature during the whole experiment.
The THz spectrum was obtained using the THz time-domain spectrometer (Advantest Corp., TAS7500SP, Tokyo, Japan), as shown in Figure 1d. A pair of GaAs photoconductive antennas as the THz emitter and detector were driven by a femtosecond laser with the center wavelength of 800 nm. The emitted THz radiation was collimated and focused on samples by parabolic mirrors. Then, the signal transmitted through the sample was collected by THz detector. The spectrum was measured from 0.3 to 2.5 THz with a frequency resolution of 7.6 GHz and dynamic range of 70 dB. All the experimental results were obtained using transmission mode at room temperatures. The relative humidity was always kept below 3% by purging the drying air during the measurements in order to prevent the effect of the absorption of atmospheric vapor. The final spectrum of each sample was the average of five measurements. The time domain signals of each sample and reference (without sample) were converted to frequency domain signals through the Fourier transform. Then, the THz spectrum was the result of the sample frequency signal divided by corresponded reference [24].
Theoretical Calculation
Quantum chemical computation of theoretical structures was carried out using the DFT. The initial structures of the CBZ-NIC and CBZ-SAC co-crystals were obtained from the Cambridge Crystallographic Data Centre (CCDC), as shown in Figure 2. The CCDC number of CBZ-NIC and CBZ-SAC are 1544195 and 1562073, respectively. The simulated low-wavenumber Raman and THz spectra of the CBZ-NIC and CBZ-SAC co-crystals were calculated with the B3LYP functional and 6-311++G (d, p) basis set. Lorentzian line shapes were convolved into the calculated vibrational modes using a full-width half-maximum (FWHM) value of 4.0 cm −1 [25,26].
PXRD Analysis
The PXRD patterns for the CBZ-NIC and CBZ-SAC co-crystals with their individual constituents were shown in Figure 3. It was obvious that there were significant characteristic diffraction peaks in the 10-35° range. The PXRD patterns of CBZ-NIC and CBZ-SAC co-crystals with their individual constituents were consistent with the literature, which verified the reliability of our prepared co-crystals [3,5]. Figure 4 showed THz spectra of CBZ-NIC and CBZ-SAC co-crystals with their individual constituents in the 0.3-2.5 THz region, where the positions of all characteristic peaks were labeled. It can be seen from Figure 4a that CBZ as an API has three characteristic peaks at 1.23, 1.82 and 2.04 THz, in which the peaks at 1.23 and 1.82 THz show strong THz absorption characteristic, while the peak at 2.04 THz is relative weak. The NIC has one very narrow strong THz absorption peak at 1.06 THz, one strong peak at 1.93 THz and three weak peaks at 0.60, 1.70 and 2.39 THz, respectively. The THz spectra of CBZ and NIC were in good agreement with the previous work except an obvious characteristic peak of NIC at 2.39 THz [5]. The SAC exhibits three weak peaks at 1.13, 1.89 and 2.01 THz,
PXRD Analysis
The PXRD patterns for the CBZ-NIC and CBZ-SAC co-crystals with their individual constituents were shown in Figure 3. It was obvious that there were significant characteristic diffraction peaks in the 10-35 • range. The PXRD patterns of CBZ-NIC and CBZ-SAC cocrystals with their individual constituents were consistent with the literature, which verified the reliability of our prepared co-crystals [3,5].
PXRD Analysis
The PXRD patterns for the CBZ-NIC and CBZ-SAC co-crystals with their individual constituents were shown in Figure 3. It was obvious that there were significant characteristic diffraction peaks in the 10-35° range. The PXRD patterns of CBZ-NIC and CBZ-SAC co-crystals with their individual constituents were consistent with the literature, which verified the reliability of our prepared co-crystals [3,5]. Figure 4 showed THz spectra of CBZ-NIC and CBZ-SAC co-crystals with their individual constituents in the 0.3-2.5 THz region, where the positions of all characteristic peaks were labeled. It can be seen from Figure 4a that CBZ as an API has three characteristic peaks at 1.23, 1.82 and 2.04 THz, in which the peaks at 1.23 and 1.82 THz show strong THz absorption characteristic, while the peak at 2.04 THz is relative weak. The NIC has one very narrow strong THz absorption peak at 1.06 THz, one strong peak at 1.93 THz and three weak peaks at 0.60, 1.70 and 2.39 THz, respectively. The THz spectra of CBZ and NIC were in good agreement with the previous work except an obvious characteristic peak of NIC at 2.39 THz [5]. The SAC exhibits three weak peaks at 1.13, 1.89 and 2.01 THz, Figure 4 showed THz spectra of CBZ-NIC and CBZ-SAC co-crystals with their individual constituents in the 0.3-2.5 THz region, where the positions of all characteristic peaks were labeled. It can be seen from Figure 4a that CBZ as an API has three characteristic peaks at 1.23, 1.82 and 2.04 THz, in which the peaks at 1.23 and 1.82 THz show strong THz absorption characteristic, while the peak at 2.04 THz is relative weak. The NIC has one very narrow strong THz absorption peak at 1.06 THz, one strong peak at 1.93 THz and three weak peaks at 0.60, 1.70 and 2.39 THz, respectively. The THz spectra of CBZ and NIC were in good agreement with the previous work except an obvious characteristic peak of NIC at 2.39 THz [5]. The SAC exhibits three weak peaks at 1.13, 1.89 and 2.01 THz, as shown in Figure 4b. However, the measured spectral properties at 1.89 and 2.01 THz were different from that in Ref [5], where a broadband characteristic peak at 1.98 THz appeared. sorption characteristics and two weak peaks appear at 1.46 and 2.22 THz, as shown in Figure 4b. It is obvious that those absorption features of the co-crystal do not derive from the superposition of THz spectra of their individual constituents. It could be inferred that the inter-molecular interactions between the CBZ and CCFs result in the structure change of the unit cells between the raw materials, which can be characterized by THz spectroscopy [26]. In addition, it should be mentioned that the new THz characteristic peaks of CBZ-NIC and CBZ-SAC co-crystals were detected compared with the results in the reference [5]. It may be due to the difference of spectral resolution and measurement range for experimental systems. Tables 1 and 2. As shown in Figure 5a, the CBZ-NIC co-crystal has five THz absorption peaks at the positions of 0.32, 0.82, 1.62, 2.07 and 2.56 THz in the theoretical spectrum, which are essentially in agreement with the experimental result.
THz Absorption Spectral Characteristic and Analysis of CBZ-NIC and CBZ-SAC Co-Crystals
The experimental peak at 0.41 THz corresponds to the vibrational mode calculated at 0.32 THz, which arises from CBZ and NIC molecules' collective twisting vibration. The experimental peak at 0.63 THz can be attributed to the simulated mode at the position at 0.82 THz, arising from the collective out-of-plane rocking vibration of the CBZ and NIC molecules. The characteristic peak at 1.56 THz corresponds to the theoretical spectrum at 1.62 THz, which is due to the strong collective out-of-plane rocking vibration of CBZ and the weak collective in-plane rocking vibration of NIC. The experimental spectral feature at 1.91 THz can be attributed to the calculated vibration mode at 2.07 THz, which is caused THz spectra of co-crystals exhibit different characteristics from their individual constituents. From Figure 4a, the CBZ-NIC co-crystal has two strong THz peaks at 1. Figure 4b. It is obvious that those absorption features of the co-crystal do not derive from the superposition of THz spectra of their individual constituents. It could be inferred that the inter-molecular interactions between the CBZ and CCFs result in the structure change of the unit cells between the raw materials, which can be characterized by THz spectroscopy [26]. In addition, it should be mentioned that the new THz characteristic peaks of CBZ-NIC and CBZ-SAC co-crystals were detected compared with the results in the reference [5]. It may be due to the difference of spectral resolution and measurement range for experimental systems.
The DFT theoretical spectra of CBZ-NIC and CBZ-SAC co-crystals are shown in Figure 5. For better comparison, the THz spectra of CBZ-NIC and CBZ-SAC in Figure 4 were depicted again in Figure 5. At the same time, all of these vibrational mode distributions are presented in Tables 1 and 2. As shown in Figure 5a, the CBZ-NIC co-crystal has five THz absorption peaks at the positions of 0.32, 0.82, 1.62, 2.07 and 2.56 THz in the theoretical spectrum, which are essentially in agreement with the experimental result.
The experimental peak at 0.41 THz corresponds to the vibrational mode calculated at 0.32 THz, which arises from CBZ and NIC molecules' collective twisting vibration. The experimental peak at 0.63 THz can be attributed to the simulated mode at the position at 0.82 THz, arising from the collective out-of-plane rocking vibration of the CBZ and NIC molecules. The characteristic peak at 1.56 THz corresponds to the theoretical spectrum at 1.62 THz, which is due to the strong collective out-of-plane rocking vibration of CBZ and the weak collective in-plane rocking vibration of NIC. The experimental spectral feature at 1.91 THz can be attributed to the calculated vibration mode at 2.07 THz, which is caused by the NIC molecule's strong collective twisting vibration and the CBZ molecule's weak shearing vibration. The peak at the position 2.26 THz in the experimental result corresponds to the calculated mode at 2.56 THz, arising from the strong out-of-plane rocking vibration of N2-6C=10O and the twisting of C7-16C-17C-19C-21C-30C within CBZ molecules, and the weak collective in-plane rocking vibration of the NIC molecule.
CBZ-SAC co-crystals, the relative intensities of absorption peaks are different, and the THz characteristic peaks shifted a little. This can be attributed to the fact that the theoretical calculation was performed at an absolute 0 degree temperature, whereas the experimental spectrum was obtained under room temperature [27]. It is demonstrated that THz spectroscopy is sensitive to the collective molecular vibration, weak molecular vibration and skeleton vibration.
Additionally, the absorption peak of the CBZ-NIC co-crystal at 1.09 THz and the absorption peak of the CBZ-SAC co-crystal at 1.46 THz in the measured spectra does not appear in the corresponding theoretical spectra. It might be that the theoretical calculation is only of a single molecule unit, and ignores intermolecular forces within solid-state crystalline unit cells [27]. As shown in Figure 5b, the CBZ-SAC co-crystal also has five THz absorption characteristic peaks at the positions of 0.23, 0.86, 1.65, 2.00 and 2.50 THz in the theoretical spectrum. The experimental THz peak at 1.04 THz can be attributed to the calculated vibration mode at 0.86 THz, which is due to the collective in-plane shearing vibration of the CBZ and SAC molecules. The characteristic peak at 1.68 THz corresponds to the calculated spectrum at 1.65 THz, which is caused by the strong bending vibration of CBZ, the in-plane rocking vibration of O2=S1=O3 within the SAC, and the weak out-of-plane rocking vibration of SAC. The experimental peak at 2.22 THz corresponds to the theoretical spectrum at 2.00 THz, which is caused by the bending vibration of C45-C36-C34-C32-C31-C22 belonging to the CBZ molecule. The experimental spectral feature at 2.35 THz corresponds to the simulated peak at 2.50 THz, arising from the out-of-plane rocking vibration of N19-21C=O18 and the twisting vibration of C45-C36-C34-C32-C31-C22 of the CBZ molecule. It is noted that, comparing the experimental and theoretical spectra of the CBZ-NIC and CBZ-SAC cocrystals, the relative intensities of absorption peaks are different, and the THz characteristic peaks shifted a little. This can be attributed to the fact that the theoretical calculation was performed at an absolute 0 degree temperature, whereas the experimental spectrum was obtained under room temperature [27]. It is demonstrated that THz spectroscopy is sensitive to the collective molecular vibration, weak molecular vibration and skeleton vibration.
Additionally, the absorption peak of the CBZ-NIC co-crystal at 1.09 THz and the absorption peak of the CBZ-SAC co-crystal at 1.46 THz in the measured spectra does not appear in the corresponding theoretical spectra. It might be that the theoretical calculation is only of a single molecule unit, and ignores intermolecular forces within solid-state crystalline unit cells [27]. Figure 6 shows the experimental low-wavenumber Raman spectra of CBZ-NIC and CBZ-SAC co-crystals with their individual constituents, where the position of all characteristic peaks were labeled. It is obvious that the CBZ molecule has five Raman characteristic peaks in the 0-100 cm −1 region, which contains one strong peak at 39.6 and 90.4 cm −1 , and three relatively weak peaks at 47.9, 65.3 and 74.0 cm −1 . The low-wavenumber Raman spectrum of NIC has three characteristic peaks at 26.1, 50.5 and 74.8 cm −1 , in which the peak at 26.1 cm −1 exhibits strong and sharp characteristic, the peak at 74.8 cm −1 is relative weak and the peak at 50.5 cm −1 is very weak, as shown in Figure 6a. From Figure 6b, SAC shows three strong characteristic peaks at 24.3, 50.1 and 56.9 cm −1 , a strong shoulder peak at 64.7 cm −1 , and two weak peaks at 79.4 and 90.7 cm −1 . For CBZ-NIC and CBZ-SAC co-crystals, they exhibit different Raman features from their individual constituents, as shown in Figure 6a,b, respectively. A comparison of the theoretical DFT calculation and the experimental Raman spectral results are shown in Figure 7. The characteristic vibrational peaks of the CBZ-NIC and CBZ-SAC co-crystals shown in the low-wavenumber Raman spectra are summarized in detail in Tables 3 and 4, respectively. The CBZ-NIC co-crystal has five low-wavenumber Raman characteristic peaks at the position of 9.2, 27.4, 53.7, 73.8 and 91.3 cm −1 in the theoretical spectrum, as shown in Figure 7a. The peak at the position 23.4 cm −1 in the experimental result corresponds to the peak at 27.4 cm −1 in the theoretical spectrum, which is caused by the collective out-of-plane rocking vibration of CBZ and NIC molecules.
Low-Wavenumber Raman Spectral Characteristic and Analysis of CBZ-NIC and CBZ-SAC Co-Crystals
The experimental peak at 51.9 cm −1 can be attributed to the simulated mode at the position 53.7 cm −1 , arising from strong collective out-of-plane rocking vibration of CBZ molecules, and weak collective in-plane rocking vibration of NIC. The experimental peak at 73.0 cm −1 corresponds to the calculated the peak at 73.8 cm −1 . This peak is due to the bending vibration of C7-16C-17C-19C-21C-30C within CBZ molecules, and weak in-plane rocking vibration of NIC molecules.
CBZ-SAC co-crystal has seven low-wavenumber Raman characteristic peaks at the positions of 8.7, 21.5, 28.6, 54.2, 65.5, 91.1 and 99.6 cm −1 in the theoretical spectrum, as shown in Figure 7b. The characteristic peak of the CBZ-SAC co-crystal at 29.2 cm −1 in the experimental result corresponds to the peaks calculated at 28.6 cm −1 , which is due to the collective in-plane shearing vibration of CBZ and SAC molecules. The experimental spectral feature at 50.8 cm −1 can attributed to the simulated result at 54.2 cm −1 , which arises from a combination of CBZ molecules' strong bending vibration and the in-plane rocking vibration of O2=S1=O3 of SAC molecules. The experimental Raman peak at 64.6 cm −1 corresponded to the calculated vibrational mode at 65.5 cm −1 arises from the bending vibration of C45-C36-C34-C32-C31-C22, which belongs to CBZ molecules. These results show that the low-wavenumber Raman spectroscopy can characterize the collective vibration of molecules of different types, weak molecular vibration and molecular skeleton vibration with low-frequency Raman characteristic.
It should be noted that there is still a significant gap between the experimental and the calculated results of the two co-crystals. The CBZ-NIC co-crystal characteristic peaks at 9.2 and 91.3 cm −1 and the CBZ-SAC co-crystal peaks at 8.7, 21.5, 91.1 and 99.6 cm −1 in the theoretical spectra do not appear in the corresponding experimental spectra. Furthermore, the CBZ-NIC co-crystal peak at 43.6 cm −1 and the CBZ-SAC co-crystal peaks at 37.1, The CBZ-NIC co-crystal exhibits three strong Raman characteristic peaks at 23.4, 43.6 and 73.0 cm −1 , and two weak intensity peaks at 33.5 and 51.9 cm −1 in the 0-100 cm −1 range. The CBZ-SAC co-crystal has six peaks, which consists of three strong peaks at 29.2, 50.8 and 76.1 cm -1 , one peak at 64.6 cm −1 and a peak at 41.1 cm −1 with a shoulder at 37.1 cm −1 .
The results indicate that the CBZ co-crystal is a new compound with hydrogen bonds, π-π stacking and other inter-molecular interaction between the starting parent materials. Additionally, the CBZ co-crystal leads to the changes of the molecular structures of CBZ and CCFs, which can be detected by the low-wavenumber Raman spectroscopy.
A comparison of the theoretical DFT calculation and the experimental Raman spectral results are shown in Figure 7. The characteristic vibrational peaks of the CBZ-NIC and CBZ-SAC co-crystals shown in the low-wavenumber Raman spectra are summarized in detail in Tables 3 and 4, respectively. The CBZ-NIC co-crystal has five low-wavenumber Raman characteristic peaks at the position of 9.2, 27.4, 53.7, 73.8 and 91.3 cm −1 in the theoretical spectrum, as shown in Figure 7a. The peak at the position 23.4 cm −1 in the experimental result corresponds to the peak at 27.4 cm −1 in the theoretical spectrum, which is caused by the collective out-of-plane rocking vibration of CBZ and NIC molecules. 41.1 and 76.1 cm −1 in the experimental spectra do not match with the corresponding calculated result spectra. It may be deduced from two reasons. On one hand, the theoretical calculated results were obtained under the condition of 0 K, whereas the experiment results were obtained under the room temperature. On the other hand, the calculation modes used here were based on the single-molecule structure, and inter-molecular forces within solid-state crystalline unit cell were ignored. Therefore, the structure model should be optimized in future work.
(a) (b) Figure 7. Comparison of low-wavenumber Raman spectra between theoretical and experimental results of (a) CBZ-NIC co-crystal and (b) CBZ-SAC co-crystals. Table 3. Vibrational mode assignment for low-wavenumber Raman characteristic peaks of the CBZ-NIC co-crystal. The experimental peak at 51.9 cm −1 can be attributed to the simulated mode at the position 53.7 cm −1 , arising from strong collective out-of-plane rocking vibration of CBZ molecules, and weak collective in-plane rocking vibration of NIC. The experimental peak at 73.0 cm −1 corresponds to the calculated the peak at 73.8 cm −1 . This peak is due to the bending vibration of C7-16C-17C-19C-21C-30C within CBZ molecules, and weak in-plane rocking vibration of NIC molecules.
Experimental
CBZ-SAC co-crystal has seven low-wavenumber Raman characteristic peaks at the positions of 8.7, 21.5, 28.6, 54.2, 65.5, 91.1 and 99.6 cm −1 in the theoretical spectrum, as shown in Figure 7b. The characteristic peak of the CBZ-SAC co-crystal at 29.2 cm −1 in the experimental result corresponds to the peaks calculated at 28.6 cm −1 , which is due to the collective in-plane shearing vibration of CBZ and SAC molecules. The experimental spectral feature at 50.8 cm −1 can attributed to the simulated result at 54.2 cm −1 , which arises from a combination of CBZ molecules' strong bending vibration and the in-plane rocking vibration of O2=S1=O3 of SAC molecules. The experimental Raman peak at 64.6 cm −1 corresponded to the calculated vibrational mode at 65.5 cm −1 arises from the bending vibration of C45-C36-C34-C32-C31-C22, which belongs to CBZ molecules. These results show that the low-wavenumber Raman spectroscopy can characterize the collective vibration of molecules of different types, weak molecular vibration and molecular skeleton vibration with low-frequency Raman characteristic.
It should be noted that there is still a significant gap between the experimental and the calculated results of the two co-crystals. The CBZ-NIC co-crystal characteristic peaks at 9.2 and 91.3 cm −1 and the CBZ-SAC co-crystal peaks at 8.7, 21.5, 91.1 and 99.6 cm −1 in the theoretical spectra do not appear in the corresponding experimental spectra. Furthermore, the CBZ-NIC co-crystal peak at 43.6 cm −1 and the CBZ-SAC co-crystal peaks at 37.1, 41.1 and 76.1 cm −1 in the experimental spectra do not match with the corresponding calculated result spectra. It may be deduced from two reasons. On one hand, the theoretical calculated results were obtained under the condition of 0 K, whereas the experiment results were obtained under the room temperature. On the other hand, the calculation modes used here were based on the single-molecule structure, and inter-molecular forces within solid-state crystalline unit cell were ignored. Therefore, the structure model should be optimized in future work.
Comparison of THz and Low-Wavenumber Raman Characteristic of CBZ-NIC and CBZ-SAC Co-Crystals
In order to further understand the low-frequency characteristic, we compared the THz absorption spectra and low-wavenumber Raman spectra of CBZ-NIC and CBZ-SAC cocrystals, respectively, as shown in Figure Here, a little difference between THz frequency and Raman mode frequency is allowed owing to the influence of factor group splitting [28,29]. oretical vibrational modes assignment. For the CBZ-NIC co-crystal, although the position of the THz absorption peaks at 1.09 THz and 2.26 THz are close to the Raman characteristic peak at 33.5 cm −1 and 73.0 cm −1 , respectively, they are not both infrared and Raman active. Moreover, the THz absorption peak at 2.35 THz of the CBZ-SAC co-crystal does not coincide with the Raman peak at 76.1 cm −1 , though their positions are close. It is because the vibrational modes assignment of the THz absorption spectra is different from the lowwavenumber Raman peaks of the co-crystal.
Conclusions
In summary, the low-frequency vibrational characteristics of two CBZ co-crystals have been demonstrated by combining THz absorption spectroscopy and low-wavenumber Raman spectroscopy. The experimental results show that CBZ-NIC and CBZ-SAC co-crystals with their individual constituents have significant THz and low-wavenumber Raman characteristic peaks in the 0.3-2.5 THz and 0-100 cm −1 regions, respectively, which provide fingerprint spectral information of co-crystal and starting parent materials. The vibrational mode assignment of THz and Raman peaks has been analyzed using DFT calculation. The simulation shows a better agreement with the measured vibrational peak positions. Especially, the low-frequency infrared and/or Raman active of the characteristic peaks for CBZ-NIC and CBZ-SAC co-crystals were given based on the experimental and theoretical results. It could be indicated that the combination of THz and low-wavenumber Raman spectroscopy can characterize richer low-frequency vibrational properties, such as molecular collective vibration and skeleton vibration. These results provide theoretical and experimental benchmarks for vibrational spectroscopic stud- Comparing Table 1 with Table 3 for CBZ-NIC co-crystal, it is seen that Raman characteristic peak at 23.4 cm −1 and THz absorption peak at 0.63 THz both arise from the collective out-of-plane rocking vibration of CBZ and NIC molecules; the Raman peak at 51.9 cm −1 and THz absorption peak at 1.56 THz both arise from a combination of the strong collective out-of-plane rocking vibration of CBZ molecules and the weak in-plane collective rocking vibration of NIC molecules. These demonstrate that the two characteristic peaks of CBZ-NIC co-crystals are not only infrared active, but also Raman active. Furthermore, comparing Table 2 with Table 4 for CBZ-SAC co-crystals, it is obvious that the characteristic peak at 1.04 THz of the THz spectrum and the peak at 29.2 cm −1 of the Raman spectrum are both attributed to the collective in-plane shearing vibration of CBZ and SAC molecules. The Raman peak at 50.8 cm −1 and THz absorption peak at 1.68 THz both come from the strong bending vibration within CBZ molecules, in-plane rocking vibration of O2=S1=O3 of SAC molecules, and the weak out-of-plane rocking vibration of SAC molecules. The Raman characteristic peak at 64.6 cm −1 and the THz absorption peak at 2.22 THz both derive from the bending vibration of C45-C36-C34-C32-C31-C22 that belongs to CBZ molecules. Thus, it is concluded that the three characteristic peaks of CB-SAC co-crystal are both infrared and Raman active.
Moreover, it is seen from Figure 8a,b that the THz absorption peaks of CBZ-NIC co-crystal at 0.41 THz, 1.09 THz, 1.91 THz and 2.26 THz and the THz absorption peaks of CBZ-SAC co-crystal at 1.46 THz and 2.35 THz did not appear in their corresponded Raman spectrum, which had different assignments of vibration modes. It indicates that these THz characteristic peaks of CBZ-NIC and CBZ-SAC co-crystals only are infrared active. Similarly, the Raman peaks at 33.5cm −1 , 43.6 cm −1 and 73.0 cm −1 of CBZ-NIC co-crystal and the Raman peaks at 37.1 cm −1 , 41.1 cm −1 and 76.1 cm −1 of CBZ-SAC co-crystal only are Raman active but not infrared active, where no corresponding peak appeared in the THz spectra. It can be demonstrated that THz absorption spectrum and low-wavenumber Raman spectrum are complementary to each other in characterizing the low-frequency characteristic of co-crystal. Therefore, a combination of the THz spectroscopy and low-wavenumber Raman spectroscopy is able to characterize more comprehensive low-frequency information both with Raman active and infrared active characteristics, such as collective vibration, weak molecular vibration and skeleton vibration of co-crystals.
Particularly, the determination of the infrared and Raman peaks of co-crystal not only depends on the characteristic peak position of experimental spectra, but also the theoretical vibrational modes assignment. For the CBZ-NIC co-crystal, although the position of the THz absorption peaks at 1.09 THz and 2.26 THz are close to the Raman characteristic peak at 33.5 cm −1 and 73.0 cm −1 , respectively, they are not both infrared and Raman active. Moreover, the THz absorption peak at 2.35 THz of the CBZ-SAC co-crystal does not coincide with the Raman peak at 76.1 cm −1 , though their positions are close. It is because the vibrational modes assignment of the THz absorption spectra is different from the low-wavenumber Raman peaks of the co-crystal.
Conclusions
In summary, the low-frequency vibrational characteristics of two CBZ co-crystals have been demonstrated by combining THz absorption spectroscopy and low-wavenumber Raman spectroscopy. The experimental results show that CBZ-NIC and CBZ-SAC cocrystals with their individual constituents have significant THz and low-wavenumber Raman characteristic peaks in the 0.3-2.5 THz and 0-100 cm −1 regions, respectively, which provide fingerprint spectral information of co-crystal and starting parent materials. The vibrational mode assignment of THz and Raman peaks has been analyzed using DFT calculation. The simulation shows a better agreement with the measured vibrational peak positions. Especially, the low-frequency infrared and/or Raman active of the characteristic peaks for CBZ-NIC and CBZ-SAC co-crystals were given based on the experimental and theoretical results. It could be indicated that the combination of THz and low-wavenumber Raman spectroscopy can characterize richer low-frequency vibrational properties, such as molecular collective vibration and skeleton vibration. These results provide theoretical and experimental benchmarks for vibrational spectroscopic studies in many fields. For example, this technique can be used in the real-time reaction monitoring of crystal form, phase or structural formations during the formulation of chemicals and polymers.
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2022-06-02T15:12:20.021Z
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2022-05-27T00:00:00.000
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12006520
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pes2o/s2orc
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v3-fos-license
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A Coupling Kinetics Model for Pollutant Release and Transport in the Process of Landfill Settlement
A coupling kinetics model is developed to simulate the release and transport of landfill leachate pollutants in a deformable municipal solid waste landfill by taking into account of landfill settlement, seepage of leachate water, hydrolyse of insoluble and degradable organic pollutants in solid phase, biodegradation of soluble and degradable organic pollutants in solid phase and aqueous one, growth of aerobic and anaerobic microorganism, and consumption of dissolved oxygen. The release and transport of organic pollutants and microorganisms in landfills in the process of landfill settlement was simulated by considering no hydraulic effect. Simulation results demonstrated that the interaction between landfill settlement and the release, transport and biodegradation of landfill leachate pollutants was significant. Porosity and saturated hydraulic conductivity were not constants because of the landfill settlement, which affected the release, transport and biodegradation of landfill leachate pollutants, and furthermore acted on the landfill settlement. The simulation results accorded with the practical situation, which preliminarily verified the reliability of the mathematical model and the numerical program in this paper.
Introduction
The release and transport of landfill leachate is a complex process, affected by landfill settlement, fluid movement, biodegradation and temperature changes, so a complete model which describes the release and transport of landfill leachate pollutants must contain mechanical, hydraulic, gas transport, temperature and biodegradation models, but it is almost impossible to realize a five-field coupling simulation, so this is often simplified to two or three field coupling model.
Many researchers have studied the multi-field coupling problems of landfill leachate transport, and new models have been developed based on more detailed mathematical descriptions of the landfill and incorporating other aspects of interest apart from hydrology, such as the biological and physicalchemical degradation and settlement. Demirekler et al. [1] developed a three-dimensional mathematical model to estimate the quality and quantity of the landfill leachate produced. The effect of overburden stress was considered. Lobo et al. [2,3] has reported the first version of MODUELO. The development of the second version and the Meruelo Landfill (Spain) simulation results were presented in Lobo et al. [4][5][6]. Chanthikul et al. [7] developed a mathematical model of BOD5 concentration without and with leachate recirculation. Durmusoglu et al. [8] developed a one-dimensional multiphase numerical model to simulate the vertical settlement involving liquid and gas flows in a deformable MSW landfill. McDougall [9] developed a hydro-bio-mechanical model for settlement and other behavior in land filled waste. Fellner and Brunner [10] established a 2-dimensional 2-domain model for simulating the leachate generation from MSW landfills. A flow field consisting of a vertical path (channel domain) surrounded by the waste mass is defined using the software HYDRUS-2D. One-dimensional advectiondispersion transport modeling was conducted as a conceptual approach for the estimation of the transport parameters of fourteen different phenolic compounds and three different inorganic contaminants migrating downward through the several liner systems in Gamze et al. [11]. Gaetano et al. [12] presents a 1D mathematical model for the simulation of the percolation fluxes throughout a landfill for MSW, which considered the landfill divided in several layers evaluating the inflow to and outflow from each layer as well as the continuous moisture distribution. But, there are still one or more defects in most models as follows: (a) The pollutants were considered as a single solute, whether it is soluble or insoluble, degradable or non-degradable was not definitely differentiated; (b) Solid-phase pollutants and its dissolution to aqueous phase weren't taken into account; (c) The effect of oxygen on biodegradation was neglected and the transition from aerobic degradation to anaerobic one wasn't taken into account; (d) the effect of settlement on porosity, saturated hydraulic conductivity and the transport of pollutant was neglected. A coupling kinetic model was developed to simulate the release and transport of leachate pollutants in a deformable MSW landfill taking into account of hydrolyse and dissolution of solid-phase pollutants, oxygen consumption and transition of aqueous-phase pollutant biodegradation from anaerobic stage to aerobic one, and other behaviors such as convection and hydrodynamic dispersion, adsorption/desorption and growth of microorganism. A case study was given by considering none hydraulic action for studying the change law of water quality and quantity, which preliminarily verified the reliability of the mathematical model by comprising with the practical situation.
Basic Assumptions
The release and transport of organic pollutants in landfill is a complicated process which is accompanied by physical behavior and chemical and microbial reactions. It can be barely described by a completely correct model. The development of the simulation model must be based on some suitable assumptions. The assumptions of the models in this study are as follows: (a) Landfill gas is released rapidly after generation, so the landfill leachate transport is considered as a single phase flow; (b) MSW particles are incompressible, but degradable; (c) The simulated landfill was taken as a biochemical reactor. Organics transport and transform under a series of physical, chemical and biological actions, such as convection and hydrodynamic dispersion, hydrolyse, dissolution, adsorption/desorption and biodegradation; (d) Density and viscosity coefficient of landfill lecheate are constants.
Mass-Conservation Equation
Based on the mass conservation principle the mass-conservation equation of solid phase is:
Mechanical Model
The Merchant model was used to simulate landfill settlement. It was constructed by a Hooke elastomer and a Kelvin model in series. Kelvin model was constructed by a Hooke elastomer and a Newton viscosity mode in parallel. The creep equation is: where ij e is the deviator strain tensor So the effective stress can be written as: ; ij is the Kronecher symbol; and: Furthermore, the effective stress principle can be described by: Geometric equation and stress equilibrium equation are: , , The stress equilibrium equation represented with displacement can be obtained by plugging Equation (7) and Equation (8) to Equation (9): , , , The velocity of solid phase is: Equation (1) and Equation (7) (or Equation (10)), Equation (8) and Equation (11) are the basic equations of landfill settlement model. The liquid pressure p was contained in it, so the hydraulic model must be developed for obtaining p .
Hydraulic Model
Based on the mass conservation principle the continuity equation of aqueous phase is: where w is the liquid phase density [ML −3 ]; w Q is the source/sink term [ML −3 T −1 ]; w w r s is the absolute velocity of aqueous phase [13] [LT −1 ]; and wr xi v is the relative velocity of aqueous phase to the solid phase [LT −1 ].
During settlement, the solid particles as well as the liquid move simultaneously. Hence, it is necessary to state Darcy's law relative to solids movement. That is: is the relative permeability.
VG function [14] is used to describe the water retention curve: where a , n , are parameters; r and s are the residual and saturated volumetric moisture contents, respectively. So the relative permeability can be written as: ij k and w Q [15] are calculated by: where A , B , 0 Q , 1 A , 2 A , 1 B and 2 B are parameters; s is the particle density of MSW [ML −3 ].
Conceptual Framework
Organic biodegradation in landfills can be divided into two stages: (1) aerobic biodegradation and (2) anaerobic biodegradation. The first one always occurs in the initial landfill stage, and it can be also divided into two stages: (1) the hydrolysis stage of insoluble macromolecular organics to soluble and small molecular ones and (2) the biodegradation of soluble organics to H 2 O and CO 2 , etc. When the oxygen is consumed, biodegradation enters the anaerobic stage. In this stage, macromolecular organics are hydrolyzed to small molecular ones, and then decomposed to CH 4 and H 2 O by anaerobic microorganisms after the acidification process.
Based on above biodegradation process, organic pollutants in landfill can be classified as insoluble and degradable ones (IDS), soluble and degradable ones (SDS), and adsorbed ones (AS) in solid phase; and soluble and degradable ones in aqueous phase (SDA). Microorganism includes aerobic and anaerobic ones in aqueous phase (AM and ANM) and hydrolysis ones in solid phase (MS). The model which describes the pollutant release and transport in landfill can be developed by using the mass conservation principle, including hydrolysis of IDS, dissolution and biodegradation of SDS, adsorption/desorption and aerobic and anaerobic biodegradation of SDA; growth and death of AM, ANM and MS, and consumption of dissolved oxygen (DO). The biodegradation process of organics was shown in Figure 1.
Hydrolysis of IDS
The hydrolysis of insoluble macromolecular organics (IDS) to soluble and small molecular ones can be described as first order reaction:
Dissolution of SDS
The dissolution of SDS is closely related to the water content and the pollutant concentrations in solid and aqueous phase. It is described by [16]: where
Biodegradation
The decomposition and stabilization of MSW in landfill is essentially a microbial metabolic process. The depletion of the substrate and microorganism growth can be described by Monod kinetics [17], hence for MS accumulation: is the stoichiometric yield coefficient for MS (biomass produced per unit amount of electron donor utilized) [MM −1 ].
When the dissolved oxygen (DO) exists, and its concentration is low, the cell growth rate for AM and ANM can be represented by the following double Monod models [18]: where where The MS, AM and ANM decay are given by: where [19]. So the adsorption rate is described by:
Governing Equations
Based on the mass conservation principle and considering landfill settlement and hydrolysis of macromolecular organics, the governing equation for IDS can be described by: The SDS governing equation considering landfill settlement, hydrolysis of IDS and dissolution and biodegradation can be given by: The MS governing equation considering landfill settlement, growth and decay of MS is described as: The AS governing equation is: The SDA transport model considering convection and hydrodynamic dispersion, dissolution of SDS, aerobic and anaerobic degradation and adsorption/desorption is given by: The equations for AM and ANM by considering convection and hydrodynamic dispersion, growth and decay are given by: The governing equation for DO is:
Numerical Solution Method
The Merchant model was obtained by Lagrangian description. Hydraulic model and pollutant release and transport model were obtained by an Eulerian description. Total settlement in untreated landfilled MSW has been estimated to range between 25% and 50% of initial fill height [20]. So the upper boundaries of fluid and pollutant transport regions are obviously moving, and the small deformation assumption isn't suitable. The coupling of these two types of model may lead to the moving boundaries of Eulerian describing models, so the Arbitrary Lagrangian-Eulerian (ALE) method was used to the model solution for solving the moving boundary problem. Due to space limitations, the solution process can be seen in the authors' another work [15].
Results and Discussion
An ideal landfill should have an effective seepage control system. After closure, it is in a relative independent state and can't be affected by the external hydraulic environment. In this study, the change law of the main physical and chemical variables and its effect on the pollutant transport was analyzed in an ideal landfill. The simulated landfill had a rectangular vertical section of 15 m in height and 20 m in width. All boundaries were impervious. The upper boundary can move freely, and the others are all fixed. The parameters are given in Table 1. Physical and mechanical parameters were determined by testing, and biological parameters were obtained by parameter inversion. The results are shown in Sections 3.1-3.10. In Figures 1 and 2, z is the space coordinate of a certain particle at the initial time, that is, the corresponding space coordinate z at initial time was used to represent a certain particle. In the other figures, z is the space coordinates of a certain particle when the MSW was filled for 30 years, that is, the corresponding space coordinate z at 30 years after MSW was filled was used to represent a certain particle. Figure 2 shows that the settlement occurred in almost 2 years. It's about 85% of total settlement. The total settlement was about 2.6 m, which was about 17.3% of initial fill height. The simulation results fitted well with the observed data from a similar landfill cell in Wuhan Jinkou landfill in China which was observed from 2001 to 2010, and it accorded with the reported settlement law [21,22]. Figure 3 shows that porosity decreased at first and then increased with time due to the landfill settlement and organic biodegradation. The 85% settlement that occurred in 2 years led to the MSW compression and the decrease of porosity. After 2 years, the effect of biodegradation on porosity was more and more obvious. The organic biodegradation led to the reduction of solid mass, and the porosity presented an increasing trend. When the MSW was filled for 30 years, the porosities at top and bottom increased to 0.55 and 0.458 again, respectively. Meanwhile, the decrease of the MSW porosity at bottom due to the landfill settlement was more obvious; however the increase of the one at top due to organic biodegradation was more obvious. In addition, the porosity presented an increasing trend with the fill height increasing. Figure 4 shows that the change of saturated hydraulic conductivity was similar to that of porosity, and the effect of displacement was significant. It decreased at first and then increased taking two years as a turning point. Increase was the main trend of Ks of the upper MSW, and the lower ones had a decreasing trend. The Ks at z = 0.73 m decreased from 0.8 m·day −1 , which was the initial value, to 0.32 m·day −1 , and then tended to be stable; and the one at z = 12.4 m showed a small decrease at first, and then increased until the maximum value.
Pressure Head
It's seen from Figure 5 the effect of settlement on pressure head wasn't significant, although the water transport was closely related to landfill settlement, porosity and saturated hydraulic conductivity. The water mainly moved from top to bottom under gravity action and the upper pressure head decreased with time and the lower one increased by taking 5 m-6 m as separatrix.
The pressure head at the bottom reached 0.6 m when MSW was filled for 5 years, and increased gradually with time. When MSW was filled for 30 years, the MSW below 2.5 m was saturated, which was equivalent to 16.7% of the landfill height. Thus, although without the effect of groundwater and surface water invasion, the landfill leachate generated by MSW itself was large, and can't be neglected when designing the seepage control system and leachate treatment system. Figure 6 shows that because it's assumed that the hydrolysis rate of IDS was only related to its own concentration, and the whole landfill cell has the same initial IDS concentration values, the calculated IDS concentration was only the decaying exponential function of time and independent of fill height and other parameters.
SDS
SDS concentration in this landfill increased at first and then decreased with time as seen in Figure 7. This change process was closely related to the dissolution and biodegradation of SDS and the hydrolysis of IDS. Because of the fast hydrolysis of IDS in the early period of the landfill, the SDS concentration increased rapidly. Over time, IDS concentration gradually decreased, and SDS continuously dissolved from solid phase and was biodegraded, so the SDS concentration decreased year by year. The inflexion point was about the eleventh year.
The difference of SDS concentrations between every height was small and is more obvious before the MSW was filled for 15 years. Because the pore water of lower waste was tending to be saturated gradually, and the dissolution of soluble organic pollutants was accelerated, there was a little difference between upper waste and lower one after the MSW was filled for 15 years.
MS
The MS concentration increased year by year, and the difference between every height was small as seen in Figure 8. 3.8. SDA Figure 9 shows the simulation results of SDA fitted well with the observed data from the Wuhan Jinkou landfill in China, which verified the accuracy of the model and parameters. These data were observed from 2001 to 2010. The suspended particles in water samples were filtered out and the SDA content was represented by chemical oxygen demand (COD) of the sample. SDA concentration increased at first and then decreased. At the early period, because of the higher SDS concentration and its dissolution to aqueous phase, the SDA concentration increased rapidly. Meanwhile, the water content of the lower waste was relatively higher, which accelerated the dissolution of SDS, so the SDA concentration in lower waste was higher than the upper one. The rapid dissolution of SDS in the early period also led to the higher SDA concentration in upper waste than the lower one in the later period of landfill.
ANM
It is seen from Figure 10 that the ANM concentration change was similar to that of MS in Figure 8, but it had a significant difference along height. The ANM concentration at z = 0.73 m was almost three times of the one at z = 12.40 m at utmost. This is mainly because the higher organic concentration in the lower waste provided sufficient nutrients for microorganisms and promoted their growth, while at later periods the SDA concentration in lower waste became lower, the ANM concentration presented a decreasing trend combining with its own decay. Figures 11 and 12 show that the change of DO was consistent with AM, which concentrations presented a rapid decreasing trend with time, and reached 0 when MSW filled for 200 days. The values for the 1,001th day to 10,000th day were omitted in Figures 11 and 12 because they were the same as on the 1,000th day.
Conclusions
A coupling kinetic model of landfill leachate pollutant release and transport in the process of landfill settlement was developed, which contained three sub models-landfill settlement model, hydraulic model and pollutant release and transport model. Landfill settlement, convection and hydrodynamic dispersion of leachate, hydrolysis, dissolution, adsorption/desorption, biodegradation of pollutant and other behaviors were considered. The release and transport of` pollutants and microorganisme in a landfill was simulated by considering no hydraulic action. The total settlement in this landfill cell was about 2.6 m, which was about 17.3% of initial height, and 85% almost occurred within 2 years. The simulation results fitted well with the observed data, and accorded with the reported settlement law. The changes of porosity and saturated hydraulic conductivity were closely related to settlement and biodegradation. They all presented a decreasing trend at first, and then increased with time. The leachate generated by MSW itself can saturated 16.7% of the landfill, so when designing the seepage control system and landfill leachate treatment system, this must be fully considered. The soluble and degradable organic pollutants in solid phase and aqueous phase presented an increasing trend at first and then decreased with time, respectively, due to the release of pollutants from the solid phase. The peak value of the latter could reach 30 kg·m 3 . The microorganisms in the solid phase and aqueous phase presented an increasing trend with time.
|
2014-10-01T00:00:00.000Z
|
2012-09-27T00:00:00.000
|
{
"year": 2012,
"sha1": "12d52f87c4461ff832458c45905afefeab342c7b",
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"oa_url": "https://www.mdpi.com/1660-4601/9/10/3437/pdf",
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|
595262
|
pes2o/s2orc
|
v3-fos-license
|
Acute pancreatitis with saw palmetto use: a case report
Introduction Saw palmetto is a phytotherapeutic agent commercially marketed for the treatment of benign prostatic hyperplasia. Evidence suggests that saw palmetto is a safe product, and mild gastrointestinal adverse effects have been reported with its use. We report a case of acute pancreatitis, possibly secondary to the use of saw palmetto. Case presentation A 61-year-old Caucasian man with a history of benign prostatic hyperplasia and gastroesophageal reflux disease developed epigastric pain associated with nausea 36 hours prior to presentation. He denied drinking alcohol prior to the development of his symptoms. His home medications included saw palmetto, lansoprazole and multivitamins. Laboratory results revealed elevated lipase and amylase levels. An abdominal ultrasound demonstrated a nondilated common bile duct, without choledocholithiasis. Computed tomography of his abdomen showed the pancreatic tail with peripancreatic inflammatory changes, consistent with acute pancreatitis. Our patient's condition improved with intravenous fluids and pain management. On the fourth day of hospitalization his pancreatic enzymes were within normal limits: he was discharged home and advised to avoid taking saw palmetto. Conclusion It is our opinion that a relationship between saw palmetto and the onset of acute pancreatitis is plausible, and prescribers and users of saw palmetto should be alert to the possibility of such adverse reactions.
Introduction
Serenoa repens (W. Bartram) Small, more commonly known as saw palmetto or scrub palmetto, is a lowgrowing palm endemic to the Southeastern United States [1]. Ecologically, saw palmetto is used for nesting, protective cover and as a food source by wildlife. The medicinal value of the fruit among humans has been described in scientific literature since the 1800s for the relief of prostate gland swelling [2].
The liposterolic extract of saw palmetto has antiandrogenic activity in human prostatic cell lines [3]. Furthermore, it inhibits binding of dihydrotestosterone (DHT) to its receptor [4] and prevents the conversion of testosterone into DHT by inhibiting the activity of 5alpha-reductase [5], exhibiting a similar mechanism to the Food and Drug Administration (FDA) -approved medication finasteride. In vitro studies have shown that it also inhibits cyclooxygenase and 5-lipoxygenase pathways, thereby preventing the biosynthesis of inflammation-producing prostaglandins and leukotrienes [6].
As a phytotherapeutic agent, saw palmetto is currently being commercially marketed for the treatment of benign prostatic hyperplasia (BPH). A systematic review of randomized control trials of saw palmetto, involving 2939 men, reported similar improvement in the urologic symptoms of BPH and urinary flow measures compared with finasteride. Furthermore, it was associated with fewer adverse effects [7]. However, a more recent double-blind randomized control trial of 225 men, comparing a saw palmetto and a placebo group, showed no significant difference in their American Urological Association Symptom Index scores or maximal urinary flow rate, with the incidence of side effects similar between the two groups [8]. The latest Cochrane review with 5222 subjects from 30 randomized trials also confirmed that saw palmetto was well tolerated, but was no better than placebo in improving urinary symptoms, peak urine flow, or prostate size for men with BPH [9].
Amidst conflicting data on its utility as treatment for BPH, current evidence suggest that saw palmetto is well-tolerated by patients and is not associated with serious adverse events. Concomitantly, most adverse events reported were mild, infrequent and reversible. A systematic review of adverse events by Agbabiaka et al. from monopreparations of saw palmetto suggests that adverse events associated with its use are similar to those occurring in placebo groups. Adverse effects that have been frequently reported include abdominal pain, diarrhea, nausea and fatigue, headache, decreased libido and rhinitis [10].
More serious adverse effects have also been reported in isolated case reports, including protracted cholestatic hepatitis after the use of Prostata, an herbal preparation used for prostatic hypertrophy with saw palmetto as presumably the most active ingredient [11], and acute pancreatitis. To date there have been two case reports of acute pancreatitis associated with saw palmetto use [12,13]. Acute pancreatitis accounts for more than 200,000 hospital admissions annually in the United States and its hospitalization rate is rising [14]. It ranks third in the list of hospital discharges for gastrointestinal related diseases. Mortality from acute pancreatitis is less than 5% overall, but severe cases cause prolonged hospitalization and significantly higher mortality [15]. We report a case of acute pancreatitis, possibly secondary to the use of saw palmetto, and review the other cases in the literature of this condition.
Case presentation
A 61-year-old Caucasian man with a history of BPH and gastroesophageal reflux disease, who has been in his usual state of health until 36 hours prior to presentation, developed epigastric pain characterized as dull, constant, non-radiating, aggravated by positional changes without any alleviating factors and associated with nausea. He denied any similar episode of abdominal pain in the past. On physical examination, our patient was febrile at 38.5°C and tachycardic; his abdomen was soft with epigastric and periumbilical tenderness and minimal guarding. He occasionally drank a bottle of beer every two to three weeks but denied drinking alcohol recently, had a remote smoking history, and denied any illicit drug use. His home medications included saw palmetto, which he had been taking for the past three years, lansoprazole and multivitamins. His BPH was initially treated with tamsulosin by his urologist, however he experienced dizziness with this medication and was unable to tolerate it. He was then prescribed saw palmetto, which offered relief for his BPH symptoms.
A complete blood count was unremarkable except for leukocytosis at 14.1 × 10 3 cells/mm 3 . An abdominal ultrasound demonstrated a common bile duct, measuring 0.5 cm in diameter, without cholelithiasis ( Figure 1). Computed tomography (CT) of his abdomen with contrast showed that his pancreatic tail was indistinct with peripancreatic inflammatory changes, consistent with acute pancreatitis (Figure 2). Our patient was diagnosed with acute pancreatitis and treated with supportive care, which included intravenous fluids and pain management. Our patient's pain improved, his diet was slowly advanced, and home medications were resumed with the exception of saw palmetto. On the fourth day of hospitalization, his pancreatic enzymes were within normal limits; he was discharged home with a lipase of 32 units/L and advised to avoid taking saw palmetto.
Discussion
The most common causes for pancreatitis in adults are cholelithiasis and excessive alcohol use, accounting for 35-40% and 30% of cases, respectively. Other causes include anatomic variants of the pancreas, mechanical obstruction to pancreatic juice, hypertriglyceridemia, Figure 1 Abdominal ultrasonography showed no evidence for gallstones; common bile duct was not dilated and measured 0.5 cm. hypercalcemia, drug induced, toxins, trauma, ischemia, infections and autoimmune conditions [16]. Many medications also have been identified as a probable cause of acute pancreatitis. The first to report a case of druginduced acute pancreatitis was Zion et al. in 1955; they described a case of hemorrhagic pancreatitis associated with cortisone therapy [17]. Drug-induced pancreatitis is rare, although more than 100 drugs have been implicated in causing this condition. It is rarely accompanied by clinical or laboratory evidence of a drug reaction, such as eosinophilia and rash [18]. Definite association of drugs with acute pancreatitis include aminosalicylates, L-asparaginase, azathioprine, didanosine, estrogen, furosemide, pentamidine, sulfonamide, tetracycline, thiazides, valproic acid, vinca alkaloids and 6-mercaptopurine [16].
A detailed history and physical examination along with routine radiological evaluation consisting of ultrasound and/or CT of the abdomen can detect the underlying etiology of acute pancreatitis in approximately 80% of patients. If this initial investigation is unrevealing, the patient is classified as having idiopathic acute pancreatitis [19].
In our patient, cholelithiasis, hypertriglyceridemia, infection and trauma were ruled out as possible causes of pancreatitis. In addition, as per the history of our patient, he had no recent alcohol use and consumption was very minimal.
Though confirmation of microlithiasis and anatomic variants of the pancreas such as papillary stenosis and sphincter of Oddi dysfunction are most accurately obtained using endoscopic ultrasonography, magnetic resonance cholangiopancreatography or sphincter of Oddi manometry [20], these procedures were not pursued in our patient because of his clinical presentation and subsequent improvement.
The required extensiveness of a search for the etiology in a patient with a first episode of unexplained pancreatitis is still a matter of debate [21]. A retrospective study which looked at patients presenting with a first episode of unexplained acute pancreatitis showed that only about 3.2% (one in 31 cases) would suffer another attack during a median follow-up of 36 months [22], suggesting that extensive investigation for unusual causes of pancreatitis may not required after the first episode of unexplained pancreatitis. Furthermore, invasive testing is associated with procedure-related complications and the relationship between some of those findings and the etiology of the pancreatitis is not always clear [23]. Based on local expertise, advanced evaluation is definitely indicated in patients with a severe initial attack of acute pancreatitis or with two or more attacks [19].
An association between acid suppressing drugs and acute pancreatitis has not been clearly supported by cohort and case control studies. Although proton pump inhibitors like omeprazole, pantoprazole and rabeprazole have been implicated in drug-induced pancreatitis, no case report for other proton pump inhibitors like lansoprazole has been described [24,25].
Apart from our patient and two other case reports, no other data on saw palmetto-induced acute pancreatitis have been reported. Our case and previous case reports (Table 1) reveal consistent resolution of symptoms and pancreatic enzymes following discontinuation during brief hospitalization (three to four days) which is consistent with the short half-life of saw palmetto [26]. To the best of our knowledge, this is the first reported case of acute pancreatitis with a normal alanine transaminase level, which goes against the probability of gallstone pancreatitis [27]. In a recent review by Badalov et al. of reports on drug-induced acute pancreatitis from 1955 to 2006, they classified reported medications into four classes based on the published weight of evidence for each agent and the pattern of clinical presentation. Class I included medications in which at least one case was proven by a re-challenge with the drug. Class II included drugs with a consistent latency in 75% or more of the reported cases. Class III included drugs that had two or more case reports published, but neither a re-challenge nor a consistent latency period. Class IV drugs were similar to class III drugs, but only one case report had been found [18]. Based on the aforementioned classification, saw palmetto could be placed as a Class III agent for druginduced acute pancreatitis.
The mechanisms of action for drug-induced acute pancreatitis are based on theories extracted from case reports, case-control studies, animal studies and other experimental data. In general, some potential mechanisms of action for drug-induced acute pancreatitis include pancreatic duct constriction, cytotoxic and metabolic effects, accumulation of a toxic metabolite or intermediary and hypersensitivity reactions [28]. A mechanism for saw palmetto-induced pancreatitis has not been thoroughly established. One theory suggests that it occurs through its estrogenic effects by stimulating estrogen receptors; it then induces a hypercoagulable state that leads to pancreatic necrosis [29]. However, as in the two previous case reports, pancreatic necrosis was not observed in our patient.
It is also important to note that the United States FDA regulate dietary supplements under a different set of regulations than those covering "conventional" foods and drug products, where the dietary supplement manufacturer is responsible for ensuring that a supplement is safe before it is marketed. FDA is responsible for taking action against any unsafe supplement product after it reaches the market; hence the lack of a standard premarketing regulation for dietary supplements [30].
Conclusion
Current data show that the risk of drug-induced acute pancreatitis is low and it is imperative to rule out more common causes before attributing the event to a certain medication. Three case reports of probable saw palmetto-induced pancreatitis have been described and all the patients had been taking the medication for BPH symptoms. Even with a relatively safe profile as shown by studies on patients taking saw palmetto, the risk of having an adverse reaction exists and warrants immediate withdrawal of the drug and further investigation to prevent serious consequences.
Except for the fact that our patient took saw palmetto, there was no established cause of his acute pancreatitis. Our case highlights the importance of taking a detailed medication history including phytotherapeutic agents in all patients and prompt discontinuation of a probable offending drug to ensure patient safety.
Consent
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
|
2016-05-31T19:58:12.500Z
|
2011-08-25T00:00:00.000
|
{
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"Medicine"
]
}
|
221697513
|
pes2o/s2orc
|
v3-fos-license
|
Investigation of the effect of training given to high ranking soldiers on the level of knowledge regarding sexually transmitted diseases
Objective: In this research, the effect of the training given to the senior soldiers on the level of knowledge about sexually transmitted diseases was investigated. Material and methods: This research, which is planned as an semi-experimental study with pretest-posttest, without control group, was conducted between March and April 2020 with the soldiers working in garrison located in the city center of Ağrı. Results: The pre-test of the soldiers of the rankings was a total score mean of 11.66 ± 4.18, and the level of knowledge was low. The total score mean of the post-test Sex Knowledge Level for the Sexually Transmitted Disease of the ranking soldiers was 16.26 ± 3.78 and the level of knowledge was found to be high. A statistically significant difference was found between the total score means of the pre-test and post-test, Sexually Transmitted Disease Knowledge Level of high-ranking soldiers (p <0.05). Conclusion: It has been determined that the training given to senior soldiers increases the level of knowledge of sexually transmitted diseases, which is low. It is recommended to work in larger groups.
Introduction
Sexually transmitted diseases (STDs) are an important public health problem that negatively affects public health [1,2]. STDs; These are infectious diseases that usually start with acute symptoms and signs after sexual intercourse and often become chronic [3]. Chlamydia, human papilloma virus, trichomonas, syphilis, gonorrhea, fungus, herpes, granuloma inguinale, lymphogranuloma venereum, scabies, pediculosis, hepatitis B, hepatitis C, AIDS are some of these diseases [4]. Sexually transmitted infections (STIs) often lack symptoms or mild symptoms that do not bother the person, causing the disease to spread rapidly. For this reason, the disease spreads silently in the society and affects a wide population [1]. The incidence of STI has increased in recent years. Conditions such as inadequate sexual education programs in developing countries, the age of sexual intercourse in developed and developing countries, having more than one sexual partner, having sexual intercourse for money, not using condoms during sexual intercourse, joint use of tools used for manicure, pedicure, razor , piercing and tattooing, and the use of dental instruments without sterilization are among the risk factors for STI. [5][6][7]. According to the data of the World Health Organization, more than 1 million people are caught with STIs every day in the world. Approximately 500 million people get chlamydia, gonorrhea, syphilis and trichomoniasis infections every year [8].
46.8 million of 450 million people in the 15-49 age group living in the European region, in which Turkey is located, are estimated to have curable STI. [9]. One of the situations that increase infection most is likely to be due to the lack of knowledge of young people [10]. There are a limited number of studies to increase the level of knowledge on education about sexually transmitted diseases. This study was carried out with the aim of effecting the training given to the soldiers on the level of knowledge about sexually transmitted diseases.
Study Design
This research, which was planned as a semi-experimental study with pretest-posttest, without control group, was conducted between March and April 2020 with the soldiers working in garrison located in the city center of Ağrı.
The target population of the study; It formed 382 high-ranking soldiers in the 12th Mechanized Infantry Brigade Command. The sample of the study was composed of individuals from the ranks in the 12th Mechanized Infantry Brigade Command, who met the research criteria (being a ranking soldier, serving in the garrison now) and agreeing to participate in the study.
Those who had to be on duty during data collection and training, those who had previously been trained in this subject were not included in the study. Priori power analysis was performed to determine the sample volume.
In the power analysis, it was determined that at least 24 individuals should be reached in order to reach 80% power in the 95% confidence interval at 0.05 significance level.
Collection of Data
By explaining the purpose of the study, after obtaining verbal permission from those who voluntarily agreed to participate in the study, pre-test application of the Knowledge Level Questionnaire for Sexually Transmitted Diseases and Introductory Information Form prepared by the researchers were collected. Later, a professor who is a specialist in the field of Sexually Transmitted Diseases was provided with face-to-face education and brochures were distributed. What are sexually transmitted diseases, causes of infection, symptoms, methods of diagnosis, treatment and prevention are mentioned. The training lasted 4 hours with 3 breaks. Posttest data was collected from the group where the pretest was performed two weeks later. In the collection of research data, Introductory Information Form and Knowledge Level Test for Sexually Transmitted Diseases were used.
Introductory Information Form
It consists of 7 questions created by the researchers and containing the identifying features of the soldiers (age, gender, marital status, educational level) and their experiences about sexually transmitted diseases [1,6,7].
Knowledge Level Questionnaire Form for Sexually Transmitted Diseases
A questionnaire form for sexually transmitted diseases was created by scanning the literature [1,2,10]. In the questionnaire consisting of 25 questions, there are True (1 point), False (0 point), I don't know (0 point) options and the scale is scored in the range of 0-25. Questions were created by taking the expert opinion. The high total score of the survey means that the level of knowledge is high. In our study, the Kuder-Richardson 20 value of the questionnaire was found 0.72.
Evaluation of the Data
The analysis of the data was done on the computer using the SPSS statistical software. Descriptive statistics, Kolmogorov-Smirnov, Independent Samples t test and Paired Samples t test were used to evaluate the data.
Ethical Principles
Consent was obtained from Ağrı İbrahim Çeçen University Scientific Research Ethics Committee and written permission was obtained from the institution where the study would be conducted. Verbal permission was obtained from those who wanted to participate in the research by making necessary explanations to the individuals included in the research.
Results
All of the individuals participating in the study are male, 69.0% are single, 81.0% are graduates of higher education, 76.2% are not trained on Sexually Transmitted Disease (STD), 100% have not STD on their own and family. It was determined that there are no individuals who have STD disease. And the mean age of the group was found to be 31.43 ± 5.08 (Table 1).
The pre-test of the rank soldiers was 11.66 ± 4.18 and the level of knowledge was low. The total score mean of the post-test Sex Knowledge Level for Sexually Transmitted Disease of the ranking soldiers was 16.26 ± 3.78 and it was determined that the level of knowledge was high (Table 2). A statistically significant difference was found between the total score averages of the Level of Knowledge Regarding Sexually Transmitted Disease pre-test and post-test of the ranking soldiers. (p <0.05) ( Table 2). The ranking soldiers' pre-test knowledge level about Sexually Transmitted Disease total score mean was found to be statistically significantly higher in the individuals whose marital status was married and trained on STD (p <0.05). The ranking soldiers' Post-test knowledge level about Sexually Transmitted Disease was statistically significantly higher in the individuals whose marital status was married and trained on STD (p <0.05) ( Table 3).
Discussion
In the literature, training studies on the knowledge level of sexually transmitted diseases for soldiers, who are at risk groups and senior soldiers, are not sufficient. In this study, training was provided to determine the knowledge levels of soldiers about some selected infectious diseases such as tuberculosis, tuberculosis, malaria, AIDS, typhoid, which are considered important for public health and health management. It protects themselves and their close friends about sexually transmitted diseases with the training given to the senior soldiers, but it is also important for them to raise their rank-free soldiers.
The ranking soldiers' pre-test and post-test knowledge level of the Regarding Sexually Transmitted Disease total score mean were statistically significantly higher among the individuals whose marital status was married. (p <0.05).
X X
In the study conducted by Kırmızıtoprak and Şimşek for sexually transmitted diseases, it was found that those whose marital status was married had higher level of knowledge about sexually transmitted disease [11].
The pre-test and post-test knowledge level of Regarding Sexually Transmitted Disease total score mean of the ranking soldiers was found to be statistically significantly higher in the individuals trained in STD. (p <0.05). In the study conducted by Siyez and Siyez, Çalışkan et al., Kaymak et al. on sexually transmitted diseases, it was found that training on STD increased the mean score [5,7,12].
It has been determined that the pre-test STD knowledge level of the soldiers in the rank is low and the post-test STI knowledge level is higher. A statistically significant difference was found between the total score means of the Level of Knowledge Regarding Sexually Transmitted Disease pre-test and post-test of the ranking soldiers. (p <0.05). In the training given by Kökcü and Elçioğlu to the Turkish Armed Forces (TAF) employees, the change in the level of knowledge about sexually transmitted infections was examined, and at the end of the study, the STD mean attitude score increased from 4.00 ± 0.50 before training to 4.32 ± 0.47 after training and a significant increase of 8% was achieved in the score (p <0.001) [13]. In the literature, it was found that education was important in many studies [14][15][16][17][18][19]. It is thought that it is of great importance to educate and inform individuals in advance in order to take necessary measures to prevent sexually transmitted diseases.
CONCLUSIONS
It has been determined that the training given to the ranking soldiers increases the knowledge level of regarding sexually transmitted diseases, which is low. Prevention methods that are valid for the control of sexually transmitted infections are health education and health management. These trainings are of great importance in informing themselves and the soldiers who have just completed their adolescents under their commands with the training given to the senior soldiers in the scope of health protection, which is one of the priorities of public health.
|
2020-08-27T09:08:16.068Z
|
2020-08-18T00:00:00.000
|
{
"year": 2020,
"sha1": "3b7c310807d2cf172b2a4b025c48e8dd00597ef8",
"oa_license": "CCBYNC",
"oa_url": "https://www.aejog.com/index.php/aejog/article/download/38/16",
"oa_status": "GOLD",
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"extfieldsofstudy": [
"Psychology"
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|
203379856
|
pes2o/s2orc
|
v3-fos-license
|
Evaluation and pilot implementation of essential interventions for the management of hypertension and prevention of cardiovascular diseases in primary health care in the Republic of Tajikistan
Background: Non-communicable diseases (NCDs) are the leading cause of death worldwide and are a major burden in Tajikistan. The health system of Tajikistan is still shaped by the country's Soviet legacy and the pace of reform has been slow, with high patient out-of-pocket expenditure. The aim of this study is to determine the feasibility of implementing and evaluating essential interventions for the management of hypertension and prevention of cardiovascular disease in primary health care in Tajikistan. Methods and analysis: A pragmatic, sequential mixed methods explanatory design, composed of quantitative and qualitative strands will be used with greater weighting of the quantitative strand. A single geographic district was nominated by the Ministry of Health and chosen for implementation. All primary health care centres in the district that meet inclusion criteria will be included; half will be randomly assigned to the intervention arm and half to the control arm. The overall process is organized into seven steps: (1) refresh clinical decision-making tools including open source WHO PEN and HEARTS resources; (2) update training package for primary health care workers; (3) collection of baseline data; (4) training staff in intervention clinics; (5) implementation of protocols and implementation coaching; (6) collection of follow-up data after 12 months; (7) evaluation of results and sharing experience. Ethics and dissemination: Ethical review and approval have been obtained. Findings will be disseminated at the participant level, national level through a national conference of key stakeholders, and internationally through publication in an open-access peer review journal.
Introduction
Non-communicable diseases (NCDs) are the leading cause of death worldwide, causing 41 million deaths in 2016 1 . Half were due to cardiovascular diseases (CVD) 2 . Primary health care (PHC) systems are an essential component required to tackle this global burden 3 . While strong PHC systems based on the principles of family medicine contribute to achieving universal health coverage, better health outcomes and economic and social development, many nations lack PHC capacity 4,5 . To help national governments develop primary health care capacity for NCDs, the World Health Assembly endorsed the WHO Global Action Plan for the Prevention and Control of NCDs 2013-2020 6,7 .
A country of the former Soviet Union, Tajikistan is located in Central Asia; the capital city is Dushanbe. Ranking among the poorest in the WHO European Region, Tajikistan had a gross domestic product (GDP) of 796 USD per capita in 2016 8 . The WHO Regional Office for Europe continues to support Tajikistan to implement the Global Action Plan, including support for the development, testing, and national scale-up of evidence-based clinical guidance and health policy for the prevention and management of NCDs.
Non-communicable diseases in Tajikistan
NCDs are a major burden in Tajikistan. The age-standardized NCD mortality rate was 685.3 per 100,000 in 2015 9 . In 2016, the probability of dying prematurely (between the ages of 30 and 70 years) from CVD, cancer, diabetes, or chronic respiratory disease was 25.3%; like most populations, this rate is higher for men than women 10 .
The Ministry of Health of Tajikistan and the World Health Organization Regional Office for Europe recently completed a population STEPS survey (unpublished by WHO) which indicated that 25.7% of men are current users of tobacco (including smokeless tobacco) although rates are low amongst women (0.2%) 11 . For cultural reasons, alcohol use is relatively low with only 9.4% of men and 0.2% of women identified as current drinkers, and lifetime abstention is very high. The prevalence of obesity is 11.9% in women and 15.4% in men, and around half (46.7%) the adult population are overweight.
The prevalence of raised blood pressure (BP) (defined as systolic BP ≥ 140 mmHg and/or diastolic BP ≥ 90 mmHg or currently taking medication for raised blood pressure) among the adult population is 32.2%. Less than half of men (44.3%) have never had their blood pressure measured; the same is true for 24.7% of women. Nearly all (96.9%) adults have never had cholesterol measured. One-in-seven (13.8%) people aged 40-69 years have a 10-year fatal or non-fatal CVD risk of over 30% (including those with an existing CVD).
Primary health care in the Tajikistan
The health system of Tajikistan is still shaped by the country's Soviet legacy and the pace of reform has been slow 12 .
The country has the one of the lowest total health expenditure (THE) per capita in the WHO European Region, estimated to be 6.88% of GDP in 2014, and the proportion of THE that is financed privately through out-of-pocket payments (OOP) is 61.69%; second highest in the WHO European Region 8 . Although formally free, OOP expenditure for PHC is common and expenditure on pharmaceuticals is the biggest financial burden.
Recent reforms have aimed to strengthen PHC, but success has been mixed. In 2010, Tajikistan launched the National Health Strategy for the period 2010-2020 13 , which recognized the importance of health system strengthening, and highlighted the development of PHC based on family medicine practice as a top priority. The National Programme on the Development of Family Medicine 2011-2015 in Tajikistan had the goal of ensuring the sustainable development of PHC according to the principles of family medicine. Some substantial improvements were achieved, including trainings for the health workforce, the review of clinical protocols, promotion of quality of care, increasing capacity in family medicine, improvements in evidence-based practice, and greater availability of resources. Nevertheless, challenges have been highlighted, including insufficient pace and scale of initiatives, and the scope of work and distribution of family doctors across the country 14 . Although some computers have been installed in district health care facilities to introduce electronic submission of statistical reports, the vast majority of rural health clinics rely on paper records which need to be completed manually, hindering reliable data collection, processing and analysis 12 .
In Tajikistan, health services are delivered at five levels: rural (village), district (rayon), city, oblast (regional), and republican (national). In villages, PHC services are provided in rural health centres (RHCs) with a family medicine doctor or health houses with feldshers, family medicine nurses and midwives.
Essential interventions to manage hypertension and prevent CVD in primary health care In order to build capacity in PHC and ultimately prevent premature mortality from major NCDs, from 2013 to 2015 Tajikistan endeavoured to adapt and pilot the World Health Organization Package of Essential NCD Intervention for Primary Healthcare in Low Resource Settings (WHO PEN) 5 . WHO PEN includes simplified clinical protocols which together cover the integrated management of hypertension and diabetes, as well as education and counselling on healthy behaviours aimed to prevent CVD. The central strategy of this integrated approach is the use of total cardiovascular risk assessment to stratify and target individuals at high CVD risk, a process considered to be a "best buy" intervention by the WHO 15 .
These early efforts led to limited success. A national PEN steering group was established in 2012, facilities were assessed for access to essential medicines and technologies, national clinical guidelines were updated to include the WHO/ISH risk prediction chart 16 , and family doctors and nurses from 9 pilot facilities received two-day training on the WHO PEN protocols during 2013/14. Apart from a "refresh" training for selected clinics two years later, there was no further intervention, and the lack of systematic monitoring and evaluation made it difficult to demonstrate any significant change in clinical practice, disease detection, disease control rates or clinical outcomes.
In 2016, Tajikistan agreed to embark on a pilot of the global WHO HEARTS initiative supported by WHO 17 . Work on this was able to proceed in earnest in 2018 with the publication of the HEARTS technical package which aimed to provide a strategic approach to improving cardiovascular health in countries 18 . It comprises six modules and an implementation guide to support Ministries of Health to strengthen CVD management in primary health care settings.
Led by the Ministry of Health and Social Protection, the WHO PEN steering group was expanded to include chief specialists, district and rural health facility managers and clinicians, the Republican Clinical and Training Center of Family Medicine and the Service of State Supervision for Medical Activities and Social Protection of the Population of the Republic of Tajikistan. The national steering group is supported by an international team of experts coordinated jointly by the WHO Regional Office for Europe and the WHO Country Office in Tajikistan. Tajikistan has a particular interest in systems monitoring, and in developing sustainable and scalable approaches, building upon other initiatives in PHC reforms and quality improvement.
Aim and objectives Aim
The aim is to determine the feasibility of implementing and evaluating essential interventions for the management of hypertension and prevention of CVD in PHC in Tajikistan.
Objectives
In order to achieve this aim, the objectives are to: 1. Determine the baseline performance of PHC services with respect to essential interventions for the management of hypertension and prevention of CVD; 2. Assess the ability to conduct practical trainings for primary care doctors and nurses and implement Tajikistanadapted HEARTS tools, in one pilot region of Tajikistan; 3. Estimate the change in clinical practice with respect to essential interventions for the management of hypertension and prevention of CVD after 12 months of implementation; 4. Determine the feasibility of collecting quantitative data required for future studies of effectiveness from routine clinical data.
Methods and analysis
As part of a larger programme of implementation research, we have previously developed an approach to evaluating and piloting essential interventions for NCDs in low-resource settings, and have adapted this methodology to suit the context of Tajikistan 19 .
Overview of process and design
The general approach to developing, piloting, and evaluating essential interventions for the management of hypertension and prevention of CVD in PHC in Tajikistan are outlined in the following seven steps. A more detailed methodological description is presented in the section that follows.
Step One: Refresh clinical decision-making tools. Tajikistan national guidelines for PHC exist in a printed book format, and these books are available and used by family doctors. The national guidelines for hypertension (updated in 2018) endorse the use of WHO/ISH CVD risk charts, and some family doctors were previously trained on the use of WHO PEN. However, there is a lack of simple clinical decision support tools, such as laminated one-page algorithm cards for the treatment of hypertension. As such, the following tools will be adapted to be in-line with existing national guidelines, used for training, and disseminated to PHC workers in intervention clinics. •
• AUDIT and Fagerstrom Clinical Decision Support Tools
While the content of these tools is consistent with national guidelines, a ministerial order may be required to inform clinicians in the intervention clinics that it is appropriate to follow these updated versions of the already approved national guidelines.
Step • Measuring inequalities in care and outcomes by gender
• Raising population awareness of cardiovascular diseases
Step Three: Collection of baseline data. Baseline data will be collected from all clinics in the included district before implementation (i.e. training) begins. Data for quantitative indicators will be extracted by randomly sampling individual paper-based patient records from all PHC units using a standardized data collection instrument. This will be done by a small team of specially-trained national experts. After analysis, each health clinic will receive a summary report of their facility's performance with respect to key project indicators. This report will not be powered for statistical significance, but will be used as a quality improvement exercise to show how routine clinical data can be used to improve clinical practice, for example by setting improvement targets.
Step Four: Training staff in intervention clinics. All doctors and nurses from the PHC centres in the intervention arm will be invited to be trained together by a national team of experts in groups of approximately 30. It is estimated that up to 100 health workers will be trained in total. Given the human resources, the trainers will be themselves a group of family doctors and nurses, rather than narrow medical specialists or academics, so as to maintain direct relevancy to practical aspects of PHC services.
The training will be two-days; therefore it is estimated that two trainings can be conducted in one week (Monday/ Tuesday and Thursday/Friday). Thus, 60 participants can be trained per week; two weeks will be required to train all doctors and nurses in the pilot district. A ministerial order will be required to relieve the health staff from their duties to attend the training.
Step Five: Implementation of protocols and implementation coaching. Trained participants from intervention clinics will then be free to implement the clinical protocols and change their clinical practice, without incentives, for 12 months. During this time, a team of national and district experts will be created to offer support (distance and on-the-job) to the PHC centres. These teams will visit each clinic once every three months (for a total of four visits) and use a set of standardized instruments to review performance and provide constructive and timely feedback to the health facility manager and individual clinicians. The tools used during the support visits and implementation coaching will include: • HEARTS Treatment Supervision Form -with minor adaptations to local context In addition to clinic visits every three months, representatives from each clinic will be brought together after 6 months of implementation for a workshop. This will be an opportunity for the project management team to provide support based on the needs identified in clinic visits, to encourage target setting, quality improvement and peer support, as well as gather more information about implementation barriers.
Step Six: Collection of follow-up data. After 12 months, using the same method and data collection instruments used to collect baseline quantitative data (Step Three), data will again be extracted from randomly selected individual paper-based patient records from both intervention and control health centres.
As was done at baseline, each health clinic will receive a summary report of their facility's performance with respect to key project indicators comparing baseline to follow-up.
One-on-one semi structured interviews will be conducted with doctors, nurses, and health facility managers in the intervention clinics at 12 months follow-up, and these qualitative data will be analysed thematically for explanatory themes.
Step Seven: Evaluation of results and sharing experience.
The findings of the quantitative and qualitative analyses will be integrated in a final report and shared with key stakeholders, including health staff from the participating primary health care centres. The results will also be shared at a national conference and in an open-access peer reviewed journal, in order to inform the future development of primary health care capacity of Tajikistan and other similar settings.
Methodological design
A pragmatic, sequential mixed methods explanatory design, composed of quantitative and qualitative strands will be used ( Figure 1). The quantitative strand will be weighted more than the qualitative strand. A mixed methods design was chosen because it allows for the use of qualitative data to enlighten and explain the quantitative findings, but due to limited capacity to conduct qualitative research in Tajikistan, the qualitative strand will be weighted less than the quantitative strand.
Due to resource constraints, and several ongoing PHC reform projects, a single district was nominated by the Ministry of Health and chosen for implementation (rather than multiple districts). All PHC in the district that meet inclusion criteria will be included; half will be randomly assigned to the intervention arm and half to the control arm. Baseline data will be collected from all clinics in the intervention arm and the control arm using the same standardized data extraction form.
Selection of primary health care centres
The national working group initially nominated four districts for consideration which had participated in other projects: while three of these are subordinate districts ("Rayons") of Dushanbe, the 4 th (Vakhsh) is nearby to Dushanbe in the Oblast of Khatlon (Figure 2).
In order to provide more intensive support and ensure the quality of data collection, it was decided to focus on only one district. All PHC centres and the associated family doctors and nurses will be invited to take part in the training and pilot implementation. Shahrinav was chosen after considering its size, exposure to previous interventions, proximity to Dushanbe, year-round road access, and by recommendation from the Ministry of Health.
Within Shahrinav district, all PHCs were compared against the following exclusion criteria: (1) City Health Centre designation; (2) presence of narrow specialists (i.e. any physicians with specialization other than family medicine) within the clinic; (3) clinics with a family doctor to patient ratio of 1:4000 or more; or (4) clinics where information on the number of family doctors and/or family doctor to patient ratio is not available. If one or more of these four exclusion criteria were met, the clinic will not be included in the intervention or control arm.
Comparison
Clinics allocated to the control arm will not receive the intervention; clinicians will proceed with usual care. The performance of individual health facilities will be compared from baseline to follow-up. To compare change in performance between intervention and control arms, the data from all intervention clinics will be aggregated together and compared to aggregated data of all control clinics.
Quantitative indictors
Given the emphasis of hypertension management, including total CVD risk approach, indicators were developed to be calculated from a sample of hypertensive patients. These include indicators along the hypertension care pathway, including detection of hypertension, CVD risk assessment, risk factor modifying prescriptions, and BP control. Table 1 shows the primary and secondary indicators, the question the indicator seeks to answer, and the respective numerator and denominator definitions which will be used in the calculations.
Data collection and management
Quantitative data collection tool. We developed a standardized data collection tool to collect the anonymized data required for calculation of each indicator (Table 2). This tool will also be digitized such that it can be used on a computer (offline) or smartphone (online). We estimate it take 15 minutes per unique health record in order to extract data. Since there is a lack of electricity and mobile phone reception in many of the clinics, it is likely that data will have to be extracted first on paper and then later input to a database using an online data form. Data extractors will be blinded to the allocation of health care facilities to intervention or control for baseline data collection, but blinding is not possible for follow-up data collection because after the intervention period, allocation will be apparent when data collectors visit clinics and look at patient records.
Method of randomly sampling patient records.
Each family doctor owns and maintains a single register (list of patients) for hypertensive patients (henceforth "hypertension register").
The hypertension register will be used to randomly select patient records. A computerized random number generator will be used to randomly select patients from the register. The selected patient chart will then be found and checked to see if it meets two inclusion criteria: (1) the patient must have visited the clinic at least once in the previous 12 months; and (2) the patient must have been 18 years or older 12 months prior to the date of data selection. If both criteria are met, the record will be used for data extraction. If the record does not meet inclusion criteria, it will be returned and a new record will be randomly selected. This process will be repeated until the sample size for each family doctor has been met ( Figure 3).
Sample size.
A total of 400 patient records will be sampled at baseline and follow-up from each of the intervention and control arms (n=800). The sample is based on a power calculation for the primary indicator (proportion of hypertensive patients whose blood pressure is controlled), estimated Proportion of hypertensive patients with a WHO PEN risk score of > =30%, whose blood pressure is controlled Patients with a true risk score of >=30% whose last documented blood pressure reading was controlled (SBP <130 and DBP 80 mmHg) Patients with a true risk score of >=30%
Hypertension register
Is the blood pressure of lower risk patients controlled?
Proportion of hypertensive patients with a WHO PEN risk score of <30%, whose blood pressure is controlled Patients with a true risk score of <30% whose last documented blood pressure reading was controlled (SBP <140 and DBP 90 mmHg) Patients with a true risk score of <30% Hypertension register Table 2. Standardized data collection form used to extract data from individual patient records.
Data collection question Answer
What is your name? (Name of person extracting data)
Date of Data Extraction (MM-DD-YYYY)
Write the Clinic Name Is this a duplicate extraction?
If it is a duplicate extraction, enter the number you and your extraction partner have assigned to this file. based on a WHO report that the control level in low and middle income countries is generally about 20-25% 13 . A 10% point difference between the intervention and control arms could be detected having 310 to 350 observations in each group using 0.05 type I error rate and 0.2 type II error rate. Sampling will be stratified by gender and selected in a 1:1 ratio of men to women (200 men and 200 women per arm).
In the event that there are too few patients of a given gender, patients from the opposite gender will be substituted.
Data analysis.
The change in indicators from baseline to follow-up will be calculated. Subgroup analysis by age, gender, and other demographic features may be done as deemed appropriate.
Qualitative data collection.
Semi-structured interviews A purposive maximum variation sample of doctors and nurses from the intervention clinics will be invited to take part in semi-structured interviews at the end of the 12-month implementation phase. All participants will be required to provide written informed consent. Using an interview guide that will be adapted from previously published work 21,22 , interviews will be conducted one-on-one by members of the research team, audio recorded, and transcribed verbatim. Transcripts will be analysed thematically using framework thematic analysis, by two or more members of the research team, with oversight from an experienced qualitative researcher 23 .
Patient and public involvement
Neither patients nor the public were involved in the methodological design.
Strengths and limitations of this study
• We report methodology to adapt, pilot, and evaluate the WHO HEARTS technical package in a low-resource setting in a low-income country that can be used as an example for other jurisdictions with a high burden of cardiovascular diseases • Our methods focus on evaluating the effect of the WHO HEARTS interventions in a real-world health system and therefore are expected to yield practical information to inform the national scale up of essential interventions for non-communicable diseases • The sample size is based on one primary indicator and it is therefore possible that some of the secondary indicators will be statistically underpowered • Due to the practical and resource constraints, data collectors could not be blinded to follow-up data collection, and this could potentially bias the effect estimate • Although an important stakeholder in implementation, patient perspectives were not included in the evaluation design
Current study status
Data collection has been completed and analysis is underway.
Ethics and dissemination
Ethical review and approval This project has been reviewed and granted ethical approval from the Republican Clinical Center for Family Medicine under the Ministry of Health and Social Protection of the Republic Tajikistan. As per the ethical approval review and the nature of this project, consent was not obtained from individual patients for their anonymized routine clinical data to be used.
Dissemination of findings
Findings will be shared at the participant level (intervention clinics), national level through a national conference of key stakeholders, and internationally through publication in an open-access peer review journal. At the national level, the results will be used to assess the appropriateness and feasibility of national scale-up and mainstreaming into clinical practice. The raw data used in this project will not be made publicly available.
Data availability
No data are associated with this article.
Dagfinn Aune
Department of Epidemiology and Biostatistics, Imperial College, London, UK This is a study protocol for evaluation and pilot implementation of interventions for the management of hypertension and prevention of cardiovascular diseases in primary health care in Tajikistan. A pragmatic, sequential mixed methods explanatory design, composed of quantitative and qualitative strands will be used. A single geographic district was chosen for implementation and all primary care health care centers that meet inclusion criteria will be included. Half will be randomized to the intervention group and the other half to the control group. The overall process is organized in seven steps: refresh clinical decision-making tools -WHO PEN and HEARTS, update training package for health care workers, collection of baseline data, training staff in intervention clinics, implementation of protocols and implementation coaching, collection of follow-up data after 12 months, and evaluation of results and sharing experience.
It would have been interesting to know a bit more about the data that will be collected. Will you collect clinical measurements (weight, height, waist measures, blood pressure) and take blood samples (cholesterol, glucose etc.)? Will you collect and data on dietary intake, smoking, physical activity and other lifestyle factors? Will there be any repeated measurements during the study follow-up?
What exactly will the intervention entail?
I see now some of the information is in Table 2. You might want to have more detailed data on smoking -including never, former and current smoking, cigarettes per day and duration, time since quitting in former smokers.
Is the rationale for, and objectives of, the study clearly described? Yes
Is the study design appropriate for the research question? Yes P3, paragraph 4: Is the difference in the prevalence of obesity between men (15.4%) and women (11.9%) statistically significant? Please state if it is. I also find it unusual that the prevalence is higher in men than in women which is the opposite of the situation in most other countries. Are you able to offer any explanations for this?
4.
Please explain what is meant by Soviet legacy in the health system. What specific aspects of the health system can be attributed to the fact that the country was part of the Soviet Union? As mentioned earlier, my feeling is that this reference to the past should be done away with unless a very clear and specific link can be shown.
5.
It would be useful to provide background information on the numbers of health care workers in the country as well as Shahrinav district and the doctor/nurse to patient ratios.
6.
Overview of process and design section (p4).
Step 1: Refresh clinical decision making toolsapart from having laminated one-page algorithm cards for clinicians to use in the treatment of hypertension, has consideration been made to convert these into smart phone applications? Is this feasible in Tajikistan?
7.
Please include more detail on how the random allocation of clinics to control and intervention arms was/will be conducted. Were representatives of all the clinics present to observe the randomization taking place?
8. Table 1: Is it possible to include magnitude of change in BP in mmHg as a secondary outcome? Proportion of controlled hypertensives is a rather crude measure.
9.
Data analysis-This section needs to be expanded from the current two sentences as it will be critical to understanding the effect of the project. Is there a statistical analysis plan in place or is it being developed? For the primary outcome of proportion of hypertensives controlled, what test will be used and what confounders will be included in the model? Rather than comparing the change in indicators from baseline to follow up as the authors write, I would expect that the primary comparison would be proportion of controlled hypertension in intervention group versus proportion of controlled hypertension in control group at follow up. This is because the allocation to control versus intervention group is a randomized process and I would expect the proportions of controlled hypertensives in both trial arms to be essentially the same at baseline. It is important to have these statistical issues well thought out before data collection is completed.
10.
Dissemination of findings: The authors should in the interests of open science consider having the data associated with the project made available to other researchers to reanalyze and build on their work. This can be through managed access or fully open processes.
11.
Is the rationale for, and objectives of, the study clearly described? Yes Is the study design appropriate for the research question?
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2019-09-19T09:10:37.174Z
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2019-09-13T00:00:00.000
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Mechanism of error-free DNA synthesis across N1-methyl-deoxyadenosine by human DNA polymerase-ι
N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the syn conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a “foothold” and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity.
Scientific RepoRts | 7:43904 | DOI: 10.1038/srep43904 We show here by steady state kinetic analysis that Polι exhibits an ~100 fold higher catalytic efficiency for insertion of the correct nucleotide T relative to the incorrect C opposite 1-MeA. We also present ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. We show that template 1-MeA adopts the syn conformation in both structures, though with significant differences. dTTP and dCTP insert differently opposite template 1-MeA with dTTP participating in Hoogsteen base pairing, while dCTP is largely disordered, consistent with multiple conformations. Together, our kinetic and structural studies show that Polι can not only accommodate lesions such as 1-MeA with impaired W-C edges, but that it can maintain discrimination between correct and incorrect incoming nucleotides opposite the lesion.
Results
Kinetic Analysis. We carried out steady state kinetic analyses to determine the catalytic efficiency (k cat /K m ) and fidelity of Polι for nucleotide insertion opposite 1-MeA (Table 1). Polι inserts T opposite 1-MeA with an ~5-fold higher catalytic efficiency than opposite undamaged A; however, it also inserts incorrect nucleotides opposite 1-MeA more efficiently than opposite undamaged A. For example, whereas no insertion of C was detected opposite undamaged A, Polι inserts C opposite 1-MeA with a k cat /K m of 0.1 min −1 μ M −1 . Importantly, although Polι inserts a C opposite 1-MeA, it does so with a 100 fold lower efficiency than correct T. To understand the ability of Polι to discriminate between T and C opposite 1-MeA, we determined the crystal structures of human Polι bound to a template-primer duplex with 1-MeA as the templating base and dTTP or dCTP as the incoming nucleotide.
Crystals of Polι 1-MeA.dCTP diffracted to 2.0 Å and clear electron density was visible for the templating 1-MeA in the initial F o -F c and simulated annealing Fo-Fc omit maps. However, the electron density for the incoming dCTP was not as well-defined as for the dTTP in the Polι 1-MeA.dTTP structure, despite the substantially higher resolution (2.0 Å vs. 2.6 Å) of the Polι 1-MeA.dCTP structure (Fig. 3e). Strong F o -F c density (above 3σ ) was visible only for the γ -phosphate of dCTP and partial electron density was observed for what would be considered as the base. To help improve the density, we performed iterative rounds of refinement and water picking with Phenix 21 and Coot 22 . However, no significant improvement in the electron density for the incoming dCTP was observed. We rationalized that the absence of well defined electron density was suggestive of multiple conformations for the dCTP, with only the γ -phosphate ordered and held in place by interaction with positively charged amino acids from the fingers domain (see below). The Polι 1-MeA.dCTP ternary complex was refined to 2.0 Å resolution (R free 23.9%; R cryst of 21.6%) and contains Polι residues 26-350, 356-371, 378-397 and 403-414, DNA nucleotides 4-11, 1 Cl − ion and 291 water molecules.
Overall Arrangement. In both the Polι 1-MeA.dTTP and Polι 1-MeA.dCTP complexes, a Polι molecule binds to each replicative end of the double-ended template-primer (Fig. 2). The two molecules are related by a crystallographic two-fold axis and thus make identical contacts with the template-primer. Polι has the familiar right-handed architecture with palm (residues 25-37, 99-224), fingers (38-98), and thumb (225-288) domains, and the PAD (polymerase associated domain; residues 298-414) unique to Y-family polymerases 17,18,23,24 . The palm domain forms the floor of the DNA binding cavity and contains the active site residues (Asp34, Asp126 and Glu127) that catalyze the nucleotidyl transfer reaction, whereas the fingers domain drapes over the template 1-MeA in both structures (and over dTTP in the Polι 1-MeA.dTTP structure). The thumb domain and the PAD are connected by a long linker that spans the width of the DNA. The thumb skims the minor groove on one side of the DNA duplex whereas the PAD occupies the major groove on the other side. The majority of Polι -DNA interactions are mediated by the PAD, wherein the main chain amides on "outer" β -strands of the PAD β -sheet make a series of hydrogen bonds with the template and primer strands.
The Polι 1-MeA.dTTP ternary complex. The structure reveals how the 1-MeA.dTTP nascent base pair is accommodated in the active site of Polι (Fig. 3a). In previous structures of Polι with template purines (A or G), the steric restraints imposed by the narrow active site of the polymerase are overcome by the template being pushed from the anti into the syn conformation by the incoming dNTP 17,18,23 . Rotation of the template 1, N 6 -etheneodeoxyadenosine (ε dA) to the syn conformation has also been observed in the structures of Polι with template ε dA and incoming dTTP or dCTP 19 . Template 1-MeA is similarly observed in the syn conformation, presenting its Hoogsteen edge for hydrogen bonding with dTTP which remains in the anti conformation ( Fig. 3b and c). The 1-MeA and dTTP bases are almost coplanar and two putative hydrogen bonds are established between the N6 and N7 atoms of 1-MeA with the O4 and N3 atoms of T (2.8 Å and 3.2 Å respectively). The 1-MeA.T base pair is isomorphic with the A.T and ε dA.T base pairs in the structures of Polι dA.dTTP and Polι εdA.dTTP , respectively. Superimposition of the Polι 1-MeA.dTTP structure with that of Polι dA.dTTP and Polι εdA.dTTP reveals almost perfect overlap between the common N6 and N7 atom of the templating bases and the O4 and N3 atoms of the incoming dTTP.
Incoming dTTP is anchored at one end of the dNTP binding cavity by hydrogen bonding interactions between its γ -phosphate and the side chains of Tyr68 and Arg71 from the fingers domain and Lys214 from the palm domain ( Fig. 3a). At the other end, Hoogsteen base pairing with 1-MeA secures the base of dTTP in the binding pocket. The α -and β -phosphates are fixed by interactions with the side chains of Asp126 and Thr65 and with the backbone atoms of Leu35 and Phe38. The dTTP sugar packs against the aromatic ring of Tyr39, and makes a hydrogen bond between its 3′ OH and the main chain amide of the Tyr39. A single Mg 2+ ion (metal B) is coordinated by the triphosphate moiety of dTTP, as well as the active site residues Asp34 and Asp126. Overall, Polι 1-MeA.dTTP is well poised for catalysis with a 3′ -OH modeled at the primer terminus located ~3.1 Å from the dTTP α -phosphate and aligned more or less linearly with respect to the scissile Pα -O3′ bond (~162°).
The Polι 1-MeA.dCTP ternary complex. Overall, as in the Polι 1-MeA.dTTP structure, template 1-MeA is rotated about its glycosidic bond to the syn conformation and presents its Hoogsteen edge to the dNTP binding pocket (Fig. 3d and e). However, relative to the template in the incoming dTTP complex, template 1-MeA in Polι 1-MeA.dCTP is inclined towards the DNA helical axis by ~10° and protrudes into the minor groove, partially occluding the dNTP binding site (Fig. 3f). As a result of this inclination, the N6 atom of 1-MeA moves by ~1.0 Å into the dNTP binding cavity relative to the incoming dTTP ternary complex.
Another notable difference between the structure of Polι 1-MeA.dCTP and Polι 1-MeA.dTTP is in the conformation of the catalytic residue Asp126 (Fig. 3f). In the binary complexes of Polι with template purines, Asp126 is involved in hydrogen bonding interactions with solvent molecules that occupy the vacant dNTP binding pocket, as well as a putative hydrogen bond with 3′ OH group modeled at the primer terminus 23 . In Polι 1-MeA.dTTP (and other ternary structures with purine templates), the side chain of Asp126 undergoes an ~30° rotation to establish new interactions with the backbone carbonyl of Leu35, the metal ion at site B, and with the α -phosphate of incoming (Fig. 3f) [17][18][19]23 . By contrast, in Polι 1-MeA.dCTP , Asp126 remains in the same conformation as in the binary structures. The electron density for incoming dCTP is very weak with only the density for γ -phosphate clearly visible. The "base" of dCTP is poorly defined (Fig. 3e). Attempts to model dCTP using the conformation of dTTP as a guide leads to steric overlap between its N4 amino group and the N6 amino group of 1-MeA. The dCTP C5′ sugar atom and the α -phosphate also clash with the side chain of Asp126. Efforts to relieve these steric clashes by small movements of the base, sugar and phosphate groups of dCTP lead instead to steric clashes with Val64, Thr65 and Tyr39 from the fingers domain or with the template 1-MeA. These steric clashes are aggravated by the narrow active site of Polι [17][18][19]23,25 and protrusion of the template 1-MeA into the dNTP binding pocket by ~1.0 Å. Taken together, the weak electron density for incoming dCTP and the binary like conformation of Asp126 suggest a disordered dCTP with only its γ -phosphate anchored in place while the sugar and base sample a range of conformations. Discussion Alkylating agents modify DNA by adding alkyl groups to both the ring nitrogens and the exocyclic oxygen atoms, generating adducts that have cytotoxic effects 5,6,8 . 1-MeA is a common adduct generated by the transfer of methyl group to the N1 nitrogen atom of deoxyadenosine. If left unrepaired, 1-MeA presents a strong block to replicative polymerases due to its inability to participate in W-C base pairing. We show here by steady state kinetics that Polι , a Y-family polymerase, is capable of TLS across 1-MeA, and that it incorporates the correct T with an ~100 fold higher efficiency than the incorrect C. We also derive a structural framework for the ability of Polι to accommodate the 1-MeA adduct, and a basis for the selection of correct from incorrect incoming nucleotide.
In all previous ternary structures of Polι with a template purine, the template is in syn and dNTP is in anti conformation [17][18][19]23 . The Polι active site cleft is narrower than in other polymerases, which effectively pushes the template purine into a syn conformation when the incoming dNTP binds. The resulting C1′ -C1′ distance across the nascent base pair reduces to < 9 Å, favorable for Hoogsteen base pairing. Thus, given the constraints of the Polι active site cleft, it is not surprising that 1-MeA is also pushed in to the syn conformation for Hoogsteen base pairing with incoming dTTP (which remains in the anti conformation). Importantly, the complex is competent for catalysis with the scissile Pα -O3′ bond of incoming dTTP aligned favorably with respect to a 3′ OH modeled at the primer terminus.
By contrast, although the dCTP γ -phosphate occupies the same position as the γ -phosphate of dTTP in the Polι 1-MeA.dTTP structure, the rest of the molecule is disordered (Fig. 3e). The ~100 fold lower efficiency of Polι in inserting C relative to T opposite 1-MeA can be rationalized by the inability of dCTP to gain a firm "foothold" opposite 1-MeA. Incoming dCTP does not offer the same hydrogen bonding opportunities opposite 1-MeA as dTTP, and its N4 amino group sterically impinges on the N6 group of 1-MeA. Accordingly, it seems to adopt a range of conformations that is not conducive to catalysis. Also, Asp126 remains in a binary-like conformation and prevents the α -phosphate of dCTP from aligning properly with respect to the primer terminus for catalysis.
Although Polι is inefficient at incorporating the incorrect C (and incorrect A) opposite 1-MeA, it does so more efficiently than opposite undamaged template A (Table 1). We suspect that this is because the methyl group at N1 favors the imino tautomer of 1-MeA 26 . In its imino tautometric form, the N6 imino group of 1-MeA would be in a position to establish a putative N6(1-MeA)… N4(dCTP) or N6(1-MeA)… N6(dATP) hydrogen bond, enabling Polι to incorporate C or A opposite 1-MeA more readily than opposite A.
In conclusion, we present here the first kinetic and structural analysis of the ability of Polι to replicate through the 1-MeA adduct. 1-MeA is highly cytotoxic because a methyl group at N1 atom impairs W-C base pairing and presents a strong block to normal DNA replication. By pushing 1-MeA in to the syn conformation, Polι can carry out effective TLS opposite 1-MeA via Hoogsteen base pairing with correct incoming T.
Structure Determination and Refinement. X-ray data on cryocooled crystals were measured at Brookhaven National Laboratory (BNL beamline X-25) and Advanced Photon Source (APS, beamline 24-ID-E). Data sets were indexed and integrated using HKL2000 28 . The Polι 1-MeA.dTTP and Polι 1-MeA.dCTP structures were solved by molecular replacement (MR), using the Polι A.dTTP complex as a search model (2FLL, with template and incoming dNTP omitted). The first round of refinement and map calculation was carried out without the template and the incoming nucleotide. Initial electron density map showed unambiguous density for the template 1-MeA in both the structures, which was then included in the model for subsequent refinement. Iterative rounds of refinement and water picking were performed with Phenix 21 and model building with program Coot 22 . All models have good stereochemistry, as shown by MolProbity 29,30 with > 99% of the residues in the most favored regions of the Ramachandran plot and 0.8% in the disallowed regions. Figures were prepared using PyMol 31 .
DNA Polymerase Assay. DNA substrates consisted of a radiolabeled oligonucleotide primer annealed to a 75nt oligonucleotide DNA template by heating a mixture of primer/template at a 1:1.5 molar ratio to 95 °C and allowing it to cool to room temperature for several hours. The template 75-mer oligonucleotide contained the sequence 5′AGC AAG TCA CCA ATG TCT AAG AGT TCG TAT AAT GCC TAC ACT GGA GTA CCG GAG CAT CGT CGT GAC TGG GAA AAC-3′ and was either undamaged A or harbored a 1-MeA at the underlined position. For steady-state kinetic analyses of nucleotide insertion opposite the undamaged A or 1-MeA, a 44 mer primer 5′ GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GGC AT-3′ was used annealed to the above mentioned 75 mer templates.
Scientific RepoRts | 7:43904 | DOI: 10.1038/srep43904 Steady-State Kinetic Analysis. Steady-state kinetic analyses for deoxynucleotide incorporation were performed as described 32 . Polι (0.02-0.2 nM) was incubated with primer:template DNA substrate (10 nM) and increasing concentration of dNTPs for 10 min, at 37 °C. Gel band intensities of the substrate and products of the deoxynucleotide incorporation reactions were quantified by using a PhosphorImager and the IMAGEQUANT software (Molecular Dynamics). The observed rate of deoxynucleotide incorporation, v obs was determined by dividing the amount of product formed by the reaction time and protein concentration. The v obs was graphed as a function of the deoxynucleotide concentration, and the data were fit to the Michaelis-Menten equation describing a hyperbola: v obs = (k cat [E] × [dNTP])/(K m + [dNTP]). From the best fit curve, the apparent K m and k cat steady-state kinetics parameter were obtained for the incorporation of dNTP by the Polι and the efficiencies of nucleotide incorporation (k cat /K m ) were determined.
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2018-04-03T03:38:59.563Z
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2017-03-08T00:00:00.000
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Natural Deep Eutectic Solvents (NADESs) Combined with Sustainable Extraction Techniques: A Review of the Green Chemistry Approach in Food Analysis
Usual extraction processes for analyzing foods, supplements, and nutraceutical products involve massive amounts of organic solvents contributing to a negative impact on the environment and human health. In recent years, a new class of green solvents called natural deep eutectic solvents (NADES) have been considered a valid alternative to conventional solvents. Compared with conventional organic solvents, NADES have attracted considerable attention since they are sustainable, biodegradable, and non-toxic but also are easy to prepare, and have low production costs. Here we summarize the major aspects of NADEs such as the classification, preparation method physicochemical properties, and toxicity. Moreover, we provide an overview of novel extraction techniques using NADES as potential extractants of bioactive compounds from foods and food by-products, and application of NADEs in food analysis. This review aims to be useful for the further development of NAES and for broadening the knowledge of these new green solvents in order to increase their use for the extraction of bioactive compounds and in food analysis.
Introduction
The analysis of foods is a comprehensive process of extraction, identification, and quantification of several classes of compounds from natural matrices. The detection and quantification of primary metabolites (sugars, amino acids, vitamins, and lipids), contaminants (toxins, heavy metals, and allergens), and secondary metabolites (polyphenolics, flavonoids, terpenes, and alkaloids) is a crucial practice for ensuring the safety and quality of foods and related functional products. Due to the variable structure of food analytes, a gap in a universal method suitable for the extraction and analysis of all compounds is lacking. Moreover, conventional extractants are usually made of organic solvents and common extraction techniques usually require a long extraction time to exhaust the matrix [1]. The actual discussions about climatic changes provide a growing awareness of the scientific and industrial community to reduce the environmental impact by using sustainable processes. In general, the main principles of "green chemistry" are based on the design of processes aimed to reduce energy consumption and the use of eco-friendly solvents with less toxicity to the environment and human health. In this view, ionic liquids (ILs) and deep eutectic solvents (DES) replaced the use of traditional organic solvents in several analytical applications of the last years.
The development of ionic liquid (ILs) solvents started in the 90s. Despite their nontoxic behavior as pure solvents, several steps of purification should be used to produce ILs that negatively affect economic and environmental sustainability [2]. Thus, deep eutectic solvents (DES) were born due to their non-toxic handling, wide versatility of use in different sectors, Generally, the subgroup of NADESs uses HBD and HBA made by sugars, alcohols, organic acids, amino acids, or amines naturally occurring in plant metabolism or eucaryotic cellular systems [9]. NADESs have been indicated as possible solvents present in living cells, thus explaining the presence of compounds at much higher concentrations than what is soluble in aqueous solutions [10]. Furthermore, NADESs reduce physicochemical constraints of metabolite transport and cellular processes through the formation of liquid microenvironments [11]. The mostly used HBA are chlorine chloride (ChCl) and betaine for their low cost, non-toxicity, and biodegradability [8,12] but also proline, glycine, alanine, histidine, lidocaine, acetylcholine chloride, and nicotinic acid are currently used as eco-friendly and biocompatible molecules [13]. Natural carboxylic acids (gallic acid, benzoic acid), hydroxycinnamic acid derivatives (coumaric acid and caffeic acid) different sugars (xylitol, glucose, fructose), organic acids (oxalic acid, malic acid), and fatty acids (stearic acid, oleic and linoleic acid) are the used as natural HBD combined with HBA [13].
The advantage of these solvents is related to chemical properties, such as low melting points, low volatility, nonflammability, low vapor pressure, polarity, chemical and thermal stability, and miscibility solubility [5][6][7]14,15]. Additionally, a convenient atom economy and a low environmental impact are related to their low costs and high yields of production. Their assembly through intermolecular hydrogen bond interactions does not involve chemical reaction hence there is no production of secondary compounds reducing the need for further purification steps, and no waste is usually produced [7,[15][16][17]. The current review is focused on the topic of natural deep eutectic solvents (NADES) Generally, the subgroup of NADESs uses HBD and HBA made by sugars, alcohols, organic acids, amino acids, or amines naturally occurring in plant metabolism or eucaryotic cellular systems [9]. NADESs have been indicated as possible solvents present in living cells, thus explaining the presence of compounds at much higher concentrations than what is soluble in aqueous solutions [10]. Furthermore, NADESs reduce physicochemical constraints of metabolite transport and cellular processes through the formation of liquid microenvironments [11]. The mostly used HBA are chlorine chloride (ChCl) and betaine for their low cost, non-toxicity, and biodegradability [8,12] but also proline, glycine, alanine, histidine, lidocaine, acetylcholine chloride, and nicotinic acid are currently used as ecofriendly and biocompatible molecules [13]. Natural carboxylic acids (gallic acid, benzoic acid), hydroxycinnamic acid derivatives (coumaric acid and caffeic acid) different sugars (xylitol, glucose, fructose), organic acids (oxalic acid, malic acid), and fatty acids (stearic acid, oleic and linoleic acid) are the used as natural HBD combined with HBA [13].
The advantage of these solvents is related to chemical properties, such as low melting points, low volatility, nonflammability, low vapor pressure, polarity, chemical and thermal stability, and miscibility solubility [5][6][7]14,15]. Additionally, a convenient atom economy and a low environmental impact are related to their low costs and high yields of production. Their assembly through intermolecular hydrogen bond interactions does not involve chemical reaction hence there is no production of secondary compounds reducing the need for further purification steps, and no waste is usually produced [7,[15][16][17]. The current review is focused on the topic of natural deep eutectic solvents (NADES) combined with different extraction techniques to design processes that reduce energy consumption, ensure the use of safe alternative solvents, and optimize high extraction efficiency of target molecules occurring in several fields of foods analysis.
Physicochemical Properties of NADES for the Extraction Process
The intermolecular interactions, mainly hydrogen bond interactions, between the HBA and the HBD are responsible for the physicochemical properties of the DES [14,18]. By changing either the HBA/HBD ratio or one of the components, the NADES can be specifically tailored for different applications. This is a major advantage in terms of achieving desirable properties and improving extraction efficiency [7,17]. Viscosity, polarity, density, and pH condition are the main physicochemical properties affecting the extraction of natural compounds from food matrices. The addition of water as a third component of the system can modulate the conditions of the NADESs for the extraction of several compounds and chemicals for food analysis applications.
Generally, the high viscosity of HDA and HDB mixtures cause a low mass transfer phenomenon affecting the extraction efficiency. Temperature can drastically reduce the viscosity but a mediation with the thermolability of natural compounds must be considered. Added water, reported in a range of 10% to 80% [19], disrupts the hydrogen bond interaction between HAD and HDB modulating the viscosity of the system. Data reported by Zhekenov et al., 2019 suggest a maximum limit of 50% molar fraction of water beyond the system act as a solution [20].
Polarity is the key property to solubilizing metabolites in a solid-liquid extraction. Fixing the choline chloride (ChCl) as HBA, the use of different HBD can affect the polarity of the system. Craveiro et al., 2016 reported the use of sugars (glucose, sucrose, xylose) and organic acids (citric acid, tartaric acid) generating different polarity systems. In particular, NADES composed of ChCl and organic acids resulted more polar than those combined with sugars [21]. The water addition can also affect the polarity of the NADES [22,23]. HBD used for the preparation of NADES act both as hydrophilic and lipophilic components. Components with electronegative groups can form dipole−dipole interactions with polar solvents explaining the hydrophilic properties [24]. On the other hand, natural lipophilic compounds commonly used in NADES are characterized by a polar moiety forming dipoledipole interactions with polar solvents and a hydrophobic moiety with a tendency to aggregate in aqueous solution to minimize the area of contact between water and nonpolar molecules. Several NADES systems are described by the hydrophilicity/lipophilicity balance phenomenon. For instance, fatty acid-and terpenes-based natural compounds acting as HBD have been reported for NADES [25][26][27].
Complete profiling of complex food matrices by using organic solvents allows the extraction of hydrophilic components or lipophilic components separately while the ignored components that remain in the extraction residues are discarded from the analysis. The advantage to use hydrophilic or hydrophobic NADES is the efficient enhancement of bioactive components with various polarities. The density of hydrophilic or hydrophobic solvents can change allowing a double-phase separation. The two-phase system formed with hydrophilic NADES and hydrophobic NADES can resolve the problem of extraction selectivity and could simultaneously extract the bioactive compounds with various polarities from plant materials. In 2015, Van Osch et al. developed a NADES two-phase system and evaluated the recovery of volatile fatty acids from dilute aqueous solutions [26]. A two-phase aqueous system (NADES-salt solution) has also been established to extract proteins [28][29][30][31] and non-polar anthraquinones [30]. Moreover, Jun Chao et al., 2018 developed a two-phase NADES system (ChCl-LA1/Ch-M/MCO) to extract bioactive metabolites with different polarities from Ginkgo biloba leaves [16].
Preparation of NADES
The NADESs can be produced from different natural compounds such as ChCl, sugars, amino acids, and polyols. These compounds are in solid form and only after mixing, in a specific combination and molar rates, do they turn into a liquid state [32]. Five methods are reported to prepare NADES: thermal mixing, vacuum evaporation, freeze-drying, ultrasonication, and microwave.
•
Thermal mixing is a simpler and faster approach that involves mixing and stirring at a temperature of 50-80 • C of two components with water until a colorless liquid is formed [22,33,34]. • Vacuum evaporation is also similar; the components are dissolved in water and evaporated at about 50 • C in rotary evaporation, and finally, the resulting liquid is placed in a desiccator to reach a constant weight [22,34]. • Freeze-drying involves dissolving the components in water, then freezing and drying them, resulting in a viscous, transparent solution [34,35].
•
Microwave-assisted synthesis exploits the production of microwaves that upon interaction with precursors generate collisions between molecules and between the hydrogen bond donor and hydrogen bond acceptor components due to dipole rotation resulting in dielectric heating that speeds up the synthesis time [36,37]. According to Popovic et al., 2022, microwaves could be one of the fastest methods for the preparation of some NADES, taking even less than a minute [33]. However, because of the possible overheating caused by the technique, it is advisable to divide the process into several cycles of a few seconds interspersed with cooling pauses [38]. The entire preparation is carried out in closed systems with controlled pressure and temperature.
•
Ultrasound-assisted is a little-explored but effective way of preparing NADESs. The cavitation process promotes, through the release of heat and pressure exerted because of bubbles implosion, the interaction between the hydrogen bond acceptor (HBA) and the hydrogen bond donor (HBD) [34]. According to when described by Santana et al., 2019, the preparation of NADES by ultrasound can also be performed by heating the mixture around 50 • C [34]. This approach requires several minutes with intermediate times between microwaves and the remaining techniques described above.
The preparation of NADES through the different methods ( Figure 2) generates clear solvents without the presence of precipitates at room temperature with the same properties [33,34]. The principal difference consists in the timing of the process. Ultrasound and microwave methods offer high speed and efficiency of preparation compared with thermal mixing, vacuum evaporation, and freeze-drying; however, the mixing and stirring method is still widely used because it is very simple, easy to perform and allows the production of high volumes of solvent.
Use of NADES as Green Solvent in the Extraction Techniques
The use of NADES as extraction solvent is often combined with techniques such a ultrasound-assisted liquid extraction (USAE), microwave (MAE), or pressurized liqui extraction (PLE). This is mainly due to the properties of NADES used as alternative so
Use of NADES as Green Solvent in the Extraction Techniques
The use of NADES as extraction solvent is often combined with techniques such as ultrasound-assisted liquid extraction (USAE), microwave (MAE), or pressurized liquid extraction (PLE). This is mainly due to the properties of NADES used as alternative solvents to classical solvents for lower toxicity and higher extraction efficiency, making them suitable for green chemistry extraction techniques that aim to reduce cost, risk, extraction time and environmental impact (Table 1) [33,39]. Numerous studies employing such solvents on food matrices for the recovery of various bioactive compounds are reported in the literature. Mostly, Bajkacz et al., 2017 and Rashid et al., 2023 optimized a method for recovering phenols respectably from soybean and apple by-products by using different types of NADES and ultrasonic techniques. The results demonstrated how the combination of ultrasonic with NADES solvents can be an innovative method of phenol recovery due in part to the Hinteractions formed between NADES and phenols; furthermore, the extraction efficiency was implemented by adding 30% water to the NADES mixture, which from the results proved to be the best condition [2,39]. Still, Loarce et al., 2011 have used NADES as modifiers for PHWE extraction, significantly improving anthocyanin recovery compared to water alone while keeping the process green and sustainable [40]. The process was optimized using water with 30% NADES composed of choline chloride and oxalic acid at a temperature of 60 • C as solvent. Another example is the study conducted by Fan and Li., 2022 that through the coupling of NADES and microwaves, finally these solvents were also used with microwave hydrodistillation for the extraction of essential oil from A. sinensis. The combination of NADES with microwaves that promote plant cell rupture compared to conventional methods allows implementation of the recovery of molecules by reducing while maintaining the process with low environmental impact and high efficiency [41]. 47.4 ± 1.6 mg/g c a mg total phenol /gEXT, b mg gallic acid equivalent /gEXT, c mg malvidin-3-glucoside equivalent /gEXT.
Toxicity and Sustainability
The green chemistry approach requires minimizing the risks and hazards associated with the process and its impact on the environment and human health. It is precisely for this motive that numerous scholars have begun to move toward the use of safer and more environmentally friendly solvents. At this juncture are NADES, which are considered nontoxic, environmentally sustainable, and biodegradable, thanks mainly to the fact that they are made up of compounds of natural origin. Choline chloride (ChCl), for example, one of the main HBAs, is widely used for the preparation of NADES and used commercially on a large scale as an additive for chicken feed, in addition, NADES are also used for the solubilization of drugs for oral dosing in rats, and the synthesis of biodegradable polyesters with antibacterial properties [42]. The emergence of NADES is related to their very low toxicity, which allows them to be used more safely than the previous ionic solvents (ILs), which, on the other hand, can have similar toxicity to the organic compounds they replace [43].
The toxicity of NADES has long been evaluated by considering only the single toxicity data of all the components used in the preparation, which are reported to be safe [44]. However, more recent studies have indicated that these molecules when mixed may show higher toxicity than the single components, related to the structure of the deep solvents, due to synergistic effects between the individual elements. Hayyan et al., 2013 conducted some experiments to evaluate the toxicity and cytotoxicity of ChCl and phosphonium-based deep solvents [45]. The models used for toxicity evaluation were two strains of Gram+ bacteria Bacillus subtilis and Streptococcus aureus and two Gram-strains Escherichia coli and Pseudomonas aeruginosa, while cytotoxicity was considered on Artemia salina. The results show that phosphonium-based deep eutectic solvents have higher toxicity on all bacterial strains, highlighting a possible antibacterial effect, unlike choline chloride-based NADES. However, for both types of HBAs, the mixture of NADES was more toxic than the individual components, even for cytotoxicity, the ammonium-based deep eutectic solvents showed a greater effect on Artemia salina than the individual components. Justifications for this behavior may be due to several factors such as hydrogen bonding between HBD and the salt anion. It is known that the delocalization of charge that occurs during hydrogen bonding makes the mixture more toxic; in fact, substances with delocalized charges are more toxic than chemicals with localized charges. The lack of oxygen and high viscosity that impairs the movement of A. salina may also influence by modifying the toxicity of the mixture. Further experiments conducted by Hayyan et al., 2013 also hypothesize that NADES can interact with cell surfaces and that their accumulation and aggregation may cause increased cytotoxicity [45]. Still, Radosevic et al., 2015 evaluated different aspects of the toxicity of different deep eutectic solvents based on ChCl, in particular, they considered phytotoxicity on wheat, toxicity on fish and human cells, and biodegradability using wastewater microorganisms through closed bottle test [46]. The results show low to moderate cytotoxicity on cells with cell inhibition comparable to that of industrial solvents, inhibition in wheat germination was also not observed with oxidative stress manifestation only at high amount addition, all NADES tested were classified as biodegradable. NADES thus show a good correlation between biodegradability and toxicity with some advantages such as low vapor pressure and low flammability making them preferable to the organic solvents they are supposed to replace. These results, therefore, showed that natural deep eutectic solvents based on choline chloride have a potential green profile and a very good prospect for use; however, considering the different toxicity observed for some mixtures, especially those based on phosphonium, further studies are needed to understand and clear their impact on the environment and organisms [45].
Food Analytics
Green chemistry plays an important role in the development of a sustainable process for food manipulation. The optimization of food analysis is a compromise between the reduction of solvents, toxic for human health and environmental pollution, reagents, and energy along with the necessity for high sensitivity, precision, and accuracy for validated analytical methods. NADES have been currently used in green chemistry procedures for sample preparation and analytical workflow focusing on achieving accurate results and prioritizing the sustainability of the processes [47]. In the current review, NADES applications in several fields of food analysis are discussed in procedural workflow steps. Green analytical chemistry plays an important role in the development of a sustainable process for food manipulation. The optimization of sustainable analytical procedures is a compromise between the reduction of solvents, toxic for human health and environmental pollution, reagents, and energy along with the necessity for high sensitivity, precision, and accuracy for validated analytical methods.
The extraction is the core procedure for the recovery of phytochemicals, bioactive compounds from natural sources. The aim to optimize the extraction procedure by choosing solvents, experimental conditions, and extraction techniques is to provide the highest amount of target compounds with minimal contamination of other undesired compounds [48]. Solid-liquid extraction with organic solvents is a conventional procedure used for the recovery of phytochemicals enabling the release of solutes from a solid matrix to the liquid phase usable for further wet analysis as HPLC combined with several UV or MS detectors for compound identification [48]. Nevertheless, pollution enabling, and the toxicity related to the use of these organic solvents is strongly discouraged in the food, cosmetic, and pharmaceutical industries [49]. Natural deep eutectic solvents (NADES) satisfied the environmental consciousness by designing sustainable extractions strategies following the basic principles of green chemistry [50]. Beyond the representation of a sustainable alternative for the extraction of target compounds, these solvents signify an opportunity to develop functional foods or nutraceuticals, using naturally occurring components, colorants, and preservatives [49].
Chromatography is a crucial tool for the analysis of complex food matrices assisting the pre-concentration, separation, and isolation steps in the usual workflows. NADESs are used as stationary phases or eluent in several chromatographic procedures assisting the separation of target compounds from natural products. Tang et al., 2015 used NADES-based stationary phase sorbents. Functionalized spheres were packed in a 250 × 4.6 mm column creating a high-performance size-exclusion chromatography (HP-SEC) system for the separation of metabolites with different shapes and sizes by using water as a mobile phase to separate three polysaccharides alginic acid, fucoidan, and laminarin [13]. [53].
Despite the interesting approaches of NADES in analytics, the interference of extractants constituents in testing samples causes a complex pattern of signals reducing the performances of the characterization and deconvolution of the data files. A treatment of the extract by NADES removal, aimed to improve the quality of signals and analysis, could be necessary. In view of green methodology for the extraction of target compounds, NADES can be recycled. Normally, distillation is the current method used for the recycling of organic solvents for the easy recovery of target compounds. The evaporation of NADES extractants is a difficult task because of low vapor pressure and peculiar physiochemical properties causing a problem for industrial applications [54,55]. The possibilities for target compounds recovery and NADES recycling include liquid-liquid extraction (LLE) using another solvent, solid-liquid extraction using a macroporous resin, and the addition of antisolvents [56][57][58]. NADES exhibit unusual solvation properties with protic solvents (methanol, ethanol, or water) due to hydrogen bonding, whereas aprotic ones (toluene, [59]. Another purification technique is Solid-Phase Extraction (SPE) by using adsorption cartridges. In SPE purification, less polar analytes are adsorbed onto the resins and then it is necessary to wash the NADES out with water followed by the elution of the target analytes with an alcoholic solvent (e.g., ethanol) [56]. Instances of flavonoid isolation through SPE were performed by Wang and Wang, 2019. After optimized ethylene glycol-choline chloride-UAE extraction, authors tested the adsorption and desorption of flavonoids from Safflower on five types of macroporous resins for flavonoids isolation, finding that the highest adsorption capacity was produced by NKA-2 followed by S-8 [60]. On the contrary, Zhuang et al. 2017 selected LX-38 as the best macroporous resin for the recovery of the flavonoids from the DES with a satisfactory yield of 98.92% [61]. Similarly, Bi et al.,2020 performed the flavonoid separation by NADES-MAE followed by direct macroporous resin adsorption and desorption process. Using the optimized parameters, the direct separation of baicalin, wogonoside, baicalein, and wogonin enriched fractions obtained from NADES extraction solution was efficiently achieved using macroporous resin ME-2 with recovery yields around 80-85% [62]. Panìc et al., 2019 proved that the presence of >50% (v/v) of water ruptures the NADES structure, a simple dilution allowed an anthocyanin recovery of 99.46%, and a highly efficient solvent recycling (yield 96.8%) when authors diluted the grape-pomace extract in 80% of water. The cleanness of the NADES after recycling was proved using NMR spectrometry and no significant differences between freshly synthesized and recycled NADES in 1H NMR spectra were observed. These results implicate that NADES structures should be disturbed prior to recycling via dilution with >50% amount of water (v/v) to release anthocyanins and make them available for better adsorption on macroporous resins [63].
Extraction of Bioactive Compounds from Natural Sources
Sustainable and highly efficient extraction of bioactive natural compounds from natural sources is considered an important task for the development of nutraceuticals and food supplements. The biodegradability, low toxicity, and adjustable solvent properties of NADES along with their extraordinary solubilizing power for natural products of diverse polarity, accent the interest in their use for the extraction of bioactive compounds from natural sources. Coumaric acid derivatives, flavonoids, anthocyanins, and resveratrol are only a few metabolites ranked in the several classes of phenolics contained in functional foods exerting antioxidant properties. Several studies reported a higher efficiency for the extraction of phenolics from different matrices as reported in Table 2. Luoxuan Lin in 2022 optimized the extraction of phenolic acids from orange peels by using different ratios of choline chloride, glucose, and water. The DPPH assay was used to evaluate the antioxidant properties of extracted phenolic acids. The optimized condition for improved antioxidant activity was obtained at a molar ratio of 5:2:5 (ChCl/D-(+)-glucose/H2O) [64]. Zannou et al., 2022 studied the incidence of a ChCl/acetic acid system on the recovery of total phenolics from bitter melon (Momordica charantia) monitored by TPC, TFC, DPPH, and FRAP assays. The tests showed a higher amount of phenolics in agreement with the optimal antioxidant activity for the extracts obtained in the conditions reported in Table 2. The amount of gallic acid, vanillic acid, epicatechin, and quercetin-3-O-glucoside, evaluated by chromatographic analysis, was higher in the considered NADES than in other used extracts [65]. Guo et al., 2019 selected a choline chloride/citric acid/glucose (1:1:1) NADES system for a high-yield extraction of anthocyanins from mulberry by using innovative high-speed homogenization (HSH) and cavitation burst extraction (CBE) [66]. Zengin et al., 2022 performed a comparative LC-ESI-QTOF-MS study of the bioactive compound profiles of Cytinus hypocistis extracted by three different choline chloride, proline, and xylitol-based NADES systems compared with conventional solvents, as detailed in Table 2. The correlation of prepared extracts with bioactivity was performed in terms of phenolic and flavonoid content yield along with specific enzyme inhibition. TPC, TFC, DPPH, and ABTS assays were performed for the evaluation of antioxidant activity while cholinesterase (AChE and BChE), tyrosinase, and glucosidase inhibitory assays were performed for specific target interaction. The study highlighted the improved ability of NADES systems in bioactive content yield and enzyme inhibitory bioactivity compared to traditional solvents [67]. Vieira et al., 2022 focused on the advantage of the NADES characterized by different polarity and viscosity to form a biphasic system for the combined extraction of polar rosmarinic acid and non-polar carnosic acid and carnosol compounds from Rosmarinus officinalis [68]. The NADES selective extraction of bioactive molecules from soursop leaves (Annona muricata L.) was investigated by Castro Leal et al., 2022. HPLC-DAD monitoring of the main antioxidants rutin and catechin showed the extraction capacity of all the selected NADES systems revealing those composed of glycerol and xylitol as hydrogen bond donors presented the higher extraction indexes [69]. Chen et al., 2022, investigated for the first time NADES for extracting agents of pectins from mango peels. Two novel green solvents were screened betaine-citric acid (Bet-CA) and choline chloride-malic acid (ChCl-MaA) coupled with the USAE preparation technique. The NADES-extracted pectins resulted higher in extraction yield, larger molecular weight, and particle size than HCl-extracted pectins characterized by Fourier transform infrared spectra (FT-IR) and thermal analysis [70]. Bajkacz et al., 2017 developed NADES extraction procedure and UHPLC-UV monitoring for the determination of isoflavones in soy-containing food samples. The optimized conditions for isoflavones extractions were selected with choline chloride/citric acid (1:1) with a 30% water content in NADES [39].
The versatile polarity modulation of NADES systems allows the recovery of a bioactive molecule with non-polar behavior from natural matrices. Several lipid-soluble compounds such as curcumin and lycopene are considered for industrial importance for both their coloring attributes but also for their bioactive properties such as antioxidant, anticancer, and immunomodulatory [71]. Alioui et al., 2022, performed different solubility tests of the non-polar curcumin by using different NADES systems with the aim to demonstrate that the studied NADESs had critical structural features in H-bonding and curcumin solubilization capabilities. Curcumin resulted more soluble in the choline chloride/glycine system than the other NADES examined [72]. The constantly increasing lycopene demand for food, pharmaceutical, and cosmeceutical applications, has led the scientific community to explore alternative approaches to extract it from natural sources. Different hydrophobic natural deep eutectic solvents (HNADES) based on terpenes (i.e., menthol and thymol) and fatty acids (i.e., decanoic acid and dodecanoic acid) were prepared at different molar ratios. Spectrophotometry and RP-HPLC-DAD were used to monitor the process efficiency of extracting lycopene from tomatoes. thymol/lauric acid system (2:1) was the optimized NADES for the selective recovery of the non-polar compound [73]. Four kinds of NADES were tested by Hong et al., 2022 for the extraction of solanesol from tobacco leaves and compared with conventional organic solvents. Solanesol was recovered from the NADES extract by microextraction with the addition of ethyl acetate and finally analyzed using high-performance liquid chromatography (HPLC). The chlorine chloride/urea NADES was selected for the improved extraction efficiency of solanesol, with a water content of 5% (w:w) [74]. Essential oils are characterized by their aroma and significant pharmacological effects as analgesic, anti-inflammatory, and cerebral activities with nutraceutical and cosmetical interest [75]. Fun and Li., 2022 studied the different NADES for essential oil extraction from Angelica sinensis radix. It was found that choline chloride and citric acid were the optimized combination for the extraction of essential oil. The higher composition of the essential oils extracted by the ChCl/citric acid system was analyzed by gas chromatography-mass spectrometry (GC-MS), highlighting the occurrence of ligustilide as the main component of Angelica sinensis essential oil [41]. * (v/v) molar ratio. "-" not provided.
Extraction of Bioactive Compounds from Agricultural Food by-Products
The awareness for the optimization of natural resources and the environmentally sound management of chemicals and all wastes throughout their life cycle is growing [49]. A big amount of food waste produced (about 1.3 billion tons of food waste per year) could provide a useful and inexpensive source to obtain high-value compounds [85]. Moreover, the use of food by-products as a source of bioactive compounds is a good opportunity to implement a circular economy and reduce the continuous increase in the production of organic wastes [86] and its consequent environmental problem. In particular, the use of by-products as a source of biomass for cosmetical, nutraceutical products, or biofertilizers can create profit and reduce the environmental impact and the costs of their disposal.
The olive oil industry produces large biomass of not-edible products, olive pomace is at least 60-65% of the total weight of olives. As well as the whole olive, pomace is considered a source of phenolic bioactive compounds. The NADES selective extraction of phenolics from this complex matrix was performed by Chanioti et Tzia 2018 ( Table 3). The NADESs obtained by choline chloride and organic acid HBD, as citric acid and lactic acid, were selected as the most promising extractants and more effective in the olive pomace phenolic compounds extraction compared to conventional solvents. The extraction technique by homogenization possessed the highest total phenolic content (TPC) and antioxidant activity of the other extracts [87]. Nelus Mayer et al., 2022 also focused their attention on the optimization of NADE extraction of phenolics on pomace olive. A comparative study between different NADES and conventional solvents obtained by ultrasound-assisted extraction technique was performed by HPLC monitoring of the hydroxytyrosol and luteolin concentration, and the determination of total phenolic content, anthocyanin content, and antioxidant capacity in samples from different cultivars and harvest seasons. Lactic acid/glucose was selected as the optimized extractant of bioactive phenolics using an ultrasound technique [88]. Olive leaves are another important waste product for the alimentary production of oil. Zurob et al., 2020, screened the extraction efficiencies of eight sugar-based and organic acid-based NADES for the recovery of hydroxytyrosol. The HPLC-UV experimental results showed that the hydroxytyrosol amount is higher in a citric acid/glycine/water (2:1:1) system than in water used as a conventional solvent for the extraction of polyphenols [89].
The recovery of anthocyanins by using NADES from grape pomace was studied by Panić et al., 2019. A comparative study of several NADES for the optimization of composition, physiochemical characteristics, anthocyanins extraction power, and solvent price was investigated and performed on a larger scale-up extraction process with NADES recycling. The highest content of all the anthocyanins, evaluated by the sum of all HPLC signals, was attributed to the ChCl/citric acid and ChCl/proline/malic acid systems by using an ultrasound microwave-assisted extraction technique [63]. In the recent work of Lanjekar et al., 2022, eight different NADES were prepared using components such as choline chloride, carboxylic acids, sugars, and alcohols to assess the extraction efficiency from mango peel by-products. The lactic acid/glucose system exerted the highest total phenol and flavonoid content [90]. * v/v ratio.
Food Safety
While natural phenolic compounds are involved in plant biological processes such as growth, reproduction, and defense mechanisms, synthetic phenolic compounds (i.e., phenol, o-cresol, and 2-chlorophenol) are precursors of nylon, and detergents used to produce polyphenoxy polymers, fertilizers, paints, or explosives. The World Health Organization, WHO, lists some of these phenolic compounds as priority pollutants due to their toxicity, so it is necessary to remove them, to a concentration below the legal limit before wastewater discharge [94]. G. Sas et al., 2019, texted six new NADES based on organic acids (dodecanoic acid, decanoic acid, octanoic acid) and menthol or thymol, as extraction solvents for these pollutant compounds. The UV-Vis spectrophotometer was used to quantify the phenolic compound concentrations in the aqueous phase after extraction. Menthol/octanoic acid and menthol/decanoic acid (both with molar ratio 1:1) presented the best results for the extraction of phenolic pollutants from water obtaining extraction yields upper than 80% for 2-chlorophenol and o-cresol compounds (Table 4) [94]. The analysis of bisphenols and alkylphenols in functional beverages, kombucha, and water kefir was investigated by Baute-Pérez et al., 2022. The main objective of their paper was to evaluate the presence of 13 alkylphenols, bisphenols, and alkylphenol ethoxylates in bottled water, kombucha, and water kefir by using NADES extractants, based on monoterpenes and fatty acids, and detected by a new vortex-assisted (VA)-DLLME-ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) as defined in Table 4. Only TOP1EO was detected in bottled waters, whereas no residues appeared in kombuchas and water kefirs. The analytical performance of the method and the obtained results demonstrated not only the applicability of the method but also that the consumption of these matrices is safe [95]. Soltani and Sereshti 2022 developed a novel green analytical procedure based on green deep eutectic solvents (DES) followed by GC-MS to analyze pesticides in tea samples. This work aims to develop a green QuEChERS approach modified based on the use of green deep eutectic solvents (DES) for two basic steps: (1) the extraction of pesticides from tea samples, and (2) the sample clean-up prior to GC-MS analysis. Among five tested hydrophilic DESs, ChCl/polyethylene glycol (1:4 M ratio) DES was selected and used as a green extractant. For the clean-up of the tea extract, magnetic 3D-graphene aerogel (3DG-Fe 3 O 4 ) was functionalized with a choline chloride/urea NADES system used as a new sorbent in the in-syringe dispersive µSPE method. A comparative GC-MS study between 3DGA-Fe 3 O 4 and the modified 3DGA-Fe 3 O 4 /NADES obtained samples showed that functionalization with natural NADES resulted in higher extraction efficiencies (1.4-1.7 fold) [96]. As discussed before, by-products generated in agro-food industries are a valuable source of numerous bioactive compounds that can be used for the prevention and treatment of several diseases or as nutritional ingredients in functional foods, for the preparation of cosmetics. The determination of pollutants in citrus and olive waste products is a crucial point for the safety of products with human use. An analytical method for the detection of pollutants in olive and citrus by-products was studied by Socas-Rodrìguez et al., 2022. A comparative GC-MS determination of pesticides in several betaine-based NADESs used as extractants for the ultrasound-assisted extraction (UAE) from olive and citrus by-products. A group of 12 pesticides (4 organophosphate pesticides, 3 organochlorinated, 1 chlorotriazine, 1 tiadiazine, 1 strobilurin, 1 pyrazole, and 1 pyrethroid), commonly used in olive and citrus crops and during storage processes, was monitored to evaluate the safety of valorized by-products with neuroprotection potential against neurodegenerative diseases such as Alzheimer's disease. The combination of betaine and 1,2-propylene glycol (in a molar ratio 1:4) provided the highest extraction efficiencies for the largest number of screened compounds [97]. Sereshti et al., 2022 developed a green analytical procedure to analyze pesticides in water samples. A new green alternative DLLME based on two novel NaDESs, thymol/myristyl alcohol (2:1) and alanine/kojic acid/water (1:2:5) coupled to GC-µECD method was conducted to determine 16 multiclass pesticides in water samples. The results showed that no detectable target analytes were observed in seawater and drinking water tap samples. However, bromopropylate was detected in river water and groundwater; β-endosulfan was found in groundwater; and fenpropathrin was seen in groundwater [98].
In addition to organic pollutants, the presence of metals in foods is currently an environmental and health problem. Cadmium (Cd) is a common and toxic ecological pollutant, which originates from swift industrial operations, the overuse of fertilizers, composts and sewage sediments, and municipal activities [99,100]. In the work of Shamsipur et al., 2022, a comparison of the analytical performances achieved from the ultrasound-vortex-assisted liquid-phase microextraction technique and previously reported techniques for the extraction and determination of Cd (II) ions was detected. A hydrophobic natural deep eutectic solvent composed of salicylic acid and l-menthol (at a 1:4 molar proportion) was used as both the complexing agent and the extraction solvent of the metal. The performed extraction technique was favorably applied to the separation and determination of ultra-traces of cadmium in several food and water samples. Among the used determination tools, graphite furnace atomic absorption spectrometry (GFAAS) was established as a highly efficient technique for the determination of the concentration of various metal ion species [101].
The determination of mycotoxins also interferes with the food safety guarantee. Zearalenone is one of the most common mycotoxins produced by Fusarium fungi [102]. Pochivalov et al., 2022, studied extraction systems applied to the mycotoxin (zearalenone) determination in cereal samples. A molar ratio between DES components (DL-menthol and 1-hexanol) was investigated in the range from 1:2 to 3:1. The studied extraction system was successfully applied to zearalenone separation from cereal samples followed by its sensitive determination. The stability of the deep eutectic solvent in an acetonitrile-water mixture was studied using gas chromatography-flame ionization detection and the Karl Fisher method. The obtained results are useful for future developments in this area and are aimed at drawing the attention of the researchers to the stability issues with hydrophobic DESs in polar solvent-water mixtures [103]. 5-hydroxymethylfurfural (HMF) is a breakdown product and is practically absent in fresh food, but it is naturally generated in sugar-containing food during heat treatments such as drying or cooking because of the Maillard reaction. It is particularly toxic to human health, but it has been regarded as an essential building block for synthesizing chemicals and biofuels. Until now, the efficient preparation of HMF from biomass is still very challenging, and it needs to overcome several steps of catalytic reactions. The work recently published by Zuo et al., 2022, aimed to provide a simple, green, and cheap route for HMF production from raw biomass and suggest a new idea for biorefinery technology. Glucose was initially selected as a model feedstock to optimize the reaction conditions in A-NADES, as well as to uncover the catalytic mechanism of the glucose isomerization and dehydration steps, with the low amount of metal chlorides tested as the catalysts. Subsequently, starch and food waste were applied as raw feedstocks to test the comprehensive utilization efficiency of the NADES system and study its benefits in relieving environmental pollution. Furthermore, they also provide a food waste disposal procedure with considerable environmental relevance owing to the immense volumes of food waste generated globally. We demonstrated that aqueous natural deep eutectic solvent ChCl/glucose/water (2:1:1) showed remarkable performance for converting glucose, starch, and food waste into 5-hydroxymethylfurfural (HMF) combined with SnCl4 catalyst. More importantly, food wastes such as rice waste and bread waste were effectively converted into HMF with a high yield of 61.3 and 54.5%, respectively, indicating the NADES system has a good potential for food waste. High HMF yields of 64.3, 64.0, 61.3, and 54.5% were obtained from glucose, starch, rice waste, and bread waste at 130 • C in the NADES/MIBK (methyl isobutyl ketone) biphasic system, respectively [104].
The selective extraction of allergens from food is required to ensure the health care of consumers with alimentary allergies and intolerances. Gluten is the main allergen commonly diffused in everyday products based on flavors of cereals such as grains such as wheat, rye, barley, and oats. In food processing, the removal of the protein occurring in the endosperm of cereals is obtained by extraction with an ethanol-water solution. The current approach could produce oxidated forms and structure degradation of the protein causing the incomplete extraction of modified peptides from the food. Lores et al. 2017 performed an ultrasound-assisted extraction in combination with the use of fructose/citric acid NADES [105]. After the extraction step, ELISA tests were performed to monitor the production of, the solubilized proteins maintained their structures well without significant changes. An inverse relationship was found between the viscosity and solubilization ability of NADES, and a solvent with a viscosity close to that of water appeared to be most effective for gluten extraction. Additionally, the presence of citric acid in the NADES can help prevent proteins from oxidation, thus eliminating the need for additional reducing agents such as β-mercaptoethanol, normally required for ethanol-water extraction. * v/v ratio. "-" not provided.
Conclusions and Future Prospective
The selection of the optimum extraction solvent is a crucial step to achieving a good extraction efficiency of target analytes, and at the same time minimizing the interfering compounds, reducing the environmental impact. The growing awareness to decrease pollution and energy dispersion suggests the replacement of organic solvents with alternative systems characterized by lower environmental impact and human toxicity. In this view, the green solvents NADES has been increasingly used as sustainable solvents in several applications of food analysis in both the research community and the food industry. The current review collects several studies about the physicochemical properties of NADES, their preparation methods, evaluation of toxicity, and their use in combination with green extraction techniques for providing a green chemistry approach to several aspects of food analysis. In addition, this review also provides an update on a series of new applications of NADES in the extraction of bioactive compounds from foods and their by-products and in the determination of contaminants in foods. However, a deeper understanding about the safety of NADES systems on human health and their chemical stability is also another aspect that deserves to be further researched.
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2022-12-25T16:05:15.663Z
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2022-12-22T00:00:00.000
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P-wave indices and left atrial mechanics as predictors of atrial cardiopathy in embolic stroke of undetermined source
Recent research has shed light on the culpability of LA (left atrial) abnormality, in the form of atrial cardiopathy, as an independent risk factor for the development of atrial fibrillation, LA thrombus and subsequent stroke. The aim of this study was to measure LA electromechanical dissociation (EMD), LA volumes, P-wave dispersion (PWD) and P-wave terminal force in V1 (PTFV1) as markers of atrial cardiopathy in patients with ESUS (embolic stroke of undetermined source), to determine whether atrial cardiopathy is an integral part in the causal pathway of ESUS. 28 patients presenting with ischemic stroke and fulfilling the criteria for ESUS were enrolled into this cross-sectional, observational study along with a control group of 28 age- and gender-matched apparently healthy individuals. On ECG, PWD and PTFV1 were measured. On echocardiography, LA EMD and LA volumes were recorded. Increased PWD (34.14 ± 9.89 ms vs. 27.32 ± 8.95 ms; p = 0.01), atrial EMD (73.32 ± 16.31 ms vs. 63.63 ± 13.59 ms; p = 0.02) and LA volumes were observed in patients with ESUS as compared to controls. A significant correlation was also found between these parameters (p < 0.01). According to the results of our study, PWD, atrial EMD and LA volumes may be novel predictors for ESUS. Our results support the notion that atrial cardiopathy is a distinct mechanism of thrombosis in ESUS patients. Further research is required to clarify its function in the causation of stroke, ESUS in particular.
a continuum, rather exist on a spectrum of events, with the implication that the presence of one may not hinge on the absolute presence of the other.
Recently, atrial cardiopathy, a condition characterized by structural, functional, and biochemical abnormalities of atria prone to fibrillation, has emerged as a possible pathogenic mechanism in ESUS.Many electrocardiographic (ECG) and echocardiographic markers have been proposed in order to detect an altered atrial substrate at an early stage 10 .' Atrial failure' is a clinical term that is also being brought into common parlance, and refers to "any atrial dysfunction (anatomical, mechanical, electrical, and/or rheological, including blood homeostasis) causing impaired heart performance and symptoms, and worsening quality of life or life expectancy, in the absence of significant valvular or ventricular abnormalities" 11 .Further research may well show a significant contribution of this condition in the pathophysiology of cardioembolic stroke.
Our study was undertaken with the aim of observing the relationship between ESUS patients and the markers of atrial cardiopathy, in turn to establish whether atrial cardiopathy may be a direct cause for the development of stroke by virtue of the changes brought about in the left atrium, and whether the presence of AF may be a consequence of these changes rather than a cause, along with stroke.Furthermore, our study would help to establish these markers as predictors for stroke and may also contribute to the development of a management algorithm with regards to secondary prevention in appropriate patients of ischaemic stroke.
Study design
Our study was a cross-sectional, observational study undertaken in Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, for a period of 20 months, enrolling 28 cases of ESUS and 28 age-and sex-matched controls.The study protocol was approved by the Board of Studies and cleared by the 'Institutional Ethics Committee' , Aligarh Muslim University, Aligarh, Uttar Pradesh, in March 2018.Informed consent was obtained from all patients or their respective legal guardians.All research was performed in accordance with the Declaration of Helsinki.
Study population
ESUS was defined as an infarct visualized by CT (computed tomography) or MRI (magnetic resonance imaging) brain that is not lacunar*, in the absence of (a) extracranial or intracranial atherosclerosis causing ≥50% luminal stenosis in arteries supplying the area of ischemia, (b) major-risk cardioembolic source, and (c) any other identified specific cause of stroke.The assessment required for the ESUS diagnosis included brain CT or MRI, 12-lead ECG, cardiac monitoring for ≥24 hours with automated rhythm detection, precordial echocardiography, and imaging of the extra-and intra-cranial arteries (catheter, MR, or CT angiography, or cervical duplex plus transcranial doppler ultrasonography).
*Lacunar stroke was defined as a subcortical infarct ≤1.5 cm (≤2.0 cm on MRI diffusion images) in the largest dimension and in the distribution of the small, penetrating cerebral arteries 1 .
Patients with lacunar infarcts, significant carotid artery disease, persistent AF, rheumatic heart disease or other structural heart disease or those with prosthetic heart valves were excluded from the study.In addition, all patients underwent 24-hour Holter monitoring to rule out the presence of AF.
ECG
• The onset of the P-wave was defined as the junction between the isoelectric line and the beginning of P-wave deflection.The offset was defined as the junction between the end of the P-wave deflection and the isoelectric line.The longest atrial conduction time measured on any of the 12 leads was defined as P maximum (Pmax) and the shortest time was defined as P minimum (Pmin).The difference between Pmax and Pmin was calculated and defined as P-wave dispersion (PWD = Pmax − Pmin) 12 .• P-wave terminal force in lead V1 (PTFV1) was defined as the duration of the negative terminal deflection of the P-wave in lead V1 multiplied by the absolute value of its amplitude.Measurements were manually made from the admission standard 12-lead ECG recorded with the subject at rest in supine position at paper speed of 50 mm/s and calibration of 10 mm/mV.Increased PTFV1 was considered a value greater than 0.04 mm•s 13 .
Echocardiography
• Time intervals from the beginning of P-wave to beginning of A´ wave from the lateral mitral annulus in tissue doppler imaging was recorded as the intra-atrial electromechanical delay (Fig. 1).Average values of these indexes obtained from 3 consecutive cardiac cycles were used for analysis 14,15 .• LA volumes using the Modified Biplane Simpson's method in the apical four-and two-chamber views were also measured, as mentioned: -LA passive volumes consisting of: Pre-atrial contraction volume (LAV preA ): measured at the onset of the P-wave on an electrocardiogram (ECG); All volumes were indexed to body surface area (BSA) and expressed in mL/m 2 .
Statistical analysis
All statistical data was analysed by using SPSS Software Version 20 for Windows.Continuous variables were expressed as mean ± standard deviation while proportions were expressed as count (percentages).Comparison of categorical variables between the groups was done by the Chi-square test while continuous variables were compared using the student 't' test for independent groups.Non-parametric tests were used wherever appropriate.Pearson's test was used for correlation analysis.Receiver operating characteristic (ROC) curve analysis was used to determine the optimum cut-off level of P-wave dispersion, EMD and LA volumes to predict ischaemic stroke.A two-tailed 'p' value of less than 0.05 was considered significant.
Conference presentation
The abstract of a previous version of this paper was selected for display in the 2018Stroke ePosters' category with the title 'Evaluation of left atrial mechanics and p-wave dispersion as markers of left atrial cardiopathy in patients with embolic stroke of undetermined source (ESUS)' in the ESC CONGRESS 2020 -The Digital Experience 16 .
Results
Among the 28 patients with ESUS recruited to the study, the mean age was observed to be 51.57± 15.92 years, out of which 61% (n = 17) were males, with maximum number of patients falling into the age group of 41-60 years (53%).The baseline demographic and laboratory characteristics were similar between the case and control groups.There were no significant differences regarding age, gender, and body surface area between patient and control groups (p > 0.05).Variables like hypertension, diabetes and smoking were also equally distributed between the study groups (Table 1).With regards to the variables of interest in our study (Tables 2 and 3), we observed that the mean P-wave dispersion in the case group was higher than that in the control group (34.14 ± 9.89 ms vs. 27.32 ± 8.95 ms; p = 0.01).Using a cut-off level of 36 ms, P-wave dispersion predicted stroke with a sensitivity of 54% and specificity of 86% (ROC area under the curve: 0.700, 95% CI 0.561-0.838,p = 0.01).However, there was no significant difference www.nature.com/scientificreports/ in the value of P-wave terminal force in V1 in the case and control groups (0.0156 ± 0.022 vs. 0.0184 ± 0.019; p = 0.61).
The mean left atrial EMD in the case group was also found to be higher than the control group (73.32 ± 16.31 ms vs. 63.63 ± 13.59 ms; p = 0.02).At a cut-off level of 79 ms, atrial EMD predicted stroke with a sensitivity of 46% and specificity of 93% (ROC area under the curve: 0.680, 95% CI: 0.539-0.822,p = 0.02).
Among the LA volumes, the pre-atrial contraction volume (23.94 ± 7.66 vs. 17.93 ± 2.18 ml/m 2 ; p < 0.01), LA minimal volume (17.01 ± 8.55 vs. 10.09± 0.97 ml/m 2 ; p < 0.01), LA maximal volume (35.51 ± 8.21 vs. 29.76± 1.92 ml/m 2 ; p < 0.01) were significantly increased in the case group as compared to the control group, while the LA reservoir volume (18.49± 2.09 vs. 19.66 ± 1.34 ml/m 2 ; p = 0.02) was found to be markedly decreased in the case group.The cut-off levels of LA volumes with regards to prediction of stroke are mentioned in Table 3.
Discussion
We observed increased values of P-wave dispersion, LA electromechanical delay and LA volumes in patients with ESUS in our study (Fig. 2).Various studies have shown that inter-and intra-left atrial EMD are likely independent predictors for the development of AF and stroke 15,17 .P-wave dispersion is a variable that has been of much interest with regards to its presence in patients suffering from cardioembolic stroke, and multiple studies have found a definite association between them 18,19 .Likewise, one study comparing PWD and atrial electromechanical delay between healthy elderly (75.4 ± 6.9 years) and a younger control group (42.7 ± 9.6 years) found increased values of both in the elderly group, without any evidence of AF in either group, which signifies that a natural progression to AF in the elderly may involve a similar pathway as that hypothesized in ESUS patients.Aging, which in itself is a risk factor for AF, was found to be correlated with increased left atrial size and impaired diastolic relaxation, which is not dissimilar to the changes also seen in patients with atrial cardiopathy.In fact, a study revealed that patients who underwent intensive vascular risk factor management after catheter ablation of AF had a significant reduction in left atrial size and a lower rate of AF recurrence than patients whose risk factors were not managed as intensively 20 .This indicates that the management of AF alone may not be the crux point for prevention of stroke, rather it could be more beneficial to interrupt the processes involved in the pathophysiology of thrombus generation, which include risk factors like ageing, obesity, alcohol, amongst others.This may also explain the association that has been found in multiple studies between markers of atrial cardiopathy and various conditions like obesity, psoriasis vulgaris, PCOS etc. [21][22][23][24][25][26] .Similar to our study, investigators have found that left atrial enlargement is an independent risk factor for ischemic stroke, especially of the recurrent form, which is more commonly seen in patients with ESUS 27 .Recent similar studies have also established this association in patients with ESUS, suggesting echocardiogrpahic parameters to be of interest in ESUS population 28 .Increased mean LA volume indices (LAVI) are associated with the cardioembolic phenotype of ischemic stroke 29 , while one study found that increased LAVI significantly related to stroke recurrence in patients with non-sustained atrial tachycardias but without previously documented AF 30 .These findings impart weight to the discovery of the association between stroke and other atrial dysrhythmias besides AF [31][32][33] .In a study including 111 patients with ischaemic stroke, increased LA fibrosis and lower LA ejection fraction, as observed on cardiac MRI, were detected with similar incidence in patients with undetermined cause of embolism and those with underlying AF, further denoting that an underlying atrial disease could very well be the origin of the embolic event 34 .
The rationale for the temporal association between stroke and AF has been paradoxically diminished in view of recent studies demonstrating a surprising absence of AF in a majority of patients monitored before the index event of cardioembolic stroke 35,36 .Furthermore, the transient nature of AF led to prolonged rhythm monitoring in patients with stroke of unknown aetiology, however long-term follow-up found that only 30% of cryptogenic stroke patients manifested any AF even after 3 years of continuous heart rhythm monitoring via an implantable loop recorder 37 .
Our study was unable to find a significant difference in the value of PTFV1 between case and control groups.P-wave terminal force in V1 has been found to be associated with cryptogenic stroke and underlying AF in multiple studies 13,38 , however it is interesting to note that a study by Sajeev et al. consisting of 435 patients with ischaemic stroke in the absence of AF and other causes, found that morphology consistent with PTFV1 on ECG occurred commonly in both the stroke/TIA and control groups.There was no significant difference in the median PTFV1 value between the stroke 3.96 mV•ms [Interquartile range (IQR) 2.78-5.58]and control 4.23 mV•ms [IQR 2.91-5.57]groups 39 .The authors noted that the measurements of PTFV1 demonstrated excellent intraobserver reliability on assessment of the same P-wave (Intra class correlation (ICC) 0.91, p b 0.001) with narrow limits of agreement 2.21 to − 2.95 mV•ms, however, a change in the P-wave assessed led to a significant reduction in reliability (ICC 0.79, p b 0.001).It is difficult to pinpoint whether the lack of correlation between ESUS and PTFV1 in contrast to the strong association found between AF and PTFV1 is due to the fact that PTFV1 represents specifically an atrial dysrhythmia state rather than an underlying diseased state; or rather due to inherent reliability issues with the marker, keeping in view the absence of a normal reference range as well as a dearth of sufficiently comprehensive methodology to allow for reproducible measurements of PTFV1, particularly in the presence of subtle baseline and beat to beat P-wave variability.www.nature.com/scientificreports/ Our study was a single-centre study with a small sample size and thus additional extrapolation of our findings are limited until further confirmation by large-scale multi-centric studies.The lack of established methodology and reference ranges for the tested parameters can only be rectified by comprehensive investigations.Further research is required to cement these findings so as to develop normal ranges, as well as guidelines regarding testing these parameters in the appropriate subset of patients with cardioembolic stroke.
An overview of the existing evidence suggests that a case can be made for atrial cardiopathy being causally linked to the development of stroke due to the underlying cardiac structural changes, irrespective of the presence of dysrhythmia.A study found increased prevalence of atrial cardiopathy in patients with ESUS, with 26.6% of ESUS patients suffering from atrial cardiopathy (defined as severe LAE on echocardiogram or PTFV1 > 5,000 μV-ms on ECG), compared to only 12.1% of stroke patients with large artery atherosclerosis (LAA) and 16.9% of those with small vessel disease (SVD) (p = 0.001) 40 .Another similar study demonstrated increased incidence of atrial cardiopathy (defined by severe left atrial enlargement (sLAE) in patients with ESUS as compared to patients with non-cardioembolic strokes 41 .A recent study postulates that transient atrial mechanical dysfunction could be a part of the pathophysiology in patients with ESUS 42 .
The concept of atrial cardiopathy may clarify why the onset of AF occurs at or around the time of incident stroke in many cases, conveying that thromboembolism and dysrhythmia both develop in parallel as part of the progression of an underlying atrial cardiopathy 43 .These considerations would also help to explain the results of a study in which only 31% of patients with both subclinical atrial fibrillation and stroke had no AF during a median 8 months of continuous heart-rhythm monitoring before the stroke and only manifested AF after the stroke 35 .
These suppositions beget the question of whether the presence of reliable indicators of atrial cardiopathy may be feasible in the diagnosis and treatment algorithm of embolic stroke and, furthermore, if they may even help to predict the development of events like AF and cardioembolic stroke (CES).In fact, a study has already demonstrated that in acute ischemic stroke patients without a pre-existing diagnosis of AF, the odds of suffering from CES is associated with changes in structural and functional measurements demonstrated on routine stroke care TTE.The most significant association was seen with increases in LA systolic diameter 44 .
Our findings demonstrate that changes in echocardiographic parameters may be reflective of a dynamic process in the structure of the atria with the consequent development of atrial cardiopathy and are independently associated with a diagnosis of ESUS and are not simply markers of pre-existing AF.Moreover, the correlation that we observed between the various study parameters also hint that these aberrations signify an overall process of atrial disease that may be manifest more readily in a specific subset of patients in whom anticoagulation may very well be an appropriate component in the management of stroke.The parameters in our study have the added advantage of being non-invasive and relatively cost-efficient, and thus could prove to be more valuable in the diagnostic workflow if employed in the relevant scenarios.
Minimal
LA volume (LAV min ): measured at the closure of the mitral valve in end-diastole; and Maximal LA volume (LAV max ): measured just before the opening of the mitral valve in end-systole.-LA active volumes measured include: LA reservoir volume (LAV max − LAV min ) LA passive emptying volume (LAV max − LAV preA ) LA contractile volume (LAV preA − LAV min )
Figure 1 .
Figure 1.Left atrial electromechanical delay measurement in a study participant.
Table 1 .
Baseline characteristics of study population.
Table 2 .
LA parameters in the case and control groups.Significant values are in bold.
Table 3 .
Receiver operating characteristics (ROC) curve of study parameters for predicting atrial cardiopathy in ESUS patients.
|
2023-11-17T06:16:43.594Z
|
2023-11-15T00:00:00.000
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14460983
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pes2o/s2orc
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v3-fos-license
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CCl4-induced hepatotoxicity: protective effect of rutin on p53, CYP2E1 and the antioxidative status in rat
Background Rutin is a polyphenolic natural flavonoid which possesses antioxidant and anticancer activity. In the present study the hepatoprotective effect of rutin was evaluated against carbon tetrachloride (CCl4)-induced liver injuries in rats. Methods and materials 24 Sprague–Dawley male rats were equally divided into 4 groups for the assessment of hepatoprotective potential of rutin. Rats of group I (control) received only vehicles; 1 ml/kg bw of saline (0.85%) and olive oil (3 ml/kg) and had free access to food and water. Rats of group II, III and IV were treated with CCl4 (30% in olive oil, 3 ml/kg bw) via the intraperitoneal route twice a week for four weeks. The rutin at the doses of 50 and 70 mg/kg were administered intragastrically after 48 h of CCl4 treatment to group III and IV, respectively. Protective effect of rutin on serum enzyme level, lipid profile, activities of antioxidant enzymes and molecular markers were calculated in CCl4-induced hepatotoxicity in rat. Results Rutin showed significant protection with the depletion of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT) in serum as was raised by the induction of CCl4. Concentration of serum triglycerides, total cholesterol and low density lipoproteins was increased while high-density lipoprotein was decreased with rutin in a dose dependent manner. Activity level of endogenous liver antioxidant enzymes; catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSHpx), glutathione-S-transferase (GST) and glutathione reductase (GSR) and glutathione (GSH) contents were increased while lipid peroxidation (TBARS) was decreased dose dependently with rutin. Moreover, increase in DNA fragmentation and oxo8dG damages while decrease in p53 and CYP 2E1 expression induced with CCl4 was restored with the treatment of rutin. Conclusion From these results, it is suggested that rutin possesses hepatoprotective properties.
Background
Exposure to toxic chemicals, environmental pollutants and drugs can cause cellular injuries through metabolic activation of reactive oxygen species (ROS) [1]. Carbon tetrachloride (CCl 4 ) has been used extensively to study hepatotoxicity in animal models by initiating lipid peroxidation, thereby causing injuries to kidney, heart, testis and brain [2][3][4], in addition to liver pathogenesis [5]. Liver is particularly susceptible to oxidative stress due to the direct release of CCl 4 metabolites and cytokines, which propagate inflammatory response [6]. CCl 4 is one of the xenobiotics that has been reported to induce acute and chronic tissue injuries [7,8] through bioactivation of the phase I cytochrome P450 system to form reactive metabolic trichloromethyl radicals (•CCl 3 ) and peroxy trichloromethyl radicals (•OOCCl 3 ). These free radicals can covalently bind to macromolecules such as proteins, lipids and nucleic acids. The double allylic hydrogen bonds of polyunsaturated fatty acid (PUFA) are susceptible to abstraction by free radicals; CCl 4 exposure induces an increase in lipoperoxide and free peroxide radical concentrations that are highly reactive and cause injury or necrosis [9,10].
An increase in unsaturated fatty acid lipoperoxide and free peroxide radical concentrations [11,12], can induce alterations in the cholesterol profile and decrease in hepatic antioxidant enzymes [13], in addition to induction of oxidative DNA damage including formation of DNA adducts, genetic mutations, strand breakage and chromosomal alterations [14]. These free radicals can cause depletion of CYP2E1 activity [15] and increase in oxo8dG concentration in tissues of experimental animals [16]. DNA fragmentation induces p53 gene expression, blocks cells in the G phase of the cell cycle, and gives additional time for DNA repair, while severe DNA damage triggers apoptosis [17]. It has been reported that CCl 4 administration increases the silver-stained nucleolar organizer region, alters its size, morphology or spreading in the nucleus, which may be utilized as an indicator of genotoxicity, neoplasia and hyperplasia to complement other histological procedures [11].
Flavonoids are a large group of polyphenolic compounds that play an important role in detoxification of free radicals and are markedly found in fruits, vegetables and medicinal plants [18,19]. Glycosidic flavonoids such as rutin are much more readily absorbed by humans than aglycones [20,21]. Rutin possesses antitumor [22], anti-inflammatory [23] and antimutagenic potential [24], besides myocardial protection [25] and immunomodulating activities [26]. Therefore, the present study was designed to investigate the hepatoprotective effects of rutin against CCl 4 -induced oxidative stress and its role in alleviation of lipid peroxidation and restoration of p53 and CYP2E1 activity.
Animals and treatment
Six week old, 24 Sprague-Dawley male rats (200-210 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25±3°C with a 12 h dark/light cycles. They have free access to standard laboratory feed and water, according to the study protocol approved by Ethical Committee of Quaid-i-Azam University Islamabad for animal care and experimentation. To study the hepatoprotective effects of rutin, rats were equally divided into four groups (six rats). Animals of group I were treated with 1 ml/kg bw of saline (0.85%) intragastrically and olive oil (3 ml/kg bw) intraperitoneally twice a week for four weeks. Rats of group II, III and IV were treated with CCl 4 (30% in olive oil) at a dose of 3 ml/kg bw intraperitoneally twice a week for four weeks. Animals of group II received only CCl 4 treatment. However, animals of group III and IV received rutin at a dose of 50 and 70 mg/kg bw intragastrically, respectively, in addition to CCl 4 treatment, twice a week for four weeks.
After 24 h of the last treatment, all the animals were weighted, sacrificed, collected the blood while liver was removed, weighted and perfuse in ice-cold saline solution. Liver samples were treated with liquid nitrogen and stored at −70°C for further studies.
Assessment of oxidative stress
For determination of oxidative stress liver tissue was homogenized in 10 volumes of 100 mmol KH 2 PO 4 buffer containing 1 mmol EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4°C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below. Protein concentration was determined by using crystalline BSA as standard. CAT and SOD activities are determined with protocol of [27,28] while phase II metabolizing enzyme, including glutathione-S-transferase (GST), glutathione reductase (GSR), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) [29][30][31][32] and thiobarbituric acid reactive substances (TBARS) contents, respectively [33].
DNA damages
Hepatic DNA damages (fragmentation % by DPA assay), DNA ladder assay [34] and number of NORs per cell [35] were determined.
Statistical analysis
To determine the treatment effects, one-way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% and 0.01% level of probability.
Body weight, liver weight
Treatment of CCl 4 caused significant reduction (P<0.01) in body weight while increased the absolute liver and relative liver weight comparatively to control group was significantly (P<0.01) restored with 50 mg/kg bw and 70 mg/kg bw treatment of rutin (Table 1).
Lipids profile
Administration of CCl 4 increased triglycerides, total cholesterol, LDL while decreased the HDL as shown in Table 2. Concentration of HDL was significantly (P<0.01) increased by rutin whereas concentration of triglycerides, total cholesterol and LDL was appreciably (P<0.01) augmented to compensate the CCl 4 -induced toxicity.
Genotoxicity studies
Exposure of CCl 4 elicited the hepatic DNA damages (% fragmentation) and number of AgNORs/cell. Percent serum level of oxo8dG was increased whereas the % activity level of p53 and CYP 2E1 was decreased in hepatic samples of rat. Treatment of rats with 50 mg/kg bw and 70 mg/kg bw of rutin restored the level of these markers (Table 3). DNA ladder assay showed conformity to the DNA fragmentation assay (Figure 1).
Indices of hepatotoxicity
Administration of CCl 4 markedly increased (P<0.01) the activity of liver serum marker enzymes such as AST, ALT, ALP and γ-GT as compared with the control group. Elevations in the secretion of these enzymes were significantly decreased (P<0.01) by 50 mg/kg bw and 70 mg/kg bw of rutin as compared to the CCl 4 group are shown in Table 4.
Assessment of oxidative stress
CCl 4 treatment in rats significantly decreased (P<0.01) the activity of CAT, SOD, GST, GSH-Px, GSR, GSH while increased TBARS contents in liver samples. The increase of lipid peroxidation caused; reduction in the activities of antioxidant enzymes and glutathione (GSH) contents were markedly attenuated (P<0.01) by administration of 50 mg/kg bw and 70 mg/kg bw of rutin in intoxicated rats (Table 5).
Discussion
The fields of dietary modification and chemoprevention show considerable effective approaches against oxidative stress and are the focus of research these days [36]. Various studies have shown that several mutagens and carcinogens cause generation of oxygen-free radicals, which play a major role in the emergence of cancer and other health disturbances [37,38]. The present study revealed that CCl 4 -induction in rats remarkably increased the level of ALT, AST, ALP and γ-GT. CCl 4 causes acute hepatocyte injuries, altered membrane integrity and as a result enzymes in hepatocytes leak out [39]. However, after treatment with rutin, the pathological increases in ALT, AST, ALP and γ-GT were significantly restored. These results indicate that rutin has the ability to protect against CCl 4 -induced hepatocyte injury, which is in agreement with a previous study [40] that reported the protective consequence of polyphenolic compounds against CCl 4 -induced liver cirrhosis. Importantly, the increased serum concentrations of triglycerides, total cholesterol and LDL, and the decreased level of HDL, were restored to normal values with rutin co-treatment. This may be explained on the basis that rutin has a strong ability to chelate multivalent metal ions, especially zinc, calcium and iron. Indeed, its ability to chelate minerals has been reported to have some protective effects, such as decreasing iron mediated free radical formation and lowering serum cholesterol, triglycerides and lipid peroxides in experimental animals [41]. Similar findings were reported in another study [42] that investigated the hepatoprotective effects of plant bioactive compounds against CCl 4 -induced hepatic injury in rats. ROS formed during the biotransformation process of CCl 4 are more reactive and toxic than the parental compound. Biotransformation of CCl 4 occurs in the endoplasmic reticulum and the isoenzyme implicated in this process is CYP2E1 [43,44]. Our results showed that the active free radical/intermediate of CCl 4 caused a reduction in CYP2E1, which was markedly restored by rutin treatment. Our results showed conformity with previous investigations, which demonstrated that the polyphenolic natural product is responsible for its protective, effects [45,46].
Results of the present study revealed that exposure of rats to CCl 4 resulted in depletion of antioxidant activities. In consonance with our results, Szymonik-Lesiuk et al. [1] reported that CCl 4 intoxication leads to changes in antioxidant enzymes and reactive intermediates involved in the bioactivation of CCl 4 that may truss to those enzymes to prevent their inactivation. Furthermore, our results correspond with [11,12], and are in agreement with an investigation following CCl 4 intoxication [47].
Glutathione provides a first line of defense and scavenges free radical oxygen species (ROS). The decreased concentration of GSH in liver may be due to NADPH reduction or GSH utilization in the exclusion of peroxides [48]. GSH-dependent enzymes offer a second line of protection as they primarily detoxify noxious byproducts generated by ROS and help to avert dissemination of free radicals [49]. GSH-Px detoxifies peroxides by reacting with GSH and converting it into GSSG, which is reduced to GSH by GSR [50]. Our study revealed that CCl 4 treatment in rats markedly changed the activity of antioxidant enzymes, which was reverted by the coadministration of rutin. Thiobarbituric acid reactive substances (TBARS), the final metabolites of peroxidized polyunsaturated fatty acids, are considered as a late biomarker of oxidative stress [51]. In our experiments, major decrease in lipid peroxidation and consequent reduction in TBARS were obtained by treatment with rutin. The increment in lipid peroxidation, as assessed by the elevated levels of TBARS following CCl 4 administration, has been well documented [12]. Data of the present study indicated that lipid peroxidation induced by oxidative stress caused DNA damage. TBARS react with the DNA strand to form the M 1 G adduct, the mutagenic pirimedopurinone adduct of deoxyguanosine [52]. Administration of rutin markedly reduced the DNA damage, which is in close agreement with a previous study [11]. This level of DNA damage decreases the expression of p53 and blocks cells in the G phase of the cell cycle, which gives the cells additional time to repair the DNA damage. However, severe DNA damage may elicit apoptosis [53]. The data revealed that CCl 4 -induction caused marked reduction in p53. This result may be explained on the basis that CCl 4 acts as a tumor promoter through increasing the intracellular concentration of ROS necrosis/regeneration and cell proliferation and/ or may be due to mutation of p53. Our results regarding p53 are in agreement with previous studies [54,55].
Conclusion
These results demonstrate that administration of rutin may be useful in the treatment and prevention of hepatic stress. Mean ±SE (n=6 number). ** indicate significance from the control group at P<0.01 probability level. ++ indicate significance from the CCl 4 group at P<0.01 probability level.
|
2016-05-12T22:15:10.714Z
|
2012-10-08T00:00:00.000
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247169259
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pes2o/s2orc
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v3-fos-license
|
Inhibition of spleen tyrosine kinase decreases donor specific antibody levels in a rat model of sensitization
Antibody mediated rejection is a major cause of renal allograft loss. Circulating preformed donor specific antibodies (DSA) can result as a consequence of blood transfusion, pregnancy or prior transplantation. Current treatment strategies are limited due to partial or transient efficacy, adverse side-effects or patient unsuitability. Previous in vivo studies exploring autoimmune diseases have shown that spleen tyrosine kinase (SYK) signalling is involved in the development of pathogenic autoantibody. The role of SYK in allogenic antibody production is unknown, and we investigated this in a rodent model of sensitization, established by the transfusion of F344 whole blood into LEW rats. Two-week treatment of sensitized rats with selective SYK inhibitor fostamatinib strongly blocked circulating DSA production without affecting overall total immunoglobulin levels, and inhibition was sustained up to 5 weeks post-completion of the treatment regimen. Fostamatinib treatment did not affect mature B cell subset or plasma cell levels, which remained similar between non-treated controls, vehicle treated and fostamatinib treated animals. Our data indicate fostamatinib may provide an alternative therapeutic option for patients who are at risk of sensitization following blood transfusion while awaiting renal transplant.
Antibody mediated rejection is a major cause of renal allograft loss. Circulating preformed donor specific antibodies (DSA) can result as a consequence of blood transfusion, pregnancy or prior transplantation. Current treatment strategies are limited due to partial or transient efficacy, adverse side-effects or patient unsuitability. Previous in vivo studies exploring autoimmune diseases have shown that spleen tyrosine kinase (SYK) signalling is involved in the development of pathogenic autoantibody. The role of SYK in allogenic antibody production is unknown, and we investigated this in a rodent model of sensitization, established by the transfusion of F344 whole blood into LEW rats. Two-week treatment of sensitized rats with selective SYK inhibitor fostamatinib strongly blocked circulating DSA production without affecting overall total immunoglobulin levels, and inhibition was sustained up to 5 weeks post-completion of the treatment regimen. Fostamatinib treatment did not affect mature B cell subset or plasma cell levels, which remained similar between non-treated controls, vehicle treated and fostamatinib treated animals. Our data indicate fostamatinib may provide an alternative therapeutic option for patients who are at risk of sensitization following blood transfusion while awaiting renal transplant.
Antibody mediated rejection (AMR) represents a significant barrier to allograft survival. The presence of preformed alloreactive donor specific antibodies (DSA) can arise from exposure to cells originating from other individuals via pregnancy, prior transplantation and blood transfusion 1 .
There is abundant research implicating the pathogenic role of DSA directed against polymorphic human leukocyte antigen class I (HLA I) and HLA class II (HLA II) in multiple organs. Non-HLA targets also play a crucial role in allograft rejection and DSA targets include minor histocompatibility molecules such as major histocompatibility complex (MHC) class I chain-related protein A (MICA) and major histocompatibility complex (MHC) class I chain-related protein B (MICB) 2 . Other commonly reported non-HLA targets are expressed on epithelial cells and endothelial cells 3,4 .
There are two main mechanisms of allograft damage by DSA. The classical complement pathway can be activated by DSA binding to target antigen on endothelium, with generation of complement components which recruit inflammatory cells to the graft or directly damage the graft through the formation of the membrane attack complex 5 . In the process of antibody-dependent cell mediated cytotoxicity (ADCC), effector cells bearing Fcγ receptors (FcγR) can interact with the crystalline fragment (Fc) of bound DSA on endothelium and trigger lysis of target cells 6 .
Over 40% of patients awaiting kidney transplant in the United Kingdom are presensitized, with a reported median wait time of 6.1 years, which is double that of non-sensitized patients 7 . Current desensitization strategies include plasmapheresis and intravenous immunoglobulin, with or without immunosuppression using the B cell depletion-agent rituximab 8,9 . However, these strategies are limited and have partial or transient efficacy, in addition to their adverse effects and unsuitability for patients with certain comorbidities 10 . It is crucial therefore to identify new, safer and more effective therapeutic targets for desensitization of patients.
Spleen tyrosine kinase (SYK) is a cytosolic non-receptor tyrosine kinase mainly expressed in haemopoietic cells. Activation of SYK and subsequent signal transduction is initiated downstream of classical immunoreceptors www.nature.com/scientificreports/ including FcγR and the B cell receptor (BCR). In the B cell, SYK signalling plays a critical role in B cell maturation and effector functions [11][12][13][14] . Increasing numbers of studies have targeted SYK for the treatment of immune and inflammatory diseases, and it has potential efficacy in the treatment of presensitized patients [15][16][17][18] . Fostamatinib is a small molecule SYK inhibitor, and through its active metabolite R406 has previously shown efficacy in the treatment of experimental autoimmune glomerulonephritis in rats, where treatment resulted in attenuation of autoantibody production 19 .
Here, in a rat model of sensitization, we demonstrate that fostamatinib treatment is able to prevent the production of allogenic antibody, even after cessation of treatment, maintaining overall non-allogenic immunoglobulin levels, whilst having no depletory effect on the levels of plasma cell or B cell populations.
Materials and methods
Animals and transfusions. Eight-week old LEW.Crl RT1 I (LEW) and F344/DUCrl RT1 lv (F344) male rats were purchased from Charles River UK Ltd (Margate, UK) and maintained in a pathogen-free animal facility at the Central Biomedical Services unit, Hammersmith Hospital Campus, Imperial College London. LEW received 800 µL heparinised whole blood from F344 via an intravenous route. All animal studies were licensed by the Home Office Science Unit. Studies and procedures were approved by Imperial College London Research Ethics committee and carried out in accordance with the regulations of the UK Animals (Scientific Procedures) Act (1986) and ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.
Treatments. SYK specific inhibitor fostamatinib was provided by Rigel Pharmaceuticals (South San Francisco, CA, USA). Fostamatinib or vehicle (0.1% carboxymethylcellulose) was administered in sensitized animals by oral gavage twice daily for a period of 14 days at 40 mg/kg from either 24 h or 7 days post-transfusion.
Detection of total serum immunoglobulin. Serum levels of total IgG and IgM were detected with Enzyme Linked Immunosorbent Assay (ELISA) from ThermoFischerScientific. Serum was diluted by 1:10,000 (IgM) or 1:50,000 (IgG).
Statistical analyses. Data were analysed with GraphPad Prism 8.0 (GraphPad Software, Inc, La Jolla, CA) and are displayed as the mean ± SEM using a Mann-Whitney U test as appropriate. A P < 0.05 was defined as significant.
Results
Transfusion results in the production of donor specific antibody. A rat model of sensitization was established by whole blood transfusion between F344 and LEW rats. T lymphocyte (CD3 + ) flow crossmatch analysis showed that transfusion between these strains elicited an alloantibody response. An initial peak in allogenic IgM levels 7 days post-transfusion (Fig. 1A), was followed by isotype switching to IgG (Fig. 1B) where levels peaked at day 17. Additionally, all DSA IgG isotypes IgG1 ( Treatment with fostamatinib inhibits allogenic antibody production. Following establishment of a sensitized model, the role of fostamatinib in DSA production was investigated. Sensitized rats were treated with fostamatinib 24 h after blood transfusion. Flow crossmatch analysis demonstrated that SYK inhibitor treatment was able to block production of significant levels of IgM ( Fig Total circulating IgG and IgM levels are not affected by fostamatinib. As fostamatinib prevented the formation of an alloantibody response, serum was analysed for total circulating IgM (Fig. 3A) and IgG levels (Fig. 3B). These levels remained comparable between treatment groups. . 4A,B), switched cells (CD45R + CD27 + IgD − ) (Fig. 4C,E) and non-switched cells (CD45R + CD27 + IgD + ) (Fig. 4D,E), were detected at similar levels in both treatment groups compared to control rats. Given that DSA production was profoundly inhibited by fostamatinib, effects on numbers of splenic plasma cells were evaluated between treatment groups and compared to control non-sensitized LEW rats (Fig. 4F,G). Unlike DSA production, plasma cell numbers (CD138 + CD45R − ) were not affected by fostamatinib therapy and both treatment groups remained at similar levels compared to non-treated non-sensitized animals.
Fostamatinib prevented DSA levels rising 5 weeks after termination of treatment. Following potent inhibition of DSA levels with fostamatinib treatment, we wanted to evaluate if this suppression was transient or maintained. Sensitized rats were treated with fostamatinib for the 2-week regimen as described previously and, upon completion of the treatment course, rats were left untreated for 5 weeks with weekly venesection for measurement of DSA levels. T lymphocyte crossmatch analysis of serum showed that 2-week fostamatinib treatment was able to maintain suppression of formation of DSA IgM (Fig. 5A) and IgG (Fig. 5B) up to 5 weeks after completion of the treatment regimen. www.nature.com/scientificreports/ Delayed fostamatinib treatment prevented IgG DSA reaching levels found in vehicle treated rats. To assess the efficacy of fostamatinib treatment initiated at a later timepoint in sensitized rats, the fostamatinib treatment regimen was delayed until 7 days post-transfusion. Results showed that fostamatinib did not block allogenic IgM production and circulating levels were comparable between treatment groups (Fig. 6A). However allogenic IgG levels were significantly reduced from day 14 in fostamatinib treated rats (Fig. 6B). Investigating this further, IgG subsets were measured, demonstrating variable results (Fig. 6C-F). Levels of IgG2b, IgG1 and IgG2a showed significant blocking of alloantibody production from days 14, 17 and 21 respectively. IgG2c showed no difference between treatment groups at any time point.
Discussion
In this study, we demonstrate for the first time that SYK inhibition with fostamatinib significantly prevents allogenic DSA production in a rat model of sensitization. Fostamatinib treatment initiated 24 h post-transfusion for the treatment regimen of 2 weeks was effective at preventing production of both allogenic IgG and IgM, and had www.nature.com/scientificreports/ no depletory effects on total IgG and IgM levels. Fostamatinib treatment remained effective even 5 weeks posttermination of treatment, with significantly lower levels of IgG and IgM detected by the T cell crossmatch test at this time point. Fostamatinib was also able to partially block DSA production when treatment was implemented 7 days post-sensitisation, after the initiation of an allogenic antibody response. Our study shows that fostamatinib had a direct effect on inhibition of alloantibody without adversely affecting B cell survival. Splenic plasma cell numbers were comparable between both treatment groups. In addition to plasma cells, we examined the effect of SYK blockade on other mature B lymphocyte populations. Memory, switched and non-switched populations of B cells in the spleen remained similar between both treatment groups. Normal B cell activation and development is therefore not being affected by fostamatinib treatment, rather the production of allogenic antibody is. SYK has a well-defined role in signal transduction downstream of immunoreceptors and is critical in mediating BCR responses and FCR responses in mast cells 20 , dendritic cells 20 , macrophages 21 and neutrophils 22 . The SYK homologue zeta chain-associated protein kinase 70 (ZAP-70) 23 is the predominant signalling molecule downstream of the TCR in T cells and natural killer cells. Studies involving SYK knockout murine models have shown the critical role of SYK signalling for B cell development and maturation, where B cell maturation is arrested from progressing at the early pro-B-cell state in murine SYK knockout models 11,24,25 . Early mechanistic data has demonstrated that, in vitro fostamatinib was able to inhibit BCR responses in primary human B cells, where CD69 cell surface upregulation induced by BCR crosslinking with IgM was inhibited with fostamatinib treatment 26 .
The role of SYK in antibody production in plasma cells is unclear, as B cells from SYK knockout mice are unable to mature beyond the pro-B-cell stage 25 . In vivo models have provided some insight and indications that SYK inhibition may be an effective target for the treatment of antibody-mediated diseases. In a rodent model of anti-glomerular basement membrane disease, R406 treatment prevented the induction of disease, and inhibited circulating and deposited autoantibody production in established disease 19 . Interestingly in this model, CD45RA + cell numbers remained the same despite decrease of autoantibody. Non-obese diabetic mice were protected against developing diabetes with fostamatinib treatment in a prevention setting. Treatment was also able to delay disease progression in glucose intolerant mice, with a measured reduction in anti-glutamic-aciddecarboxylase anti-islet antibodies 27 .
Currently there are no reported data on the efficacy of fostamatinib in reduction of alloantibody production, and to our knowledge this is the first report of its kind. In contrast to our experiments, early immunotoxicology studies where rats were immunised with KLH-Ribi antigen and treated with fostamatinib, did not show reduced KLH specific IgM and IgG 28 . Differences between the effects of fostamatinib on production of alloantibody compared to autoantibody are intriguing and require further investigation. The mechanism of inhibition of DSA production by fostamatinib in our experiments is unclear. Due to the functional action of fostamatinib, it is likely that downstream signalling molecules have been blocked from phosphorylation, which in-turn has impacted DSA production. Fostamatinib inhibition of SYK prevents phosphorylation of downstream molecules, which include phospholipase Cγ1, Akt/protein kinase B, c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase 26 . Exactly which signals contribute to immunoglobulin production requires further investigation. It is possible in our experiments that fostamatinib treatment is blocking antigen presentation to B cells via follicular dendritic cells, a process required for the progression of the B cell response. Studies have also established a crucial role for SYK signalling in the induction of the antigen presentation machinery in both B cells 27 and dendritic cells 29 . In dendritic cells, SYK signalling has shown to be crucial for immunocomplex uptake and antigen presentation 20 .
In our model, blood transfusion initially induced IgM production, with peak titres approximately 7 days post sensitization. Traditionally in the context of AMR, preformed IgM antibodies have been perceived to be nonpathogenic. However emerging studies suggest that anti-HLA IgM may make important contributions to the pathogenesis of allograft loss [30][31][32][33] . In our experiments where treatment was implemented 24 h post sensitization, DSA IgM production was blocked, indicating this might be beneficial for human presensitization. Fostamatinib 35 . In humans four IgG subclasses exist, IgG1, IgG2, IgG3 and IgG4. It is widely accepted that IgG1 and IgG3 are the most pathogenic subclasses in AMR due to their ability to activate the complement cascade 36 . IgG 3 has the highest binding efficiency to complement component C1q, and IgG1 is highly effective at complement dependent cell lysis 37 . The presence of these subclasses as a result of sensitization is associated with poor graft outcome 33 . In early treatment experiments we have shown the efficacy of fostamatinib in blocking production of all IgG subtypes, thereby resulting in prevention of downstream effects of humoral immunity, suggesting this approach could be suitable in prevention of presensitization when given concomitantly to blood transfusions or possibly de novo DSA during transplantation 38 . In addition to DSA level reduction, utilizing fostamatinib to inhibit the effector phase and ultimately influencing the pathogenicity of the DSA underpins the rationale for using fostamatinib in AMR.
Fostamatinib treatment initiated 7 days post transfusion was able to significantly reduce the levels of DSA IgG detected in the CD3 + crossmatch assay at days 14, 17 and 21. Study of IgG subsets provided variable results. IgG2c levels were similar between groups, but IgG1 and the most pathogenic subsets IgG2a and IgG2b demonstrated significantly lower levels in treated rats from at day 14. It is probable that fostamatinib treatment was inhibitory soon after administration, as demonstrated by early time point experiments, however fostamatinib is unlikely to inhibit DSA IgG that has already been made. The circulating half-life of IgG is 7-25 days and it is likely that an extended follow-up time would continue to show more pronounced repression of DSA IgG levels. The ability of fostamatinib to block de novo DSA IgG production but not existing DSA IgG could explain the substantially higher DSA IgG levels measured in late treatment experiments compared to earlier treatment experiments.
In the context of blood transfusion as a sensitizing event, there is an abundance of literature associating allosensitization with higher rates of graft rejection and lower rates of graft survival 39 . Chronic anaemia is prevalent in patients with chronic kidney disease or end-stage kidney disease and avoiding transfusion is not always feasible 40 . A causal link between blood transfusions and DSA production has been identified by Hassan et al. 41 . In patients receiving blood transfusion after renal allograft transplantation, this study found direct evidence of a de novo HLA alloimmune response elicited against the blood donor, and enhanced risk of transplant specific antibody development within these patients. These factors were significantly associated with the increased risk of AMR and allograft failure. Current treatment options including early post-transplant erythropoietin therapy and cell salvage have limited efficacy 42 .
In a pre-transplant setting, current treatment strategies for sensitized patients have partial or transient efficacy and include the use of plasma exchange, anti-CD20 monoclonal antibody therapy with rituximab, or blocking macrophage FcγR with high-dose intravenous immunoglobulin 43 . Fostamatinib treatment presents a potential alternative strategy in preventing allosensitization in both pre-transplant and post-transplant blood transfusions. In our experiments, a relatively transient period of orally given treatment was able to inhibit DSA levels even up to 5 weeks post sensitization, which is significant in a clinical context as it is likely to improve patient compliance compared to more regular or invasive treatment. Our data are limited by the moderately short follow up time, and the long-term impact of SYK inhibition on plasma cell and B cell function is unknown. Further work will be needed to investigate whether treatment with fostamatinib may have a role in treatment of sensitized rats in models of experimental transplant rejection.
LEW and F344 rats are weakly histocompatible due to differing partially at both MHC I & II and non-MHC loci 44 . Transplantation of an F344 kidney into a LEW rat is a well-characterized model of chronic antibody mediated rejection, where rejection develops over the course of a few months 45 . It will be crucial to investigate the protective potential of fostamatinib in sensitized transplanted rats from developing chronic AMR. In a rat model 46 . This study explored the efficacy of SYK inhibitor GS-492429 in acute rejection, with a splenocyte transfusion for the sensitization method. The protective results are promising for our future studies in a chronic AMR rat model. www.nature.com/scientificreports/ Fostamatinib is currently FDA approved for the treatment of thrombocytopenia in adults with chronic ITP with insufficient response to other treatment. Appropriate dosing, PKA data and a good safety profile for fostamatinib are available 47 . The data presented in this study suggest that fostamatinib should be investigated for reduction of circulating allogenic antibodies in patients with chronic kidney disease needing blood transfusion and highly sensitized patients with end-stage renal disease awaiting transplant.
Data availability
The data supporting the findings of this study is available from the corresponding author upon reasonable request.
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2022-03-02T06:23:42.399Z
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2022-02-28T00:00:00.000
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244909172
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pes2o/s2orc
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Asymptotically matched quasi-circular inspiral and transition-to-plunge in the small mass ratio expansion
In the small mass ratio expansion and on the equatorial plane, the two-body problem for point particles in general relativity admits a quasi-circular inspiral motion followed by a transition-to-plunge motion. We first derive the equations governing the quasi-circular inspiral in the Kerr background at adiabatic, post-adiabatic and post-post-adiabatic orders in the slow-timescale expansion in terms of the self-force and we highlight the structure of the equations of motion at higher subleading orders. We derive in parallel the equations governing the transition-to-plunge motion to any subleading order, and demonstrate that they are governed by sourced linearized Painlev\'e transcendental equations of the first kind. The first ten perturbative orders do not require any further developments in self-force theory, as they are determined by the second-order self-force. We propose a scheme that matches the slow-timescale expansion of the inspiral with the transition-to-plunge motion to all perturbative orders in the overlapping region exterior to the last stable orbit where both expansions are valid. We explicitly verify the validity of the matching conditions for a large set of coefficients involved, on the one hand, in the adiabatic or post-adiabatic inspiral and, on the other hand, in the leading, subleading or higher subleading transition-to-plunge motion. This result is instrumental for deriving gravitational waveforms within the self-force formalism beyond the innermost stable circular orbit.
I. Introduction
We use the same conventions and notations as [35]. We consider a Kerr background with mass M and angular momentum J. The Kerr metric in Boyer-Lindquist coordinates is used to raise and lower spacetime indices. We use geometrical units G = c = 1. Quantities are made dimensionless using the black hole mass M , including the angular momentum a = J/M 2 and the binary mass ratio η = m/M where m is the point-particle mass. The event horizon is located at the largest root of ∆ = r 2 − 2r + a 2 . We introduce the dimensionless proper time τ and define the redshift as U = dt/dτ . We consider orbits in the equatorial plane of a Kerr black hole parametrized by z µ = (t, r, π 2 , Ωdt) where Ω = dφ/dt is the orbital frequency. We denote σ = sign(Ω), i.e. σ = +1 for prograde orbits and σ = −1 for retrograde orbits. Finally, the four-velocity is v µ = dz µ /dτ = (U, dr/dτ, 0, U Ω).
The forced geodesic equation is given by where f µ is the gravitational self-force that obeys f θ = 0 on equatorial orbits. Note that we rescaled the geodesic equation with the point particle mass m. We define the slowly evolving helicoidal vector ξ ≡ ∂ t + Ω∂ φ . The normalization of the four-velocity for massive particles implies g µν v µ v ν = −1 which gives the radial first order equation of motion or, equivalently after using Eqs. (1) and (2), dr dτ 2 = e 2 − V geo , V geo (r, e, ℓ, a) ≡ e 2 − e r 2 + a 2 − aℓ 2 − ∆ r 2 + (ae − ℓ) 2 r 4 .
The derivative along proper time of the normalization condition gives the orthogonality condition f µ v µ = 0 which can be written in the two equivalent ways: The angular momentum ℓ and energy e evolve according to the forced geodesic equation as allowing to rewrite the orthogonality condition (6) as Taking the τ -derivative of Eq. (5) we obtain Equations (10) and (11) agree with the radial component of the forced geodesic equation (3), given the identity Comparing the writings of the radial self-force in Eqs. (9) and (11) and using the identities e − 1 2 ∂V geo ∂e = U g rr and 1 2 ∂V geo ∂ℓ = Ω U g rr , we deduce At early times, the inspiralling body has no causal contact with the central body and therefore it cannot change its angular momentum. From this causality argument we set a(τ ) = a constant. In summary, the conservation of the normalization of the four-velocity along the forced geodesic motion fixes the evolution of the background a to be constant along the motion at the location of the point particle. The spin of the central black hole is still evolving due to gravitational wave emission, which is encoded in perturbations of the background metric h (1) µν , h µν , . . . [37,38] and these effects appear in the self-force term f µ of the forced geodesic equation.
It will be convenient to introduce δ as the deviation from the geodesic angular velocity for circular orbits Ω geo = σ/(r 3/2 + σa) as We collectively denote all variables of interest as X ≡ (r, δ, Ω, U, e, ℓ).
III. QUASI-CIRCULAR INSPIRAL IN THE nTH POST-ADIABATIC EXPANSION
We now restrict our analysis to the small mass ratio limit η ≪ 1 and to orbits without eccentricity. Such orbits can be described in the inspiral phase using the slow proper timẽ τ ≡ η τ , (17) and the slow-timescale expansion where the collective variable X was defined in Eq. (16). Here and below, the indices in parentheses (i) label the terms appearing at order η i in the expansion. The symbol Oτ (η) refers to the limit η → 0 at fixed slow proper timẽ τ . We name the leading X (0) terms the adiabatic order or 0PA order. The nth subleading terms are denoted as nth post-adiabatic order or nPA order. We define similarly the slow Boyer-Lindquist timet ≡ η t. Equations (6) and (8) are consistent with the expansions Note that although we are taking into account the radial self-force f r in Eq. (9) the motion is quasi-circular in the sense that the radial and angular motions evolve on different timescales due to the expansion (18): indeed dφ/dτ = Oτ (1) while dr/dτ = Oτ (η), so that d log r dφ = dr/dτ r dφ/dτ = Oτ (η).
We will algebraically solve the equations and write the remaining differential equations in the nth post-adiabatic expansion. Algebraic equations are obtained at order η n while first-order differential equations come from order η n+1 due to the slow-time dependence of the quantitites X = X(τ ). As we will see in Sections III B, III C and III D the motion is governed by one differential equation controlling the evolution of the radial coordinate r (n) . All other quantities are algebraically determined.
A. Structure of the self-force
Quasi-circular orbits are entirely parametrized by the proper time along the orbit. Since the Boyer-Linquist radius is monotonically decreasing in proper time, one can parametrize as well any quantity along the orbit in terms of the radius. In particular, we can write the self-force as where F µ n are functions of r only. Using the slow-timescale expansion (18) we can expand the self-force around the adiabatic order r = r (0) , + O(η 4 ). (22) Comparing with Eq. (19) we obtain At order n the self-force will be composed of a term linear in r (n−1) and non-linear terms N L (n−1) homogeneous of degree n − 1 in the mass ratio, f µ (n) =f µ,lin (1) (r (0) )r (n−1) + f µ,non-lin The adiabatic solution without eccentricity to Eqs. (1), (2), (4), (5), (8), (10), (12) and (15) can be found straightforwardly. Equation (9) implies an exact adiabatic quasi-circular inspiral: From Eq. (7) we obtain In order to write compact expressions, it is convenient to define the following functions of r (0) : where ∆ (0) ≡ ∆| (0) = r 2 (0) − 2r (0) + a 2 . We note that the function D of r (0) admits a single root outside the horizon, which occurs at the location of the geodesic innermost stable circular orbit (ISCO) r (0) The root of this function is of particular interest as it appears in the denominator of the equations of motion (32), (38) and (44) causing the slow-timescale expansion to break down at the location of the ISCO. The roots of the function B, which also appears in the same denominators that D does, lie behind the event horizon. The algebraic part of the resolution is standard and we shall therefore be concise. Equation (12) at order η 0 gives is computed from Eq. (4). We can then solve Eqs. (1) and (2) to obtain e (0) = e (0) (r (0) ) and ℓ (0) = ℓ (0) (r (0) ). The unique solution to Eqs. (1), (2), (4) and (12) at order η 0 can then be written as Then, from Eq. (15) at adiabatic order we obtain δ (0) = 0. Note that a Z 2 "parity" symmetry exists for these equations when the signs of both σ and a are flipped. Each variable is either even or odd under parity, e.g. σ, a, ℓ (0) , Ω (0) are odd while U (0) , e (0) are even. The slow time evolution of r (0) is then deduced from either the t or φ components of the geodesic equation at linear order in η as Since D appears in the denominator, the solution breaks down at the ISCO. In the Schwarzschild case (a = 0, σ = 1), this equation reduces to Eq. (103) of [12] after using the conversion from slow proper time to slow coordinate time, dτ = U −1 (0) dt.
C. 1-post-adiabatic inspiral
In this section we obtain the equations for the quasi-circular inspiral at first post-adiabatic (1PA) order. The redshift and the orbital frequency are computed from Eqs. (4) and (12), respectively, , In the Schwarzschild case, we recover Eq. (104) of [12]. One can use Eq. (5) to relate the 1PA corrections of the energy and the angular momentum, and, use Eq. (2) to obtain ℓ (1) , Equation (1) is then obeyed. It is straightforward to compute the first-order deviation δ (1) from the radial self-force f r (1) using Eqs. (15) and (33), From Eq. (7) we obtain Following Eq. (26), the second-order self-force can be decomposed as where f µ,lin (1) (r (0) ) and f µ,non-lin (2) (r (0) ) are retarded functionals of r (0) . This form is compatible with Eq. (209) of [12]. The t-component of the geodesic equation finally gives the slow time evolution of r (1) , The functions T 1 and T 2 are given in Appendix A. In the Schwarzschild case, this equation is compatible with Eqs. (103) and (106) of [12] after using the conversion from slow proper time to slow coordinate time,
D. 2-post-adiabatic inspiral
In this section we derive the equations for the quasi-circular inspiral at second post-adiabatic (2PA) order keeping in mind the 0PA and 1PA results.
The redshift and the orbital frequency are computed from Eqs. (4) and (12), respectively, All auxiliary functions T i , i = 3, 4, . . . , 20 necessary to write the inspiral equations are given in Appendix A. One can use Eqs. (5) and (2) to write the 2PA corrections to the energy and the angular momentum, respectively, Equation (1) is then obeyed. We compute the second-order deviation δ (2) using the definition (15) and Eqs. (33) and (40), Equation (7) can be used to deduce f φ (3) in terms of f t (3) and zeroth and first-order quantities. The t-component of geodesic equation finally gives the slow time evolution of r (2) , where we have used the writing (26) of the third-order self-force.
E. n-post-adiabatic inspiral
We follow the same procedure as the one described in the previous sections. Equation (4) is first solved for U (n) while Ω (n) is computed from Eq. (12). One then solves Eqs. (5) and (2) to obtain the nPA corrections to the energy and angular momentum. Using the angular velocity and the definition (15) one can compute δ (n) . Equation (7) can be used to deduce f φ (n+1) in terms of f t (n+1) and lower order quantities. The self-force at order (n + 1) can be written using the decomposition (26). The slow time evolution of r (n) is then be obtained from the t-component of the geodesic equation at order η n+1 . It has the following structure where S r (n) is the source term.
F. Inspiral towards the last stable orbit
We will now consider the inspiral motion close to the last stable orbit (LSO) first in the adiabatic approximation (0PA), then including subleading corrections (1PA), and we will comment upon arbitrary high subleading orders (nPA). In this limit the quasi-circular inspiral is expected to smoothly match with the transition-to-plunge motion. We will denote the slow proper time at the LSO asτ * , which is either prograde or retrograde depending upon the sign of σ. The last stable orbit is defined from the radius r where after using ℓ = ℓ (0) and e = e (0) as given in Eqs. (31c) and (31d). At leading order in the small mass ratio expansion, the LSO therefore coincides with the ISCO since D| * = 0. Hence, we can either use the terminology of LSO or ISCO at this order though both concepts differ once subleading corrections in the mass ratio are considered. In the absence of radial self-force, as assumed for simplicity in the original Ori-Thorne analysis [19], Eq. (9) implies the exact quasi-circularity condition, In reality, the normalization of the four-velocity gives the orthogonality condition f µ v µ = 0, precisely Eq. (9), which leads to a deviation from quasi-circularity, as originally observed by Kesden [21], see also [25]. The motion still remains quasi-circular in the sense of Eqs. (20) and (107) below. We will refer to a deviation from Eq. (48) as a deviation from exact quasi-circularity. In order to account for deviation from exact quasi-circularity close to the LSO, we define the quantity which naturally arises in the transition motion and will be therefore helpful in the asymptotic matching. It admits the same slow-timescale expansion as (18), where δ i,j is the Kronecker delta.
Adiabatic order
As demonstrated in [19,21], the slow-timescale expansion breaks down at the ISCO in the absence of radial self-force corrections. Now, radial self-force corrections only occur starting from 1PA order, see Eq. (35). At 0PA order, the radial evolution (32) breaks down at the radial geodesic ISCO value r (0) = r (0) * since D * = 0 and other quantities remain finite and non-vanishing, which implies that dr (0) /dτ blows up.
1-post-adiabatic order
The variables at 1PA order are (r (1) , Ω (1) , e (1) , ℓ (1) , δ (1) , W (1) ) and the self-force components appearing at 1PA order are f t,φ (2) and f r (1) . We will now deduce the behavior of these fields close to the LSO. Given the expansion (52) and the solution (55), the first-order radial self-force f r (1) can be expanded as where the coefficients f r * (1),i can be written in terms of the Taylor coefficients of Eq. (52), that is, The second-order self-forces, on the other hand, depend non-linearly on r (0) and at most linearly on r (1) . They can be written as in Eq. (37). Given that they only depend on r (0) , the functions f µ,lin (1) and f µ,non-lin (2) admit the expansion Using these expansions together with the adiabatic results the differential equation for r (1) (38) takes the schematic form where S r * (1),j are the LSO-coefficients of the source S r (1) (τ ) and h * (1),i are coefficients at the LSO. In particular, The homogeneous solution is then The first coefficient q * (1),−1 is currently left arbitrary. It will be fixed from Eqs. (78) and (90) below. The subleading coefficients are easily found in terms of the coefficients h * (1),i by plugging the solution (72) back into the homogeneous equation (71). We find for i ≥ 0, We compute the particular solution using the method of variation of constants. We seek a solution of the form Substituting it into Eq. (71) we obtain that k (1) (τ ) is given by Here q * (−) (1),i are the coefficients of the reciprocal of the homogeneous solution where q * (−) (1),1 = 1/q * (1),−1 and Finally, we obtain We can finally write the asymptotic solution for r (1) asτ →τ * as The energy e (1) , angular momentum ℓ (1) , angular velocity Ω (1) and deviations δ (1) and W (1) are obtained from the solutions to the constraints at 1PA order. Given Eq. (78), they can be expanded as where the lowest order coefficients are The functions F * , G * and H * are given in Appendix A. The above expansion is compatible with imposing the condition ℓ * (1),0 = 0, which implies from Eqs. (84) and (85) that e * (1),0 = 0 and We will show that this further restriction is consistent with the transition motion in the matching performed in Section V C.
Using the above expressions for ℓ (1) and e (1) in the flux-balance equations (8), one can rewrite the second-order self-forces as We now obtain expressions which will become useful when asymptotically matching the transition motion. We can compute the second-order azimuthal self-force in two equivalent ways: either using the flux-balance equation (8) and the expansion of ℓ (1) (79), or using the solutions (55) and (78) in the Taylor-expansion as (24). We get where the lowest order coefficient is given equivalently by We will use these two expressions to compute r * (1),0 in Section V C.
n-post-adiabatic order
We further investigate the behaviour of the nth post-adiabatic (nPA) motion for n = 2, 3, . . . close to the LSO. At each nPA order the homogeneous solution to Eq. (46) is given by the same solution as the 1PA expansion (72). We then consider a specific configuration (σa, r (0) * ). We compute the source term S r (n) and its (τ * −τ )-behaviour close to the LSO, which is determined from lower order quantities that at this point are known. We assume that the order of the divergences at the LSO of the source terms originating from the non-linear terms f t,non-lin (n+1) (r (0) , . . . , r (n−1) ) are lower or equal to the order of the divergences originating from all the other known source terms. Given the behaviour of the source one can straightforwardly compute the one of the particular solution using the method of variation of constants. We find that Eq. (46) is consistent with the following solution asτ →τ * where for n even for all n = 0, 1, 2, . . . In the same manner as in the previous sections, we also computed the quantities δ (n) and W (n) that encode the deviation from a circular orbit, and which will be useful to match the transition motion. We find the behaviors where for n even and n = 0 for all n = 0, 1, 2, . . . . We recall that for n = 0 we found δ (0) = 0, see below Eq. (31). We did extensive checks up to n = 5. We conjecture that these expansions remain valid for larger n.
IV. THE TRANSITION-TO-PLUNGE MOTION IN THE nTH POST-LEADING TRANSITION EXPANSION
We consider an expansion in the rescaled transition proper time around the LSO crossing time τ * or s * = 0. Even in the presence of self-force, the radial potential is given by V geo as described in Eq. (5) and at leading order in η the LSO matches with the ISCO. The transition motion will be defined from s = −∞ (where it will asymptotically match the inspiral) up to the time s break > 0 where the transitiontimescale expansion breaks down, see Eq. (118) below. The validity of the transition equations with respect to the full equations of motion will be a smaller interval of proper time which will be assessed in Eq. (121) below.
We denote with r [0] * , ℓ [0] * and e [0] * the radius, angular momentum and energy evaluated at the ISCO. We define the variables R, ξ and Y as [19,21] Integrating the angular momentum flux-balance law (8) and comparing with Eq. (100b) we obtain In the absence of radial self-force all variables R, ξ, and Y scale as η 0 in the transition region for standard spins [19,21,25,26]. In the presence of radial self-force corrections we expand The indices in square brackets [i] label the terms appearing at relative order η i/5 with respect to the first nonvanishing leading terms in the transition-timescale expansion around r [0] * , ℓ [0] * and e [0] * . We consider the self-force along the particle worldline in the transition region. Using the transition-timescale expansion (100) and (102) in Eq. (21) we obtain The same expansion can be made for f µ or any tensorial quantity pulled back on the particle worldline. The terms ∼ η 2 start to depend upon the second-order self-force F µ 2 ; the terms ∼ η 3 start to depend upon F µ 3 , . . . We will denote the series expansion of the following self-force components as Comparing such an expansion of f φ with the one in Eq. (103) and using Eq. (101) gives where It is clear that κ * > 0 since angular momentum loss drives the transition motion. Such expansions are compatible with the field equations (8), (9), (114). During the transition motion we have the property indicating a loss in quasi-circularity with respect to the inspiral, see Eq. (20), as expected when moving towards the final plunging motion.
Introducing the rescaled variables we obtain the normalized leading-order transition equations [19][20][21] The solution x [0] is the Painlevé transcendent of the first kind and y [0] is twice its first integral [25]. As we will see below in Section IV C, in order to match the inspiral, the relevant solutions to Eqs. (116) as t → −∞ are given by The Painlevé transcendent of the first kind is then uniquely defined from t = −∞ to a finite t that we denote as t 115) and (117) to compute ξ [0] , R [0] and Y [0] (see Eqs. (122) for the results), we deduce that the transition regime breaks down when τ We can compute the asymptotic solution to Eq. (116b) as τ → τ (+) break . At leading order, and using the relation (115a) to obtain of R [0] , we get The validity condition η 2/5 R [0] ≪ 1 of the transition-timescale expansion then translates into We summarize the range of validity of the transition motion as
Early-times 0PL transition solution
We compute the asymptotic solution for the dynamical quantities in the limit of early times (s → −∞) where the transition will be shown to connect smoothly with the inspiral.
For the angular velocity from Eqs. (110) and (122a) we obtain This behavior will be matched to the inspiral motion in Section V A.
B. Structure of the general subleading transition motion
Building upon what we have done for the 0PL transition motion, we present in the following the algorithm that we use to solve the transition motion at arbitrary high subleading order. We apply the following steps: • As a consequence of Eqs. (100), U , Ω as well as δ also admit an expansion in powers of η 1/5 . The expansion of δ takes the form since the leading order is η 4/5 . Using the definitions (2) The first six such terms can be found in Appendix B.
• We consider the self-force in the transition region. In the same manner as in Eq. (103), using the transitiontimescale expansion (100) and (102) in Eq. (21) we obtain These first coefficients of the expansions of the radial and azimuthal self-force are given in Eq. (103).
• The point which requires the most attention is the solution to the equations of motion at order i (134). We extend the definition (115) for i = 0 to any i = 0, 1, 2, . . . as as well as Recalling the definition of t (115c), the equations of motion at order i (134) then take the normalized form We reduced the equations to sourced linearized Painlevé transcendental equations of the first kind. We will call these equations the linearized PT1 equations for short. In Section IV C we will first describe the two independent homogeneous solutions to Eqs. (138b). The inhomogeneous solution to (138b) will be then obtained from the method of variations of constants in Section IV D.
• The motion in the transition region can be entirely parametrized by x [i] , i ≥ 0 (or, equivalently, R [i] ) which is obtained from the equation of motion (138b). All other quantities are algebraically determined. Note that y [i] obeys the differential equation (138c), but can be also solved for algebraically using Eq. (138a). Finally, once x [i] (t) and y [i] (t) are known, it will be straightforward to compute the solutions for all the quantities (r, ℓ, e, δ, W , . . . ) that will be matched to the slow-time expansion of the inspiral.
C. Homogeneous solution to the linearized PT1 equations
In this section we describe the properties of the early-time asymptotic homogeneous solutions to Eq. (138b). The non-linear Painlevé transcendental (PT1) equation (116b) which governs the transition at leading order admits a known explicit asymptotic solution as t → −∞ which depends upon two parameters d and θ 0 . Using Mathematica, we found that the asymptotic solution takes the form or, equivalently, expanding as cases n = 2i and n = 2i + 1, where we defined Since the quasi-circular inspiral and transition have a monotonically descreasing radius, we are interested at leading order in the non-oscillating solution, which requires d = 0. We expect that oscillating solutions with d = 0 will be relevant for the inspiral and transition motion with eccentricity. For d = 0, the solution becomes which originates from the coefficients s 4i,0 , i ≥ 1 that contain d-independent terms, which are non-vanishing when d = 0. The first such coefficients are s 4,0 = The asymptotic homogeneous solution to Eqs. (138b) will be computed from ∂x [0] /∂d and ∂x [0] /∂θ 0 , x 2 lin = −6 −5/8 1 d , c lin 5 = 5174126188 and the others are easily found. The Wronskian is given by Since the approach to the LSO of the quasi-circular inspiral is consistent with the absence of oscillations as developed in Section III, we will set the homogeneous solution to Eqs. (138b) to 0 for all i = 1, 2, . . . . These fixing of initial data are matching conditions between the late-time inspiral and the transition motion.
D. Inhomogeneous solution to the linearized PT1 equations
We will now give the general method to solve the inhomogeneous linearized PT1 equations (138) in the limit t → −∞ given the structure of the sources in the transition expansion.
In order to find a particular solution to Eq. (138b) at post-leading order i we use the method of variation of constants: given the two homogeneous solutions x 1,2 lin (144), the particular solution and the coefficients p 1 [i] (t) and p 2 [i] (t) solve After an explicit evaluation, the inhomogeneous PT1 solution for sources (−t) −k/2 can be written as where the coefficients x ℓ Painlevé (k), ℓ ∈ N, can be computed from the asymptotic behavior of the Painlevé transcendent. The first coefficients are given in Table I.
Using Eqs. (148) and (152), the general solution is therefore where we defined the coefficients (17), we can now compute the asymptotic early-time solution for r. It is useful to distinguish the even and odd i indices. For i even we set i = 2n while for i odd we set i = 2n + 1. We can split the sum over i into two sums over n which both start from n = 0. We obtain In the last step we defined, for convenience, the rescaled We now turn to the solution for y [i] . According to the structure of the source terms for i = 1, 2, 3, 4, 5, 6 in Eqs. (C3), the asymptotic t → −∞ behavior of s y We conjecture this holds for i ≥ 7. As x [i] is known, the equation (138a) is an algebraic equation for y [i] . Given its complexity, we will more simply integrate Eq. (138c) using Eqs. (153) and (157). We get whereȳ [i] is an integration constant that is uniquely fixed from the constraint (138a). Since all terms in Eq. (138a) admit the fall-off behavior (158), we deduce by consistency thatȳ [i] = 0 for all i except when i = 4 + 5N where we cannot conclude from this argument, and this coefficient might be non-zero. Consistently with the zeroth-order equation (116c) we define s y,k [0] = 0 for all k ∈ N. The integrated form (158) is then also valid for i = 0. Recalling equation (100c), we are now ready to compute W (defined in Eq. (49)) as W = η 6/5 Y . Using Eqs. (102) and (136b) we obtain We are interested in the asymptotic early-time behaviour of this quantity. We consider Eq. (158) and use Eqs. (153) and (157) together with the substitution of time variables (115c), (99) and (17). Again, it is useful to distinguish the even and odd i indices. For i even we set i = 2n while for i odd we set i = 2n + 1. and we split the sum over i into two sums over n which both start from n = 0. We obtain where we have defined We obtain at leading order with u 0 [0] = − 4β * κ * 3γ * ( κ * β * α * ) 1/2 , which matches Eq. (128).
V. INSPIRAL-TRANSITION MATCHING
The adiabatic inspiral has as range of validity τ < τ * where the bound arises because the expansion becomes singular at the LSO. The LSO is approached when τ * − τ ≪ η −1 where η −1 is the radiation reaction timescale. The transition solution is valid in the range (121) and approaches the inspiral at early proper times with respect to the transition timescale τ * − τ ≫ η −1/5 . The overlapping region between the inspiral and the transition solution is with τ < τ * which is indeed a subset of the region (121). We will now match the transition solution as s → −∞ with the inspiral solution as τ → τ * in the overlapping region using the method of matched asymptotic expansions. At each order i ≥ 0, the ith post-leading transition equations of motion (116) and (138) require two initial data at t → −∞ which are determined by the late-time behavior of the quasi-circular inspiral. Since this late-time behavior is consistently described as a series in polynomial powers of the proper time difference with respect to the LSO, we already fixed in Section IV C that the two homogeneous oscillating solutions at each order i > 0 are identically zero. We shall now verify that the two asymptotic solutions as defined separately in Sections III and IV are consistent with each other.
A. Leading-order match: 0PA-0PL Making use of the relation (54), we list equivalent formulae for the coefficients of the inspiral and transition motions In order to perform the match, we first recognize Let us recall and match the relevant leading-order solutions for the adiabatic inspiral and the leading-order transition motion. For the radial coordinate r we have from the inspiral (Eqs. (16) and (55)) and the transition (Eq. (124)), respectively, where the coefficient r * (0),1 is given in Eq. (64). The angular momentum in the inspiral (Eqs. (16) and (57a)) and the transition (Eq. (125)), respectively, is given by where the coefficients ℓ * (0),2 , ℓ * (0),3 , κ * and λ * are defined in Eqs. (66), (67), (106) and (123). Using the relations (164) we are able to match We now show for completeness that also the energy e, the angular velocity Ω and the functions δ and W which can be constructed from the quantities above, are exactly matched in the overlapping region.
The solutions for the energy in the matching region are the following: for the inspiral and the transition we have, respectively, where e * (0),3 is given in Eq. (60). The match is exact once we use the relations (170). The angular velocity in the inspiral (Eqs. (57c) and (61)) is matched by the angular velocity in the transition region (129) after using the relations (170) and (164). The deviation from exact quasi-circularity W in the inspiral (63) matches with the result from the transition motion (128) after using the relations (170) and (164). The first nonzero deviation δ from geodesic circular angular velocity only appears at subleading orders in the mass ratio and will be dealt with it in Section V D.
Since quasi-circular orbits can be parametrized entirely by the Boyer-Lindquist radius r we first consider the match of this quantity. Using the slow-timescale expansion of the radius during the inspiral (18) and the asymptotic solution in the approach towards the LSO (94) we get Instead, the transition motion gives the expansion around the LSO (155) from the expansion of the Painlevé transcendental equation, In order to match the η powers of these expressions, we recognize the i index of Eq. (177) as either i = 2k for the first term and i = 2k + 1 for the second term of the k summation in Eq. (178). Then, if we match j of Eq. (177) with n of Eq. (178), the powers ofτ * −τ match given which is precisely the result (95). The matching of the coefficients is given by The structure of the match is visualized in Table II.
As a further check, we now consider the deviation from exact quasi-circularity W . Combining the slow-timescale expansion (50) and the asymptotic solution of W (i) in the approach towards the LSO (97) we obtain On the other hand, the transition motion gives the expansion around the LSO (160), In order to match these expressions, we recognize the i index of Eq. (181) as either i = 2k for the first term and i = 2k + 1 for the second term of the k summation in Eq. (182). Then, if we match j of Eq. (181) with n of Eq. (182), the powers ofτ * −τ match given which matches with the result (98). The matching conditions for the coefficients read The matching conditions (180) and (184) provide the precise match in the overlapping region (163) of the inspiral motion with transition-to-plunge motion.
D. Explicit match of the deviation from the geodesic angular velocity for circular orbits
We consider the deviation δ (15) from a geodesic angular velocity for circular orbits. For the inspiral motion, the leading-order term in both the small mass ratio expansion and the LSO expansion is (from Eqs. (18), (82) and (87)) In the transition region, this is matched by the leading term of δ [1] = 2π * This leading-order coefficient matches the one in Eq. (127) after using Eqs. (164) and (170) and recognizing This provides a non-trivial check of the matching. The deviation δ appears at leading order in the transition-timescale expansion through δ [0] . Its early time behaviour (127) matches terms in the 2PA and higher post-adiabatic inspiral, skipping the 0PA order. This is consistent with the adiabatic result δ (0) = 0.
VI. CONSTRUCTION OF THE TRANSITION-TO-PLUNGE MOTION FROM 0PA AND 1PA DATA
The aim of this section is to outline a practical scheme that provides the transition-to-plunge solution which asymptotically matches onto the quasi-circular inspiral that is only known up to adiabatic and first post-adiabatic order in the slow-timescale expansion.
The leading order i = 0 transition solution x [0] (t), y [0] (t) is the Ori-Thorne-Kesden solution, which is uniquely fixed by the requirement to have a monotonically decreasing radius. For i ≥ 1, the functions x [i] (t) and y [i] (t) can be further deduced at all transition times for low i orders from the knowledge of the sources s x [i] and s y [i] . These are given by Eqs. (C2) and (C3) using (136) and (137). The sources up to order i = 10 depend at most on f µ [9] . (This is easily seen from a scaling argument as s x [i] scales as η (4+i)/5 while f µ [i] scales as η (5+i)/5 ). Given that the self-forces f µ [i] up to i = 9 only depend upon F µ 1 and F µ 2 , see Eq. (103), they can be computed from the first and second-order self-force data. More precisely, one needs to obtain for µ = φ, r (either covariant or contravariant indices) the 8 pure numbers at the ISCO: Given that these quantities are obtained in a 0PA-1PA inspiral scheme (for the second order self-force at least the orbit average self-force), one should be able to solve the transition equations exactly up to i = 10, though the details remain to be worked out. One could then use these expressions in Eqs. (102) and (136) to deduce the transition motion R(η, s), Y (η, s). Moreover, the asymptotic matching provides further information on the transition-to-plunge motion if our assumption is correct that the non-linear sources in the transition motion are of the same order of magnitude or scale lower than the known sources, see Section III F 3. Let us start to assume that the solutions for r (0) (τ ), r (1) (τ ), W (0) (τ ) and W (1) (τ ) as defined in Eqs. (18) and (50) are known in the LSO limit, that is, we have computed the coefficients r * Finally, a smooth solution interpolating between the inspiral phase and the transition-to-plunge motion can be obtained from the asymptotically matched expansion scheme. The standard composite solution is obtained from the uniform method that consists in adding the inspiral and transition-to-plunge solution and subtracting the common matching values. Such an implementation remains to be worked out. The adiabatic, 1-post-adiabatic and 2-postadiabatic composition solutions for the radius read as, respectively, The quantities in the last bracket on the right-hand side of the above equations avoid double counting of terms. They also allow to cancel the divergences of the inspiral solution at the ISCO or, equivalently, the early-time divergences of the transition motion. The 0PA/2PL composite expansion (201) leads to an error in the radius of the order of Oτ (η) + O s (η). This composite expansion already improves the leading 0PA/0PL matching [19,21,35,36] which introduces errors of order Oτ (η) + O s (η 3/5 ). The 1PA/7PL composite expansion (202) leads to an error in the radius of the order of Oτ (η 2 )+O s (η 2 ). The 2PA/12PL composite expansion (203) would improve this error to Oτ (η 3 )+O s (η 3 ), but it would require third-order self-force data beyond the available second-order self-data (200). The numerical implementation of a waveform generation algorithm, which would allow for a precise accounting of the error made by such a scheme, is left for future work.
VII. CONCLUSION
We have considered the quasi-circular inspiral of a point particle around a rotating black hole using a slow-timescale expansion of the dynamical variables. We have worked out the expansion to post-post-adiabatic order including all self-force effects, and sketched the structure of the equations to arbitrary high order in the mass ratio. We have computed the asymptotic solution to the equations of motion in the limit of reaching the last stable orbit, where the quasi-circular inspiral is matched with the transition-to-plunge motion.
In parallel, we have considered the transition-to-plunge motion across the last stable orbit, expanding the equations of motion using a transition-timescale expansion and including self-force corrections. We proved that the solution to arbitrary high order in the mass ratio is controlled by the sourced linearized Painlevé transcendental equation of the first kind. This consolidates and extends previous analyses for equal [20,[27][28][29][30][31][32][33][34] and small mass ratios [19,21,25,26,35,36]. We have computed the asymptotic early-time behaviour of the solution to the equations of motion from the asymptotic behaviour of the Painlevé transcendent.
We have shown the existence of an overlapping region where both the slow-timescale and the transition-timescale expansions are valid. Our main result is the explicit matching between these two expansions in the overlapping region using the method of matched asymptotic expansions. We explicitly performed the leading-order matching of the adiabatic and first post-adiabatic inspiral with the transition motion, and provided a general matching scheme to all perturbative orders in the mass ratio and in the asymptotic solutions. This self-consistent inspiral-transition motion potentially provides a new tool to further calibrate using self-force theory EOB waveforms [39][40][41][42] or other waveforms [43][44][45]. (These waveforms are currently calibrated in part using extreme mass-ratio waveforms calculated using methods from [23,46].) The deviation from quasi-circularity appears in the inspiral starting from post-adiabatic order. In the transition, the deviation from the geodesic angular velocity for circular orbits needs to be taken into account starting at leading order in the expansion. However, we showed that this leading-order contribution in the transition matches to a departure from quasi-circularity during the inspiral at post-post-adiabatic order, not at adiabatic order [47].
Our treatment of the transition motion to arbitrary high perturbative orders in the mass ratio and the general matching scheme we propose provides a tool to numerically extend the quasi-circular inspiral beyond the last stable orbit. Our analysis was restricted so far to orbits without eccentricity and inclination and would need to be generalized to cover such orbits. The inclusion of finite size effects would require one further extension. (A20) We define the following parity-even expressions defined at the LSO in terms of r (0) * and σa: The functions δ [i] , i = 1, 2, . . . , 6 read as follows:
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2021-12-07T02:16:08.154Z
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2021-12-03T00:00:00.000
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pes2o/s2orc
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Dandelion Polysaccharides Ameliorate High-Fat-Diet-Induced Atherosclerosis in Mice through Antioxidant and Anti-Inflammatory Capabilities
Dandelion (Taraxacum officinale), a member of the Asteraceae (Compositae) family, is well known as the traditional medical plant. Dandelion polysaccharides, a natural active ingredient extracted from the dandelion, possess immune regulation, anti-inflammatory, antioxidant, and anti-aggregation properties. These properties suggest that dandelion polysaccharides might alleviate atherosclerosis. Using an ApoE−/− atherosclerotic mice model fed a high-fat diet, we investigated the impact and potential mechanism of dandelion polysaccharides on atherosclerosis. We observed that dandelion polysaccharides significantly reduced the levels of triglyceride, total cholesterol, and low-density lipoprotein-cholesterol in serum, while elevated the high-density lipoprotein-cholesterol level. Concomitantly, dandelion polysaccharides reduced the area of atherosclerotic lesions and necrotic core of the aortic sinus, and increased the collagen content. Mechanistic studies showed that dandelion polysaccharides were effective in reducing serum malondialdehyde levels while elevating the enzymatic activities of superoxide dismutase and glutathione peroxidase. Furthermore, dandelion polysaccharides reduced the expression of chemotactic factor Mcp-1 and pro-inflammatory cytokines (Tnf-α, Il-1β, and Il-6) in atherosclerotic lesions. Overall, these results indicated that dandelion polysaccharides may take an important part in the attenuation of atherosclerosis via its antioxidant and anti-inflammatory properties.
Introduction
Atherosclerosis (AS), characterized by atheroma or fibrous plaque formation through the accumulation of lipids and/or fibrous materials, is the leading cause of cardiovascular diseases (CVDs) [1,2].According to the Heart Disease and Stroke Statistics 2020, deaths caused by CVD will reach 22.2 million by 2030 [3].Excessive reactive oxygen species (ROS) and the inflammatory response have been widely observed in atherosclerosis [4,5].However, the etiology and pathogenesis of atherosclerosis have not been completely elucidated.Blood lipid profiles, including triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) have been considered as the causal risk factors for atherosclerotic CVDs [4,6].Several drugs, including statins, ezetimibe, and inhibitors of the proprotein convertase subtilisin/kexin type (PCSK9), have been used in clinical practice to treat atherosclerosis [7].However, long-term pharmacological therapy induced several safety concerns, such as liver damage, gallstones, myopathy, and acute renal failure [8,9].Thus, more effectiveness and safer strategy for the treatment of atherosclerosis is needed.
Functional ingredients from natural herbs have shown their potentials in the treatment of metabolic diseases [10][11][12].Dandelion (Taraxacum officinale), a nontoxic herb, has been considered as a valuable medical plant for its choleretic, diuretic, anti-rheumatic, and hepatoprotective qualities [13,14].It contains various active ingredients, which included polysaccharides, flavonoids, peptides, terpenes, and a phenolic substance [15].For example, dandelion leaf extract decreased serum lipids and insulin resistance in high-fat diet (HFD)-induced hepatic steatosis [16].Dandelion root and leaf are also shown to display hypolipidemic and antioxidative effects to suppress the atherosclerosis on cholesterol-fed rabbits [17].However, no specific ingredient was identified to exert the lipid-lowering effect so far.Dandelion flower water syrup decreased the vasoconstrictor prostanoids in the thoracic arteries of obese rats [13].Studies have shown that dandelion polysaccharides perform well in immune regulatory, anti-inflammatory, and antioxidant properties.Dandelion polysaccharides showed better antioxidant effects [18].The antimicrobial and antioxidant activities of dandelion polysaccharides prevented the metamorphism of fresh white shrimps [19].Dandelion polysaccharides were shown to attenuate CCl 4 -induced liver injury through modulating inflammatory responses and ameliorating oxidative stress in rats [20].In addition, immunomodulatory activities of dandelion extracts were capable to elevate multiple anti-inflammatory cytokines [21].Furthermore, the root extract dandelion decreased lipid peroxidation in HepG2 cells [20,22,23].
The role of dandelion polysaccharides in the treatment of atherosclerosis remains unclear [24].Because ROS and inflammation are two main factors triggering atherosclerosis, we hypothesized that dandelion polysaccharides may exert potential anti-atherosclerotic effects through antioxidant and anti-inflammatory properties.By measuring the pathological features of aorta roots, blood lipid profiles, antioxidant factors in serum samples, and inflammatory factors in aortas, we investigated the anti-atherosclerotic effect of dandelion polysaccharides in HFD-induced ApoE −/− mice.
Animals
Six-week-old male ApoE −/− mice were bought from Beijing Vital River Laboratory Animal Technology Limited Liability Company (Beijing, China).The mice were maintained under specific-pathogen-free conditions (temperature: 20-26 • C; humidity: 40-70%; pressure: 45 Pa; animal illumination: 15-20 Lux; light: 12 h/12 h light/dark cycle).To establish the animal model for atherosclerosis disease, all ApoE −/− mice were fed with HFD diet, with 41% fat content and 4.7 kcal g −1 energy (H10141, Beijing HFK Bio-Technology Limited Liability Company, Beijing, China) for 10 weeks.Then, the ApoE −/− mice were randomly assigned to a control group (saline) and experimental group (dandelion polysaccharides, 200 mg kg −1 d −1 ).Dandelion polysaccharides (≥90%, P1201031) were purchased from Beijing Kangruina Biotechnology Limited Liability Company (Beijing, China).The product was exacted from dandelion plant stems and roots by water extraction and alcohol precipitations.The purification procedures of dandelion polysaccharides were fractional precipitation, column chromatography, anion exchange chromatography, and gel chromatography.Dandelion polysaccharides powders were fully dissolved in the distilled water, and mixed without precipitation.According to the weight of each mouse, about a 200 µL volume of saline or dandelion polysaccharide solution was given in a daily oral gavage for 8 weeks.During the intervention period, weekly records were kept of both the body weight and the amount of feed consumed.All mice were sacrificed after anesthesia using 1.25% avertin intraperitoneally.Hearts and aorta tissues were harvested separately and stored in 4% formalin for histopathological study or −80 • C condition for biochemical experiments.All procedures of the study were strictly carried out according to the Guiding Principles for the Care and Use of Laboratory Animal, and were accepted by the Committee on the Ethics of Animal Experiments of China Agricultural University (Beijing, China).
Analysis of Serum Lipid Profiles
After being left at room temperature for two to three hours, the collected blood samples of mice were centrifuged for 15 min at 3000 rpm.The serums were divided and kept until use at −80 • C. Levels of total TC, TG, LDL-C, and HDL-C in serum were measured according the commercially assay kits (S03027, S03042, S03029, S03025, Shenzhen Redu Life Sciences Limited Liability Company, Shenzhen, China).
Hematoxylin-Eosin (HE) Staining for Aorta
After being immersed and fixed for more than 48 h in the 4% paraformaldehyde, the mice aortas were embedded in paraffin for serial cross-sections (5 µm thick) using the microtome.Paraffin section samples prepared were stained by the commercial HE staining kit (G1120, Beijing Solarbio Science and Technology Limited Liability Company, Beijing, China).Serial slices stained were observed by optical microscope, and then analyzed for the atherosclerosis plaques by the Image J software (version 1.8.0,National Institutes of Health, Bethesda, MD, USA).
Oil Red O Staining for Lesions in Aorta
The aortic tissues were immersed in an optimal cutting temperature compound (OCT) at −80 • C. The prepared sections (10 µm thick) were sequentially stained with modified Oil Red O solution and counterstained with Mayer hematoxylin solution using the corresponding commercial assay kit (G1261, Beijing Solarbio Science and Technology Limited Liability Company, Beijing, China).The stained slices were observed for the pathological lesions under the Leica CTR6 microscope (Leica, Wetzlar, Germany).
Masson's Trichrome Stain
Weigert's iron hematoxylin solution, fuchsin ponceau acid solution, and aniline blue solution were used to stain the prepared paraffin sections according to the Masson's trichrome stain kit instructions (G1340, Beijing Solarbio Science and Technology Limited Liability Company, Beijing, China).The optical microscope (Leica CTR6, Leica, Germany) was used to capture the Masson's trichrome stain slices.The collagen proportion of stained slices were analyzed and quantified by Image J software (version 1.8.0).
Statistical Analysis
The results of experiments were presented as mean ± standard deviation (mean ± SD).GraphPad Prism statistical software (version 8, San Diego, CA, USA) was used to conduct the statistical analysis.t-test was used to evaluated statistical differences between the control group and experimental group.p-value < 0.05 represented the statistically significant.
Characterization of Dandelion Polysaccharides
We first characterized the structure, molecular weight, and monosaccharide composition of dandelion polysaccharides in this study.In the FT−IR spectrum, the strong absorbance band at 3427 cm −1 was attributed to the stretching vibration of O-H (Figure 1).The bands at 1614 cm −1 and 1076 cm −1 indicated the stretching vibration of C-O.The band at 1384 cm −1 indicated the vibration of C-H.The weak band at 602 cm −1 was attributed to C-C stretching (Figure 1).The number average molecular weight (Mn), weight average molecular weight (Mw), and Z average molecular weight (Mz) of dandelion polysaccharides were 533, 4293, and 129,668, respectively (Table 1).The monosaccharide composition of dandelion polysaccharides was glucose, galactose, ribose, fucose, xylose, arabinose, rhamnose, mannose, glucuronic acid, and galacturonic acid in a molar ratio of 9.2:5.3:1.3:0.4:4.3:6.0:2.4:1.6:1.5:3.5 (Table 1).
The Establishement of Atherosclerotic Animal Model
In order to establish an atherosclerosis model, the ApoE −/− mice were given an HFD for 10 weeks.Then, the atherosclerotic mice were randomly assigned to gavage saline or dandelion polysaccharides (200 mg kg −1 d −1 , 200 µL) for an additional 8 weeks (Figure 2).We
The Establishement of Atherosclerotic Animal Model
In order to establish an atherosclerosis model, the ApoE −/− mice were given an HFD for 10 weeks.Then, the atherosclerotic mice were randomly assigned to gavage saline or dandelion polysaccharides (200 mg kg −1 d −1 , 200 µL) for an additional 8 weeks (Figure 2).We observed no difference between saline and dandelion polysaccharides groups in the final body weight (Figure 3A, p > 0.05).Moreover, both gram per day per mice and kcal per day per mice showed no difference in food intake between saline and dandelion polysaccharides groups (Figure 3B,C, p > 0.05).These results suggest that the treatment of ApoE −/− mice with dandelion polysaccharides showed no effect on body weight or food intake.observed no difference between saline and dandelion polysaccharides groups in the final body weight (Figure 3A, p > 0.05).Moreover, both gram per day per mice and kcal per day per mice showed no difference in food intake between saline and dandelion polysaccharides groups (Figure 3B,C, p > 0.05).These results suggest that the treatment of ApoE −/− mice with dandelion polysaccharides showed no effect on body weight or food intake.
Dandelion Polysaccharides Improved Lipid Profiles
Disordered lipid metabolism will promote atherosclerosis development [25].Therefore, we next explored whether the administration of dandelion polysaccharides might influence the blood lipid profiles (TG, TC, LDL-C, HDL-C) in atherosclerotic mice.We found that dandelion polysaccharides reduced the levels of TG (p < 0.01), TC (p < 0.05), and LDL-C (p < 0.001), while increasing HDL-C level (p < 0.001) in serum samples (Figure 4A-D).These results indicated that dandelion polysaccharides intervention significantly impacted the serum lipid profiles in atherosclerotic mice.
Dandelion Polysaccharides Improved Lipid Profiles
Disordered lipid metabolism will promote atherosclerosis development [25].Therefore, we next explored whether the administration of dandelion polysaccharides might influence the blood lipid profiles (TG, TC, LDL-C, HDL-C) in atherosclerotic mice.We found that dandelion polysaccharides reduced the levels of TG (p < 0.01), TC (p < 0.05), and LDL-C (p < 0.001), while increasing HDL-C level (p < 0.001) in serum samples (Figure 4A-D).These results indicated that dandelion polysaccharides intervention significantly impacted the serum lipid profiles in atherosclerotic mice.
Dandelion Polysaccharides Reduce Atherosclerotic Plaques in Aortic Roots
We next investigated whether the dandelion polysaccharides could reduce the plaque burden by examining the aortic roots in HFD-induced ApoE −/− mice.The aortic sinus ather-
Dandelion Polysaccharides Reduce Atherosclerotic Plaques in Aortic Roots
We next investigated whether the dandelion polysaccharides could reduce the plaque burden by examining the aortic roots in HFD-induced ApoE −/− mice.The aortic sinus atherosclerotic lesion area was decreased in mice receiving oral gavage of dandelion polysaccharides (Figure 5A,B).Moreover, we observed that the lesion area of atherosclerotic plaques was reduced by 40% after dandelion polysaccharide treatment compared with the saline group (from 43 × 10 4 µm 2 to 23 × 10 4 µm 2 ) (Figure 5C,D).To further explore whether dandelion polysaccharides could affect the progression of atherosclerosis, we classified the plaque into early, moderate, and advanced stages according to the Stary method [26].The results suggested that dandelion polysaccharidestreated mice had a higher proportion of earlier plaques, and lower proportion of moderate and advanced plaques (Figure 5E).These results suggested that dandelion polysaccharides exerted an anti-atherosclerotic effect on HFD-induced ApoE −/− mice.
Dandelion Polysaccharides Reduce Necrotic Core Area and Elevated Collagen Content of Plaques
Necrotic cores are responsible for inflammation, thrombosis, degradation of the proteolytic plaque, and physical stress on the fibrous cap, which are produced during the advanced lesion macrophages apoptosis and improper phagocytic clearance (or efferocytosis) of the apoptotic macrophages in advanced plaques [27].We found that mice treated with dandelion polysaccharides had a smaller necrotic area (decreased by 60%, from 39 × 10 4 µm 2 to 13 × 10 4 µm 2 ) (Figure 6A,B, p < 0.001).In addition, dandelion polysaccharide administration also showed a higher percentage of collagen content (41%) than those in the saline group (20%) (Figure 6C,D, p < 0.001).These results suggested that dandelion polysaccharides might lead to more stable atherosclerotic lesions in atherosclerotic mice.
Dandelion Polysaccharides Reduce Necrotic Core Area and Elevated Collagen Content of Plaques
Necrotic cores are responsible for inflammation, thrombosis, degradation of the proteolytic plaque, and physical stress on the fibrous cap, which are produced during the advanced lesion macrophages apoptosis and improper phagocytic clearance (or efferocytosis) of the apoptotic macrophages in advanced plaques [27].We found that mice treated with dandelion polysaccharides had a smaller necrotic area (decreased by 60%, from 39 × 10 4 µm 2 to 13 × 10 4 µm 2 ) (Figure 6A,B, p < 0.001).In addition, dandelion polysaccharide administration also showed a higher percentage of collagen content (41%) than those in the saline group (20%) (Figure 6C,D, p < 0.001).These results suggested that dandelion polysaccharides might lead to more stable atherosclerotic lesions in atherosclerotic mice.
Macrophages and smooth muscle cells are major cells promoting artery remodeling and atherosclerosis [27,28].To explore whether dandelion polysaccharides might impact the persistence of macrophages and smooth muscle cells, we next performed immunohistochemical staining of Mac-3 (marker for murine macrophage) and α-SMA (marker for smooth muscle cells).Compared to the saline groups, we found that the persistence of macrophages (Figures S1 and 6E) and smooth muscle cells (Figures S2 and 6F) in the atherosclerotic plaques were reduced in the dandelion polysaccharides group.The results indicated that dandelion polysaccharides inhibited the persistence of macrophages and smooth muscle cells within the atherosclerotic plaques.Macrophages and smooth muscle cells are major cells promoting artery remodeling and atherosclerosis [27,28].To explore whether dandelion polysaccharides might impact the persistence of macrophages and smooth muscle cells, we next performed immunohistochemical staining of Mac-3 (marker for murine macrophage) and α-SMA (marker for smooth muscle cells).Compared to the saline groups, we found that the persistence of macrophages (Figures S1 and 6E) and smooth muscle cells (Figures S2 and 6F) in the atherosclerotic plaques were reduced in the dandelion polysaccharides group.The results indicated that dandelion polysaccharides inhibited the persistence of macrophages and smooth muscle cells within the atherosclerotic plaques.
Dandelion Polysaccharides Reduce the Levels of Oxidant-Related Markers in Serum Samples
Previous studies demonstrated that polysaccharides possessed anti-oxidant capabilities [29].The oxidative indicators included MDA, SOD, and GSH-PX [30].MDA, a byproduct of lipid peroxidation, was a crucial signal for oxidative damage and lipid metabolism abnormalities during atherosclerosis development [31].Therefore, we measured the levels of oxidative-related markers in serum samples from atherosclerotic mice.It was found that the dandelion polysaccharides group had lower MDA levels (Figure 7A, p < 0.01).Conversely, the levels of SOD and GSH-PX were elevated after the treatment of dandelion polysaccharides (Figure 7B, p < 0.01; Figure 7C, p < 0.05).These results suggest that dandelion polysaccharides inhibited atherosclerosis partially through its anti-oxidant capacity.
Dandelion Polysaccharides Reduce the Levels of Oxidant-Related Markers in Serum Samples
Previous studies demonstrated that polysaccharides possessed anti-oxidant capabilities [29].The oxidative indicators included MDA, SOD, and GSH-PX [30].MDA, a byproduct of lipid peroxidation, was a crucial signal for oxidative damage and lipid metabolism abnormalities during atherosclerosis development [31].Therefore, we measured the levels of oxidative-related markers in serum samples from atherosclerotic mice.It was found that the dandelion polysaccharides group had lower MDA levels (Figure 7A, p < 0.01).Conversely, the levels of SOD and GSH-PX were elevated after the treatment of dandelion polysaccharides (Figure 7B, p < 0.01; Figure 7C, p < 0.05).These results suggest that dandelion polysaccharides inhibited atherosclerosis partially through its anti-oxidant capacity.
Dandelion Polysaccharides Reduce the Expression of Inflammatory Factors in Aorta
Chemotactic factor (Mcp-1) and inflammatory cytokines (Tnf-α, Il-1β, and Il-6) are released during the initial stage of atherosclerosis, where they trigger the infiltration of monocytes across the endothelium and promote the formation of atherosclerotic plaques [32].We therefore measured the mRNA and protein expressions of the chemotactic factor and inflammatory cytokines upon the administration of dandelion polysaccharides.The results suggested that the mRNA expression of inflammatory cytokines (Tnf-α, Il-1β, and Il-6) in the aortic tissues was greatly decreased in the dandelion polysaccharides group compared with the saline group (Figure 8A-C, p < 0.01).Concomitantly, the mRNA expression of the chemotactic factor (Mcp-1) was also decreased upon the intervention of dandelion polysaccharides (Figure 8D, p < 0.05).Meanwhile, the protein levels of these inflammatory cytokines and chemotactic factor in mice aortas were drastically decreased in the dandelion polysaccharides group (Figure 8E,F, p < 0.05).These results indicated that dandelion polysaccharides inhibited atherosclerosis partially through its anti-inflammatory response.
Dandelion Polysaccharides Reduce the Expression of Inflammatory Factors in Aorta
Chemotactic factor (Mcp-1) and inflammatory cytokines (Tnf-α, Il-1β, and Il-6) are released during the initial stage of atherosclerosis, where they trigger the infiltration of monocytes across the endothelium and promote the formation of atherosclerotic plaques [32].We therefore measured the mRNA and protein expressions of the chemotactic factor and inflammatory cytokines upon the administration of dandelion polysaccharides.The results suggested that the mRNA expression of inflammatory cytokines (Tnf-α, Il-1β, and Il-6) in the aortic tissues was greatly decreased in the dandelion polysaccharides group compared with the saline group (Figure 8A-C, p < 0.01).Concomitantly, the mRNA expression of the chemotactic factor (Mcp-1) was also decreased upon the intervention of dandelion polysaccharides (Figure 8D, p < 0.05).Meanwhile, the protein levels of these inflammatory cytokines and chemotactic factor in mice aortas were drastically decreased in the dandelion polysaccharides group (Figure 8E,F, p < 0.05).These results indicated that dandelion polysaccharides inhibited atherosclerosis partially through its anti-inflammatory response.
Discussion
Atherosclerosis is a chronic and progressive cardiovascular disease characterized by lipid dysfunction, oxidative stress, inflammation, migration of smooth muscle cells, formation of foam cells, covering of the fibrous cap, and accumulation of atheromatous plaques [33,34].The phytochemicals derived from natural plants have been reported to improve atherosclerosis [35,36].Dandelion polysaccharides were considered as one of the most important active compounds from the dandelion plant [37].Emerging studies have proved that dandelion polysaccharides have a broad bioactive effect, such as anti-inflammatory and antioxidant activities [15,37].These studies suggested that dandelion polysaccharides with multiple functions have a potential impact on the progression of atherosclerosis.To address this hypothesis, we explored the effect of dandelion polysaccharides on atherosclerosis in HFD-induced ApoE −/− mice.In the atherosclerotic ApoE −/− mice treated with saline or dandelion polysaccharides, we estimated the prospective impact of dandelion polysaccharides on the pathological characteristics of atherosclerotic lesions, lipid profiles, antioxidation markers in serum samples, and the expression of inflammatory cytokines in aortas.
In the present study, we used an ApoE −/− atherosclerotic mice model to examine whether dandelion polysaccharides might exert anti-atherosclerotic effects.We showed that dandelion polysaccharides ameliorated high-fat-diet-induced atherosclerosis in mice through antioxidant and anti-inflammatory capabilities.The principal finding of this investigation is that dandelion polysaccharide intervention significantly inhibited the atherosclerotic plaques formation in aorta roots, which was considered as the most important site for the pathological assessment of atherosclerotic plaques [32].Firstly, the area of atherosclerotic lesion upon the treatment of dandelion polysaccharides was reduced by 40% in
Discussion
Atherosclerosis is a chronic and progressive cardiovascular disease characterized by lipid dysfunction, oxidative stress, inflammation, migration of smooth muscle cells, formation of foam cells, covering of the fibrous cap, and accumulation of atheromatous plaques [33,34].The phytochemicals derived from natural plants have been reported to improve atherosclerosis [35,36].Dandelion polysaccharides were considered as one of the most important active compounds from the dandelion plant [37].Emerging studies have proved that dandelion polysaccharides have a broad bioactive effect, such as antiinflammatory and antioxidant activities [15,37].These studies suggested that dandelion polysaccharides with multiple functions have a potential impact on the progression of atherosclerosis.To address this hypothesis, we explored the effect of dandelion polysaccharides on atherosclerosis in HFD-induced ApoE −/− mice.In the atherosclerotic ApoE −/− mice treated with saline or dandelion polysaccharides, we estimated the prospective impact of dandelion polysaccharides on the pathological characteristics of atherosclerotic lesions, lipid profiles, antioxidation markers in serum samples, and the expression of inflammatory cytokines in aortas.
In the present study, we used an ApoE −/− atherosclerotic mice model to examine whether dandelion polysaccharides might exert anti-atherosclerotic effects.We showed that dandelion polysaccharides ameliorated high-fat-diet-induced atherosclerosis in mice through antioxidant and anti-inflammatory capabilities.The principal finding of this investigation is that dandelion polysaccharide intervention significantly inhibited the atherosclerotic plaques formation in aorta roots, which was considered as the most important site for the pathological assessment of atherosclerotic plaques [32].Firstly, the area of atherosclerotic lesion upon the treatment of dandelion polysaccharides was reduced by 40% in polysaccharide-treated mice compared with controls.These results indicated that dandelion polysaccharides greatly reduced the atherosclerotic lesion area.Secondly, dandelion polysaccharides improved the blood lipid profiles in HFD-induced ApoE −/− mice.Specifically, dandelion polysaccharides reduced the serum levels of multiple pro-atherosclerotic lipids, including TG, TC, and LDL-C.On the contrary, dandelion polysaccharides elevated atherosclerosis-protective HDL-C.These results are consistent with previous studies, showing that dandelion leave supplementation dramatically suppressed the levels of TG and TC [16,17].Since lipid dysregulation is crucial in the development of atherosclerotic plaques [38], the reduction of atherosclerosis upon the treatment of dandelion polysaccharides might partially be attributed to its improving effect on blood lipid profile [17].Thirdly, dandelion polysaccharides inhibited the serum oxidative stress in progressive atherosclerosis.Specifically, dandelion polysaccharides reduced the MDA level in serum samples and increased the SOD and GSH-PX levels.This observation was in line with earlier studies demonstrating that dandelion extract supplementation improved the lipid peroxidation [17,22].The last finding of the present study is that dandelion polysaccharides reduced the expression of the chemotactic factor Mcp-1 and pro-inflammatory cytokines.During the progression of atherosclerosis, the production of these inflammatory cytokines may promote endothelial damage [39].In line with this study, the methanolic extract of dandelion reduced the pro-inflammatory cytokine expression in the LPS-induced endothelial cell [40].
Atherosclerosis, a chronic inflammation disease of the aorta, is a progress that involves the activation of abundant immune competent cells [41].Proinflammatory cytokines and chemokines may trigger an unstable atherosclerotic plaque, subsequent atherothrombosis, and CVD events [42].Tnf-α, a pro-inflammatory cytokine, promotes aortic inflammation during the progression of atherosclerosis [43].Il-1β and Il-6 have also been considered to play an important role in atherosclerotic plaque formation and development [44].
Mcp-1, a chemotactic factor for monocytes, was connected to the persistence of lipidburden macrophages [45].Hence, the elevated expression of inflammatory cytokines and chemokines may accelerate the rupture of the atherosclerotic plaque [46].Immune modulatory therapies involving inflammation have been continually put forward to prove the possibility of anti-atherosclerosis [47][48][49].However, the current treatments had undesirable adverse effects.Dandelion polysaccharides, as a natural safety ingredient, possess excellent anti-inflammation properties [18].Therefore, we utilized this property of dandelion polysaccharides to treat atherosclerosis.As expected, we found reduced levels of Tnf-α, Il-1β, Il-6, and Mcp-1 in aortic tissues upon treatment with dandelion polysaccharides.Consisted with this study, previous studies demonstrated that polysaccharides extracted from plants modulated inflammatory cytokines and chemokines [50][51][52].Furthermore, studies also indicated that dandelion extracts exert anti-inflammatory effects.For instance, dandelion extracts had been found to reduce the LPS-induced expression of Il-1β, Il-6, and Tnf-α in rat skeletal muscle cells [53].In addition, dandelion polysaccharides reduced the CCl 4 -induced liver damage via modulating NF-kB and its regulatory inflammatory mediators in rats [20].This study also provided evidence that dandelion polysaccharides reduced the expression of chemotactic factors and inflammatory cytokines in atherosclerotic aorta.
Aside from the activation of pro-inflammation signaling pathways, as well as cytokine/chemokine expression, oxidative stress contributes to the pathogenesis of atherosclerosis [33].Specifically, MDA is an important product of lipid peroxidation in atherosclerosis [54].In contrast, the antioxidant systems SOD and GSH-PX have a reducing effect on lipid peroxidases and oxidized phospholipids.Since the pathophysiology of atherosclerosis is greatly influenced by oxidative stress [55], this study examined whether dandelion polysaccharides had an effect on oxidative stress in serum samples from atherosclerotic mice.The results showed that dandelion polysaccharide supplementation reduced the MDA level, while elevating the SOD and GSH-PX levels.This finding is in line with the observations that in atherosclerotic rabbits, dandelion root and leaf improved the activities of antioxidant enzymes in liver tissues [17].Selenized polysaccharides from dandelion roots also showed antioxidant activities in vitro [14].It has been reported that dandelion polysaccharide treatment prolonged the shelf life of white shrimps due to its antioxidant activities [18].Similarly, two novel dandelion polysaccharides (DRP-2b, DRP-3a) had a good performance on the DPPH radical scavenging activity and hydroxyl radical scavenging ability [56].Although these findings suggest that polysaccharides in dandelion may have anti-oxidant properties, they lack in vivo evidence.In this study, we provide direct evidence that dandelion polysaccharides showed antioxidant activities in HFD-induced ApoE −/− mice.Therefore, the anti-atherosclerotic effect of dandelion polysaccharides may be attributable to its antioxidant capabilities.
In conclusion, this study identifies the anti-atherosclerotic activity of dandelion polysaccharides through its antioxidant and anti-inflammatory activities (Figure 9), and suggests that dandelion polysaccharides might serve as potential agents in the intervention of atherosclerosis.
Nutrients 2023, 15, x FOR PEER REVIEW 13 of 16 polysaccharide treatment prolonged the shelf life of white shrimps due to its antioxidant activities [18].Similarly, two novel dandelion polysaccharides (DRP-2b, DRP-3a) had a good performance on the DPPH radical scavenging activity and hydroxyl radical scavenging ability [56].Although these findings suggest that polysaccharides in dandelion may have anti-oxidant properties, they lack in vivo evidence.In this study, we provide direct evidence that dandelion polysaccharides showed antioxidant activities in HFD-induced ApoE −/− mice.Therefore, the anti-atherosclerotic effect of dandelion polysaccharides may be attributable to its antioxidant capabilities.
In conclusion, this study identifies the anti-atherosclerotic activity of dandelion polysaccharides through its antioxidant and anti-inflammatory activities (Figure 9), and suggests that dandelion polysaccharides might serve as potential agents in the intervention of atherosclerosis.
Figure 3 .
Figure 3. Effects of dandelion polysaccharides on body weight and food intake in HFD-induced ApoE −/− mice.The body weight (A) and feed consumption (B,C) in dandelion polysaccharides and saline group.Results were presented as means ± SD, and n = 10 in each group.t-test, ns, p > 0.05.
Figure 3 .
Figure 3. Effects of dandelion polysaccharides on body weight and food intake in HFD-induced ApoE −/− mice.The body weight (A) and feed consumption (B,C) in dandelion polysaccharides and saline group.Results were presented as means ± SD, and n = 10 in each group.t-test, ns, p > 0.05.
Nutrients 2023 , 16 Figure 5 .
Figure 5.Effect of dandelion polysaccharides on atherosclerotic lesion in HFD-induced ApoE −/− mice.Representative images for Oil red O staining (A), HE staining (B), quantitative chart (C), and curve chart (D) of atherosclerotic lesion area by HE staining.The proportion of early, moderate, and advanced atherosclerotic plaques based on histological staging (E).The sale bar is 250 µm.Results were presented as means ± SD and n = 10 in each group.t-test, * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5 .
Figure 5.Effect of dandelion polysaccharides on atherosclerotic lesion in HFD-induced ApoE −/− mice.Representative images for Oil red O staining (A), HE staining (B), quantitative chart (C), and curve chart (D) of atherosclerotic lesion area by HE staining.The proportion of early, moderate, and advanced atherosclerotic plaques based on histological staging (E).The sale bar is 250 µm.Results were presented as means ± SD and n = 10 in each group.t-test, * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6 .
Figure 6.Effect of dandelion polysaccharides on plaques' necrotic core and collagen in HFD-induced ApoE −/− mice.Curve chart (A) and quantitative chart (B) of atherosclerotic necrotic core area.Representative images (C) and quantitative chart (D) of atherosclerotic collage area.Quantitative chart of macrophage number (E) and vascular smooth muscle cell area (F) in atherosclerotic plaque.The sale bar represents 150 µm.Results were presented as means ± SD, and n = 10 in each group.t-test, ** p < 0.01, *** p < 0.001.
Nutrients 2023 , 16 Figure 6 .
Figure 6.Effect of dandelion polysaccharides on plaques' necrotic core and collagen in HFD-induced ApoE −/− mice.Curve chart (A) and quantitative chart (B) of atherosclerotic necrotic core area.Representative images (C) and quantitative chart (D) of atherosclerotic collage area.Quantitative chart of macrophage number (E) and vascular smooth muscle cell area (F) in atherosclerotic plaque.The sale bar represents 150 µm.Results were presented as means ± SD, and n = 10 in each group.t-test, ** p < 0.01, *** p < 0.001.
Figure 9 .
Figure 9. Schematic illustration of dandelion polysaccharides in ameliorating atherosclerosis.Compared with the saline group, the dandelion polysaccharide treatment improved the serum lipids and antioxidant markers, while lowering the expression of chemotactic factors and inflammatory cytokines in atherosclerotic mice.
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2023-09-26T15:04:47.057Z
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2023-09-24T00:00:00.000
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Dynamic Analysis of Telecommunication Tower for Optimum Modal Combination and Elemental Discretization
Over the past 35 years, the growing demand for wireless and broadcast communication has spurred a dramatic increase in steel telecommunication tower construction and maintenance. Failure of such structures due to severe earthquakes is a major concern. The Indian code suggests the detailed static and dynamic analysis provisions that are to be followed for lumped mass systems like buildings. In case of continuous structures the code only suggests the static analysis provisions in details. But, due to the lack of detailed Indian codal provisions for dynamic analysis of telecommunication tower, a comparative study using response spectrum method is being carried out with the help of suitable software for different ground level conditions in case of India. According to the theoretical approach of any structural dynamics problem, the structures without lumped mass system is considered as continuous system which is further idealized as a series of small elemental segments. Furthermore, the structural analysis of these elemental segments using the concept of Finite Element Method (FEM) is being carried out with the help of the mentioned software and the results of natural frequencies, time periods of the structure are compared to obtain the optimum number of elemental discretization along with the optimum method of modal combination.
I. INTRODUCTION
In the present era, technology in communications has developed to a very large extent. The communication industries have seen a tremendous increase in the last few years which has resulted in the installation of a large number of towers to increase the coverage area and network consistency. In wireless communication networks, these towers play a significant role, hence the failure of such structure in a disaster is a major concern. Therefore the utmost importance should be given in considering all possible extreme conditions for designing these towers.
Lu, Ou, Xing and Mills (1988) [1] first ever presented the analysis of steel transmission tower and presented the structural response of the lattice tower and the detailed connection i.e. bolted connection. They have analyzed the structure considering the basic loads without using any codal provision and concluded in their paper that the Bhosale, Kumar and Pandey (2012) [5] analyzed towers with different bracing system while mounted on the rooftop. They analyzed the structure under wind and seismic loading condition with different bracing patterns and concluded that the design of roof top towers cannot be based on analytical results obtained for a similar configuration situated at ground level. As seen, the axial forces in rooftop tower are increased approximately by two to three times (max.) with respect to ground tower. By increasing the stiffness of the host structure in both the directions (X and Y), the axial forces (tensile & compression) in rooftop towers were increased by minimal amount of 5%. The axial forces in leg members under the effect of seismic load attain the highest value. Nevertheless, it has been observed that the forces in diagonal members are greater as compared to the horizontal members. Rajasekharan (2014) [7] designed the lattice tower for three heights of 30m, 40m and 50m with different types of bracings to study the effect of wind load on 4-legged lattice tower for wind zone V and VI using gust factor method. They also studied the seismic effect on the tower structures by carrying out the modal analysis and response spectrum analysis for zone II to zone V and concluded that the member stresses in bottom leg of XX braced tower are higher as compared to other tower models. The frequency of the tower with Y bracing displayed the least natural frequency since its stiffness was found to be higher due to more weight of the structure as compared to other models. It was observed that from 30m to 40m tower height, the increase in displacement is nearly linear but as the height increases from 40m to 50m there is a steep increase in the displacement in all the zones.
Sharma, Duggal, Singh and Sachan (2015) [8] presented a comparative analysis of steel telecommunication tower under combined seismic and wind load and analyzed their structures with different bracing patterns and concluded different considerations for different conditions. Specifically they have concluded that for all wind zones tower height between 25m to 35m with different bracing patterns do not show much difference in displacement. For wind zone I to IV, tower height between 35m to 45m having K-Bracing or W-Bracing gives maximum value of displacement and V-Bracing gives minimum value of displacement.
For wind zone V and VI tower height between 35m to 45m having W-Bracing gives maximum value of displacement and V-Bracing or XBX -Bracing gives minimum value of displacement. There is a steep increase in the displacement in Earthquake zone V for all considered type of bracing pattern. Results show that the increase in the displacement from earthquake zone II to VI is maximum for W-Bracing and it is minimum for K-Bracing. For all earthquake zones stress at the bottom leg members of the tower is maximum for XBX-Bracing and it is minimum for W-Bracing.
In this study, the main consideration that has been carried out is the comparative analysis among the time periods and natural frequencies of self-supporting telecommunication tower under seismic loading in different ground level conditions. The scope of this thesis is primarily focused on the comparative study of the peak-response quantities (natural frequency, time period) of the structure under seismic loads to obtain the optimum modal combination along with the optimum elemental discretization.
II. STRUCTURAL DESIGN
From the review of literature, it has been observed that the various methods of earthquake analysis are considered while analyzing a self-supporting tower including dead loads and live loads. As the structural configuration depends upon the loading intensities and variations, it also depends upon the zone and soil conditions along the loads. So, the design has to satisfy both the maximum allowable criteria for the zone and soil type considerations together.
The self-supporting towers [2] are mainly considered when the base area is limited and the required height of the tower is much higher. The guyed towers cannot be used for the limited base area as they required a large area to guy the cables and also the monopoles cannot be considered as the required height is much higher for which the wind intensity will be pretty higher. In case of the self-supporting tower it can be designed as three-legged as well as four-legged whichever is required; but in most of the cases when the height of the tower is much larger like 40m-50m or higher than that, four-legged structure is generally considered as it consists of more number of members than three-legged structure, to transmit the loads from the superstructure to the soil beneath it; so it is a basic intuition that when the applied load is same for two structure but the base areas of the structures which are subjected to the external loads varies, then in case of the larger area the developed stresses will be lower and in case of the smaller area the developed stresses will be higher. Therefore, following this basic convention, the bracing systems are used to distribute the loads among the members and to resist any kind of failure of the main legs including the bracings themselves. The towers are generally considered as a cantilever structure as a whole and as we know the bending moment of a cantilever member is highest at the fixed end and for that reason the whole structure of the tower is designed as a tapered structure whose largest area is on the fixed end and smallest area is on the free end. For designing a tapered section the legs are designed with a little angle deviation with respect to the vertical for which the legs are more effective to carry both components of force viz. horizontal and vertical.
Depending upon the loading types and the distribution of loads among the members, the sections are chosen. Generally for consideration of member of a trussed tower the angle sections are used because the truss structures have only the axial forces to be considered (no shear force or bending moment in the members) and for that reason among all the sections which are to be considered only for axial forces, the angle section has the least specific area compared to the other which is advantageous from the economic and self-weight point of view.
In a trussed tower the bracing systems [6], [10] and the connections among the members are very important aspects. The bracing systems consist of horizontal bracing as well as diagonal bracings to resist the failure of the members. The horizontal bracing members are provided to resist the buckling of the legs hence they are considered as tension members and to resist the torsional effect as well as to distribute the loads, the diagonal bracing members are used. From the structural analysis point of view of a trussed structure, as it is being known that the triangular structures have the highest stability and strength to resist the external loads the bracings are designed in such a way that the elementary structures of the bracings consist of triangular formation. The connections are to be designed very cautiously as it is the main part of the structure that holds all the members together; so the failure of connections will lead to the failure of members which will cause the total structural failure. In case of trussed towers, as there is no bending moment developed in the members, the moment connections are not needed to be considered; only the shear connections are enough to maintain the stability and strength among the members.
III. ANALYSIS METHODS
This topic reveals the basic considerations of earthquake analysis of a self-supporting trussed tower. The seismic loads are considered to act as a result of horizontal relative acceleration among the different panels which causes drifts in the structures. Earthquake is an unpredictable phenomenon for which the analysis of a structure under seismic load condition has to be done with the previous collected statistics of earthquakes for a particular zone and soil. The particular collected statistical data has been taken into account with a probabilistic approach to obtain the optimum possible safety. Therefore, basically the structures are analyzed by the previous seismic loads with the highest intensities for the certain design criteria. IS 1893:2005 (Part-4) [12] gives the provisions for static analysis of seismic load for communication towers with consideration of different zones and soil structures.
A. Response Spectrum Analysis
The dynamic analysis is defined as the analysis of structure considering the motion of the structure which depicts that all the parameters are taken as time-dependant. In case of the seismic analysis, the ground vibration is considered as random vibration which can be evaluated using probabilistic approach. The random vibration is idealized as the summation of different series or "modes" of elementary harmonic vibrations. The theoretical analysis of any stack-like structure is done by idealizing the structure as continuous system and with the help of the dynamic matrix method the elemental segments are analyzed to get the response of the whole structure for each modes of vibrations. From the first mode of vibration, the natural time period is being calculated. In case of the assignment of the seismic loads in the software viz. STAAD.Pro V8i, the calculations of the parameters that have to be made for the RS analysis are according to the provisions of IS 1893: 2002 (Part-1) and IS 1893: 2005 (Part-4). The analysis has been done using STAAD.Pro V8i and the parameters which have to be put in the software for the analysis are as follows: Zone Factor (Z) is a factor that deals with the ratio of probable average intensity of earthquakes in case of particular zones. [Cl. 6.4.2, In the dynamic analysis, the design base shear (V B ) shall be compared with a base shear (V " B ) calculated using the fundamental period T (corresponding to first mode of vibration). All the response quantities such as member forces, displacements, storey forces, storey shears and base reactions shall be multiplied by V " B /V B . This base shear multiplication factor is mentioned in the codal provision but the value that has to be given in the software is evaluated as the ratio of the calculated time period to the time period corresponding to the first mode of vibration.
IV. ANALYSIS OF THE TOWER USING RESPONSE SPECTRUM METHOD
Initially, the dead loads and the seismic loads are being considered and the combination of both of them is then taken according to the clauses of IS 1893: 2002 (Part 1) for the dynamic analysis. Then the whole structure is analyzed using STAAD.Pro V8i with the following values of the parameters: The above mentioned parameters have been used for the analysis in STAAD.Pro and the number of cut-off mode shapes are taken as 50. The eigen analysis of the tower as a continuous structure has been done by STAAD.Pro and the natural frequencies and time periods are tabulated for first ten modes. The tower structure is analysed by elemental dicretization of the members to get the mentioned results. The study of optimum method of modal combination along with optimum number of elemental discretization is being performed. From Fig. 1 to Fig. 8, the mode shapes of the tower are shown for the first eight natural frequencies. Table II to Table IV and shown from Fig. 9 to Fig. 14. The comparisons among them are listed in Table V and shown in Fig. 15 and Fig. 16. In the above comparison among the natural frequencies and time periods of the tower in different ground level conditions, it can be seen that there is no huge difference in the change of the frequencies or the time periods. Now the study proceeds to the method of discretization. The eigen analysis of the tower structure is done using the concept of dicretization for continuous structure (i.e. without lumped mass). The analysis using discretization is just a finite element method where all the members are discretized in elements. The mechanics of those elements are studied using FBD of a spring-damper-mass system to come up with the resulting response of the total structure as a whole. The accuracy of the result depends on the number of discretized elements; higher the elements greater the accuracy but along with the accuracy the structure should also be economical. For this reason, the study of optimum number of discretized elements have been carried out and listed in Table VI and Table VII with graphical representations in Fig. 17 and Fig. 18. The percentage change in three elemental members with respect to one elemental member, percentage change in eight elemental members with respect to one elemental member and percentage change in eight elemental members with respect to three elemental members are denoted as "(a)", "(b)" and "(c)" in the above table. The average changes of all the frequncies are calculated to conclude the comparative study among them. From the above table, it can be clearly observed that the average percentage change in (a) is much higher than the average percentage change in (c). And from the table, one can easily depict that the summation of (a) and (c) is equal to the value of (b). From this relations among the percentage changes, it can be concluded that the percentage change in three elemental member with respect to one, is much significant than the other two. Therefore, three elemental member will be the optimum number of elemental discretization in case of the economic and safe design of the tower using dynamic analysis of continuous structure. Table VIII with graphical visualisation in Fig. 19 and Fig. 20. The comparison among the methods have been shown to obtain the optimum method. According to the IS 1893: 2002 (Par 1), the CQC method is the most reliable and approved method for the modal combination of general building structures. In this study, it has been shown that for the analysis of telecommunication tower the peak response quantites are very much similar for SRSS and CQC method but for ABS the values are much higher. From the economic point of view the ABS method cannot be considered as optimum method. Therefore, comparing the SRSS and CQC, it has been obtained that the SRSS values are little lower than CQC values. Henceforth, from the safety point of view the CQC is considered as the optimum method of modal combination which will give an economic and safe design.
V. RESULT AND DISCUSSION
In the above analysis, the results are evaluated from the STAAD.Pro output file. The resulting graphs have been generated to draw suitable conclusions from them. The conclusions that can be made from the results and discussions are as follows: From Fig. 9 to Fig. 14, it can be concluded that there is no significant change among the natural frequencies or the time periods of the tower in different ground level conditions. In Fig. 15 and Fig. 16, it can be seen that there is some certain changes among the natural frequencies or the time periods in case of the higher modes of vibrations. In Table VII the comparisons among the percentage change of frequencies are being done along with the graphical representations in Fig. 17 and Fig. 18. The change in three elemental members with respect to one elemental member is 1.68% and the change in eight elemental members with respect to three elemental members is 0.02%. Therefore, from this result it can be concluded that the optimum number of discretization should be taken as three elements. The maximum accuracy can be achieved by taking upto three elemental discretizations. In Table VIII the comparison among the methods of the dynamic analysis is being listed along with the graphical visualization from Fig. 19 to Fig. 21. The ABS shear value is much higher than the other values. Therefore it cannot be an economical method relative to the others. The SRSS shear value is minimum among all of them, that is why it is not considered because of the strength criteria. The CQC value is the mediate value which can be treated as safe from strength point of view while it is also economical. This method is also suggested by the IS 1893:2002 (Part 1) for dynamic analysis of general buildings.
VI. CONCLUSION
The comprehensive dynamic analysis of the selfsupporting telecommunication tower is performed using STAAD.Pro V8i. The method of Response Spectrum is opted for the analysis, where the parameters of the study are different ground elevations. Due to the lack of detailed codal provisions for dynamic analysis of telecommunication tower, this structure is exposed to the theoretical dynamic analysis in which all the members have satisfied their purpose under the external excitations for each and every case. From the result of the analysis it can be concluded that there are no significant changes among the response natural frequencies or the time periods of the tower in different ground elevations. Telecommunication tower is considered as a continuous system where the discretization of the system must be carried out to analyze it. From the result and discussions it has been presented that the optimum number of discretization should be taken as three elements. Furthermore, the analysis for continuous system demands for the optimum method of modal combinations. From the results of this study it is observed that the CQC value is the mediate value which can be treated as safe from strength point of view as well as cost effective from economical point of view. Therefore, it is considered as the optimum method for the calculations of peak response quantities.
|
2019-11-14T17:07:26.912Z
|
2019-01-01T00:00:00.000
|
{
"year": 2019,
"sha1": "5e5c0abc06dd5440b4fd945b82cdff8c58d85691",
"oa_license": null,
"oa_url": "https://doi.org/10.35940/ijeat.a9720.109119",
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"Engineering"
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|
269612749
|
pes2o/s2orc
|
v3-fos-license
|
LncRNA MEG3 Restrains Hepatic Lipogenesis via the FOXO1 Signaling Pathway in HepG2 Cells
Nonalcoholic fatty liver disease (NAFLD) become a main public health concern, and is characterized by lipid accumulation in the hepatocytes. We found that overexpression of lncRNA MEG3 significantly reduced the expression of FOXO1, ACC1, and FAS, and subsequently decreased the lipid accumulation in HepG2 cells. Moreover, inhibition of lncRNA MEG3 could increase the lipid accumulation and the mRNA and protein levels of FOXO1, ACC1, and FAS. Further study showed that lncRNA MEG3 regulates the lipogenesis process by inhibiting the entry of FOXO1 into the nucleus translocation. Our study demonstrated that lncRNA MEG3 regulates de novo lipogenesis by decreasing the expression and nucleus translocation of FOXO1 in HepG2 cells, suggesting that lncRNA MEG3 could be a promising therapeutic target in lipid metabolic disorders.
Introduction
Nonalcoholic fatty liver disease (NAFLD), a critical public health concern in the 21st century, afflicts nearly 20% of individuals in developed nations.As a metabolic syndrome, NAFLD is characterized by the accumulation of lipids in hepatocytes, particularly triglycerides (TG) [1].It has been proposed that various factors, including insulin resistance, hyperlipidemia, hypertriglyceridemia, and oxidative stress, contribute to chronic inflammation in hepatocytes.This inflammation, in turn, triggers the activation of lipid metabolism signaling pathways, leading to increased lipid synthesis and subsequent hepatic steatosis and NAFLD [2,3].However, the specific mechanism underlying the genesis and development of NAFLD remains elusive.
FOXO1 belongs to the forkhead family of transcription factors which is the main target of insulin signaling and regulates metabolic homeostasis.The phosphatidylinositol 3 kinase (PI3K)/Akt pathway phosphorylates FOXO1 and mobilizes it from the nuclei to the cytoplasm, leading to inactivation of FOXO1 [4,5].In liver, FOXO1 regulation of Chrebp O-glycosylation, leads to the hepatic glucose utilization with lipid synthesis [6].However, FOXO1 nuclear retention enhances lipid uptake and lipolysis, and potentiates UCP1 expression [7].
Chronic inflammation plays a pivotal role in lipid metabolism pathogenesis, and tumor necrosis factor (TNF)-α, promoting de novo lipogenesis both in vitro and in vivo.Studies showed that TNF-α mediates lipogenic signaling and the biosynthesis of non-esterified fatty acids and triglycerides through several signal transduction pathways [8,9].TNF-α could increase serum triglycerides by inducing stimulation of hepatic lipid synthesis in an insulin independent manner and enhancing lipid droplets formation via promoting phosphorylation of c-Jun N-terminal kinase (JNK), thus activating fatty acid synthase (FAS) and SREBP-1 [10,11], resulting in an increase in de novo synthesis of fatty acids.
The lncRNA MEG3, initially discovered in glioma, is known to inhibit cell proliferation by recruiting and activating p53 through specific secondary structures [12,13].Recent research has also reported its involvement in lipid metabolism.Through promoting the ubiquitination and degradation of the enhancer of Zeste homolog 2 (EZH2), overexpression of lncRNA MEG3 suppresses lipid accumulation induced by free fatty acids (FFA), ultimately upregulating the expression of Sirtuin 6 (SIRT6) in hepatocytes [14].
In this study, we attempt to clarify the expression and regulation of lncRNA MEG3, TNF-α, and lipogenesis genes in HepG2 cells and explore possible intervention mechanism, providing new evidence for the prevention and treatment of NAFLD.
Cell Culture and Nuclear Protein Extraction
HepG2 cells were grown in minimum Eagle's medium (MEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin G sodium and 100 mg/mL streptomycin sulfate in a humidified atmosphere with 5% CO 2 at 37 °C.For TNF-α treatment, HepG2 cells were exposed to 10 ng/ml TNF-α for 24 h as described previously [15].The nuclear protein was obtaied using the nuclear cytoplasmic extraction kit (Thermo, NE-PER) according to the manufacturer's protocol.
Adenovirus Vector Construction
The adenovirus vector expressing lncRNA MEG3 (Ad-MEG3) and the corresponding control adenovirus vector (Ad-Con) were purchased from GeneChem (Shanghai).HepG2 cells were infected with adenovirus at multiplicity of infection (MOI) 100.
RNA Isolation and Real-time PCR
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, California, USA), according to the manufacturer's instructions.SYBR Green I (TaKaRa) was used for real-time PCR according to the manufacturer's instructions with the ABI 7500 qPCR System (Life Technologies, CA).The relative expression level of mRNA was normalized to β-actin.Each reaction was performed in triplicate, and analysis was conducted using the 2 -ΔΔCT method.The primer sequences were listed in Table 1.
Western Blot Analysis
Nuclear and cytoplasmic protein fractions were isolated using Nuclear Extraction kit (Abcam, Cambridge, MA, USA), according to manufacturer's protocols.Cell lysates (10-30 μg protein) were separated using 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA), blocked by 5% non-fat dry milk for 1 h and incubated with primary antibodies at 4 °C overnight.Followed by probing with horseradish peroxidase-conjugated anti-immunoglobuin G (ZSGB-Bio, Beijing, China).GelDoc XR system (Bio-Rad, Hercules, CA) was adopted in the detection and data collection.Rabbit monoclonal anti-FOXO1, Anti-FAS, and anti-β-actin antibodies were purchased from Abcam (Abcam, Cambridge, MA, USA).Antibody against ACC1 was obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).
Oil Red O Staining
HepG2 cells were fixed with 4% formaldehyde for 10 min, then incubated with prewarmed Oil Red O (Solarbio, Beijing, China) for 30 min and washed with 60% propanediol for about 10 s.The lipid droplets were observed under a microscope (Olympus, Tokyo, Japan).
Intracellular TG Measurement
The intracellular triglyceride was measured as described previously [16].Cells were washed three times with cold PBS and extracted with CHCl3/MeOH (2:1), and then added 0.05% H 2 SO 4 and centrifuged.The precipitation was transferred to 1% Triton X-100 (1:1, v/v), dried, and dissolved in deionized water, and the content of intracellular triglycerides was measured using a triglyceride enzymatic assay kit (SSYF Medical Diagnostic Products Co., Ltd., Shanghai, China).
Statistics
All data are presented as the means ± SD of the indicated number of measurements.Differences were analyzed using the Student's t-test.P < 0.05 was considered a statistically significant difference.
LncRNA, MEG3, and Lipogenesis Genes were Upregulated in TNF-α Treated HepG2 Cells
In the previous study, we confirmed TNF-α could induce hepatic insulin resistance both in vitro and in vivo [15].To further research on metabolic diseases, we still use HepG2 cells and treat them with TNF-α (10 ng/ml) for 24 h, and intracellular lipid accumulation increased significantly treated with TNF-α (Fig. 1A, B), Meanwhile, the expression of lipogenesis genes such as Acetyl-CoA carboxylase 1 (ACC1), FAS, Forkhead box protein 1 (FOXO1), as well as lncRNA MEG3 significantly increased (Fig. 1C).
Overexpression of lncRNA MEG3 Restrained Lipogenesis Gene
To determine whether lncRNA MEG3 is promoted in lipogenesis, we upregulated the intracellular lncRNA MEG3 by delivering a specific adenovirus vector (Ad-MEG3) (Fig. 2A).The overexpression of lncRNA MEG3 repressed the mRNA expression and the protein level of FOXO1, along with the de novo lipogenesis markers ACC1 and FAS.It also gives rise to upregulated phosphorylation of ACC1 (Fig. 2B, C).These results indicated that the overexpression of lncRNA MEG3 leads to inhibition of the de novo lipogenesis signaling pathway.
Inhibited lncRNA MEG3 Enhanced the Lipogenesis in HepG2 Cells Treated with TNF-α
Next, by silencing specific siRNA, we knocked down the expression of lncRNA MEG3 in HepG2 cells treated with TNF-α (Fig. 3A).The results showed that intracellular TG increased after the knockdown of lncRNA MEG3 (Fig. 3B, C).Furthermore, the mRNA and protein levels were upregulated by FOXO1, ACC1, FAS.As well as the suppression of the phosphorylation of ACC1.(Fig. 3A, D).These results suggested that inhibiting the lncRNA MEG3 could further promote the de novo lipogenesis in HepG2 cells under the stimulation of TNF-α.
LncRNA MEG3 Regulated the Expression of Lipogenesis Via FOXO1 Signaling Pathway in HepG2 Cells
In addition, to further explore whether lncRNA MEG3 regulates lipogenesis via FOXO1 signaling pathway, we deployed siRNA targeted FOXO1 and Ad-MEG3 in the following section of this study.Results displayed overexpression of lncRNA MEG3 could reverse the cellular TG accumulation induced by TNF-α (Fig. 4A, D).Combining with inhibition of FOXO1 could not further reduce the intracellular TG content.In addition, the protein upregulation of ACC1, FAS, phosphorylated AMPK and FOXO1 by TNF-α could be reversed by the overexpression of lncRNA MEG3.There is no significant difference when combine with the inhibition of FOXO1 is compared to overexpression of lncRNA MEG3 alone (Fig. 4B).We also found that TNF-α could significantly increase the nuclear fluorescence staining of FOXO1, and increased lncRNA MEG3 or inhibition of FOXO1 could significantly reduce the nuclear FOXO1 fluorescence (Fig. 4C).Moreover, the increased intracellular lipid accumulation treated with TNF-α could be reversed by lncRNA MEG3 and lncRNA MEG3 combined with siFOXO1, respectively (Fig. 4D).The increase of lncRNA MEG3 also could reverse the nuclear FOXO1 induced by TNF-α (Fig. 4E).These results suggested that lncRNA MEG3 restrained de novo lipogenesis by inhibiting the expression and nucleus translocation of FOXO1.
Discussion
LncRNA is a kind of non-coding RNA with transcripts more than 200 bp in length, most of which are unclear in function.It has been indicated that lncRNA becomes involved in chromosome recombination, gene modification, epigenetic processes, as well as synthesis and regulation of protein [17][18][19][20][21].However, there are few studies reporting the role of lncRNA triglyceride accumulation in hepatocytes.Therefore, the study of the function and mechanism of lncRNA in lipid metabolism could not only provide insights into the regulation of the human genome, but also facilitate the elucidation of the pathogenesis of NAFLD.Studies showed CRE-binding protein (CREB) binds directly to cAMP response element (CRE) site and stimulates the promoter activity of lncRNA MEG3 [22].Hence, lncRNA MEG3, as the downstream target gene of cAMP, could be intimately associated with cellular energy metabolism.Our study suggested that an elevated level of lncRNA MEG3 could be observed under the stimulation of inflammation and fatty acids in HepG2, while suppression of lncRNA MEG3 could inhibit the expression of genes associated with lipid synthesis, and overexpression of lncRNA MEG3 could increase the levels of synthesis factors of fatty acids accordingly, such as ACC1 and FAS, which revealed that lncRNA MEG3 might play a role in the lipid metabolism pathways.
TNF-α is an important lipid metabolism regulator and plays avital role in lipogenesis.Many signaling pathways might be involved in TNFα-mediated lipid metabolism.Early studies have demonstrated that TNF-α could induce the rapid stimulation of hepatic FFA de novo synthesis in normal rats.In 3T3-L1 adipocytes, TNF-α promotes NF-κB pathway resulting in IR and increase of plasma FFA in rat liver [23,24].In addition, TNF-α could also regulate a wide range of lipogenesis enzyme activities and pathways such as ERK/ JNK to cAMP, which led to the de novo lipogenesis and the accumulation of lipids [8].Marathon running exercise induced an increase in plasma FFA, IL-6, and TNF-α, which afterwards induced plasma ANGPTL4 release, FFA-induced lipotoxicity and oxidative stress [25].Our results showed that under the stimulation of 10 ng/ml of TNF-α, the intracellular TG content of HepG2 cells significantly increased, accompanied by upregulation of lncRNA MEG3 expression and increased mRNA and protein expression of lipid metabolism genes such as FAS, FOXO1, and ACC1.To clarify whether the increase in lncRNA MEG3 is due to the inhibitory factor of TNF-α which caused abnormal lipid metabolism or promoted changes in lipid metabolism pathways, we inhibited the expression of lncRNA MEG3 on the basis of TNF-α stimulation.The results showed that after inhibiting lncRNA MEG3, the TG content of HepG2 cells further increased, and the mRNA and protein expression of ACC1, FAS, FOXO1 significantly increased.It indicated that lncRNA MEG3 can be considered as an inhibitory factor of lipid metabolism.
FOXO1 is widely distributed in adult tissues and organs and becomes involved in energy metabolism and oxidative stress.An elevated expression of lncRNA MEG3 could be detected in [16].Our study also suggested that TNF-α could result in an enhanced expression of lncRNA MEG3, an increase of FOXO1 translocation to the nucleus, an elevated level of ACC1, and increased lipid deposition in HepG2, while the overexpression of lncRNA MEG3 could reverse this process in part.It could be hypothesized that TNFα can induce FOXO1 translocation into the nucleus to serve as a transcription factor, and then upregulate the expression of lipid synthesis factor ACC1 to stimulate intracellular lipid synthesis.LncRNA MEG3 is a factor that can inhibit lipid synthesis, but its elevation under the stimulation of TNF-α is not sufficient to inhibit the lipid synthesis process.Only additionally increase lncRNA MEG3 can reverse the lipid synthesis process caused by TNF-α.There are several limitations in this study, we did not investigate the molecular mechanisms of lncRNA MEG3 and FOXO1.The intracellular glycometabolism was not explored either.The present study demonstrated that lncRNA MEG3 could become involved in lipid synthesis by regulating the expression and translocation to the nucleus of FOXO1, providing new insights into the molecular mechanism and control of NAFLD.
Fig. 1
Fig. 1 TNF-α induced the expression of lncRNA MEG3 and lipogenesis genes in HepG2 cells.A Oil Red O staining of HepG2 cells treated with 10 ng/ml TNF-α for 24 h (Scale bar = 50 μm).B The RNA expression of lncRNA MEG3 and the mRNA level of lipogenesis genes.*P < 0.05, **P < 0.01 vs blank.Data are mean ± SD, N = 3 independent experiments
Fig. 4
Fig. 4 LncRNA MEG3 regulated the expression of lipogenesis via FOXO1 signaling pathway in HepG2 cells.A Oil Red O staining of HepG2 cells transfected with adenovirus vector and/or siRNA targeted lncRNA MEG3 for 48 h (Scale bar=50 μm).B Western blot assay for FOXO1, ACC1, and FAS transfected lncRNA MEG3 adenovirus vector and/or siRNA targeted FOXO1 for 48 h in HepG2 cells.**P < 0.01 vs Ad-NC, **P < 0.01 vs Ad-NC, & P < 0.05 vs Ad-MEG3, && P < 0.01 vs TNF-α, &&& P < 0.001 vs TNF-α.Data are mean ± SD, N = 3 independent experiments.C Representative [28]livers of ob/ob mice and diet-induced obese mice Overexpression of lncRNA MEG3 in primary liver cells could increase the mRNA levels of FOXO1, phosphoenolpyruvate carboxykinase (PEPCK) and G6Pase, enhance gluconeogenesis, and suppress the glycogen synthesis induced by insulin, whereas the suppression of lncRNA MEG3 could reverse this process.It has been suggested that FOXO1 can induce the expression of microsomal triglyceride transfer protein (MTP) by binding to the promoter of MTP.MTP assembles very low density lipoprotein (VLDL) together with apoB to become involved in the secretion of TG in hepatocytes.Suppression of FOXO1 could down-regulate the expression MTP and VLDL[27].Local high levels of TNF-α could activate FOXO1 in the process of endochondral ossification in patients with fractures associated with diabetes, leading to an increased mRNA expression level of apoptosis gene and chondrocyte apoptosis[28].It implied that FOXO1 could become involved in hepatic glycolipid metabolism by affecting inflammatory reaction in many ways.Mammalian ACC1 catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, an intermediate in the de novo synthesis of fatty acids
|
2024-05-08T06:17:06.741Z
|
2024-05-07T00:00:00.000
|
{
"year": 2024,
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"oa_url": "https://link.springer.com/content/pdf/10.1007/s12013-024-01278-w.pdf",
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234030969
|
pes2o/s2orc
|
v3-fos-license
|
Metal-Loaded Mesoporous MCM-41 for the Catalytic Wet Peroxide Oxidation (CWPO) of Acetaminophen
: MCM-41 based catalysts (molar ratio Si/Al = 40) were prepared by a hydrothermal route, modified by ionic exchange with different metals (Cu, Cr, Fe and Zn) and finally calcined at 550 ◦ C. The catalysts were fully characterized by different techniques that confirmed the formation of oxides of the different metals on the surfaces of all materials. Low-angle X-ray diffraction (XRD) analyses showed that calcination resulted in the incorporation of metallic Zn, Fe and Cr in the framework of MCM-41, while in the case of Cu, thin layers of CuO were formed on the surface of MCM-41. The solids obtained were tested in the catalytic wet peroxide oxidation (CWPO) of acetaminophen at different temperatures (25–55 ◦ C). The activity followed the order: > Zn/MCM-41. The increase of the reaction temperature improved the performance and activity of Cr/MCM-41 and Fe/MCM-41 catalysts, which achieved complete conversion of acetaminophen in short reaction times (15 min in the case of Cr/MCM-41). Fe/MCM-41 and Cr/MCM-41 were submitted to long-term experiments, being the Fe/MCM-41 catalyst the most stable with a very low metal leaching. The leaching results were better than those previously reported in the literature, confirming the high stability of Fe/MCM-41 catalysts synthesized in this study. HCl solution. The samples were taken at different times in maximum 2 h and they were analyzed by high-performance liquid chromatography (HPCL) in order to obtain the adsorption capacities of catalysts. The results show that adsorption equilibrium was obtained after 1 h with very low adsorption capacities.
Introduction
In recent years, global concern about the presence of pharmaceutical compounds in water has significantly increased. A wide variety of pharmaceutical compounds, such as analgesics, anti-inflammatory, antibiotics, contrast or antiepileptic agents, has recently been detected in many water resources [1]. The pharmaceutical compounds found in the environment come mainly from the elimination of these molecules and their metabolites by humans or animals under therapeutic treatment and/or and inadequate treatment of manufacturing effluents [2]. Because of their stabilities and sometimes recalcitrant behavior, they are not completely removed/degraded by conventional wastewater treatment techniques [3][4][5]. Their concentrations in water, although depending on the specific site and compound, are generally low, in the range of micrograms to nanograms per liter [3][4][5], although in the case of wastewaters coming from manufacturing plant effluents they could The main objective of this work is the preparation of different mesoporous materials containing different transition metals (Cu, Cr, Fe and Zn) using the non-calcined form of MCM-41 in order to increase the metal loading of the final catalysts. The catalysts were tested in the CWPO of acetaminophen and the proper structure-performance relationships were detailed and discussed. Figure 1 represents the X-ray diffraction (XRD) patterns at low and large angles of MCM-41 and the different catalysts. The XRD pattern of the original MCM-41 sample shows the presence of an intense peak at a 2θ value close to 2.5 • , besides to lower intensity peaks at 2θ values around to 3.7 and 4.3 • , characteristic of the reflection of (100), (110) and (200) planes of this mesoporous solid aluminosilicate. The absence of peaks at higher angles is because MCM-41 consists of amorphous silica, and thus it has no crystallinity at the atomic level [36] but only a high pore ordering. The different catalysts show clear differences in the XRD patterns. Cu/MCM-41 catalyst retains a high intensity (100) peak, besides two lower intensity peaks (110) and (200) at 2θ ≈ 3.72 and 4.30 • , respectively. This confirms that this sample maintains the hexagonal structures of the MCM-41 with a high degree of structural ordering [37]. The XRD patterns of the rest of the catalysts, Cr/MCM-41, Fe/MCM-41 and Zn/MCM-41, show much lower intensity of (100) peak and are displaced to lower diffraction angles. This can be explained by the swelling of the hexagonal mesh due to the incorporation of the metal in the MCM-41 framework (see Table 1) in agreement with previous observations [30,38]. The modification of the mesh parameters confirms that the calcination at 550 • C leads to the incorporation of the metal in the inner of the MCM-41 framework. The incorporation of a metal into the framework of the MCM-41 causes an increase in the mesh parameter depending on the size of the radius of the metal introduced in its structure [30,39]. According to the calculated values of a 0 , it is shown that all the modified materials have higher values compared to the parent MCM-41, except Cu/MCM-41 catalyst, which has an almost similar value compared to the parent MCM-41. In the large angles, all the catalysts presented new phases corresponding to the oxides of the metals added in the ion exchange process on the surface of MCM-41. Hence, the peaks at 2θ values of 35.5, 38.7, 42.3, 48,7 and 52.4 • correspond to the (-111), (111), (200), (-202) and (211) reflection phases of CuO respectively [38,40] [43,44]. This confirms that the calcination does not only lead to the incorporation of the metal into the MCM-41 framework but also forms metal oxides phases on its surface. Cu/MCM-41 catalyst has the higher intensity peaks in the region 30-60 • probably due to the formation of higher crystalline CuO outside the MCM-41 framework. This result, besides the low significant modification of the a o parameter, suggests than in the case of Cu, most of the metal was not incorporated into the Al-MCM-41 framework but deposited on the surface in CuO form. In the case of the Zn exchange, no ZnO phase was observed for the Zn/MCM-41 sample, suggesting this metal was deposited in the form of well-dispersed species with small size [36]. The nitrogen adsorption-desorption isotherms at 77 K of the the pattern MCM-41 are represented in Figure S1. All isotherms a istic of mesoporous materials with also a significant presence of m These isotherms are characterized by three steps; the first step is ca pressures (P/P0 < 0.25) which are characteristic of monolayer adsor micropore walls. The second step takes place at relative pressure The ion metal exchanged with Cetyltrimethylammonium(CTA + ) in MCM-41 before calcination increases the incorporation inside the material [45][46][47] which means more metal oxide, the amount of the metal in each catalyst was quantified by X-ray fluorescence (XRF) being summarized in Table 1 The nitrogen adsorption-desorption isotherms at 77 K of the different catalysts and the pattern MCM-41 are represented in Figure S1. All isotherms are of type IV characteristic of mesoporous materials with also a significant presence of microporosity [29,30,48]. These isotherms are characterized by three steps; the first step is carried out at low relative pressures (P/P 0 < 0.25) which are characteristic of monolayer adsorption of nitrogen on the micropore walls. The second step takes place at relative pressures between 0.25 < P/P 0 < 0.4 which is characterized by a sharp increase in nitrogen adsorption. This adsorption behavior is due to capillary condensation within the uniform micropores. The third step starts from P/P 0 > 0.4 which corresponds to the gradual increase in volume, due to the multilayer adsorption on the outer surface of the mesoporous materials. Table 1 shows the textural properties of obtained materials. It can be seen that the exchange of MCM-41 with the different metals resulted in a reduction of the porosity of the resulting catalysts [29,30]. This is strongly related to the particle blocking of the internal porosity by the incorporation of the metallic species. The increase of the pore diameter compared to the parent material is due to the incorporation of the incorporation of the metals to the structure during the heat treatment. Fe/MCM-41 exhibited the highest pore diameter suggesting a significant incorporation of this metal into the framework of the MCM-41, while Cr/MCM-41 exhibited a slight modification (in agreement with the amount of metal quantified by XRF). The catalyst Cu/MCM-41 has the higher surface area compared to the other modified samples probably due to its low metal content (see Table 1). This result confirms that CuO was deposited in the wider mesopores of MCM-41, which is in agreement with previously published works [48].
Characterization of the Catalyst
The study of the thermal behavior of the materials obtained makes it possible to determine the mass loss of each sample with increasing temperature, but also provides information about the formation mechanism of metal-loaded-MCM-41. Figure S2 shows the thermogravimetric analysis (TGA) curves under nitrogen atmosphere (inert atmosphere to avoid weight gaining caused by the presence of oxygen in air atmosphere) of as-synthesized MCM-41 (parent material) and its counterparts modified by Cr, Fe, Zn and Cu, prior to calcination. All materials presented two masses losses, the first mass loss occurs between 25 and 120 • C and it is associated to the loss of physiosorbed water. The second mass loss takes place at temperatures between 120 and 700 • C and corresponds to different stages of degradation of the surfactant CTA + .
Infrared spectroscopy was used in order to determine qualitatively the different groups of the catalysts before and after calcination ( Figure S3). After calcination at 550 • C a wide band was formed at 3387 cm −1 and another band appears at 1635 cm −1 ( Figure S3b). These bands are due to the presence of the silanol groups and also to the vibrations of physiosorbed water. This also confirms that calcination not only removes the surfactant inside the pores of MCM-41, as indicates the disappearance of the bands at 2919 and 2848 cm −1 , but also leads to the formation of higher amounts of silanol groups compared to non-calcined materials. The bands at 1221, 1032 and 786 cm −1 for the non-calcined materials have been moved to 1238, 1048 and 794 cm −1 at the higher wavenumbers, respectively. This behavior is strongly linked to the incorporation of transition metals into the framework of MCM-41 due to the calcination treatment [49].
Ultraviolet-visible (UV-vis) spectroscopy was used in order to determine the coordination nature of the metallic species supported on the surface of MCM-41. As shown in Figure S4, the bare mesoporous silica only shows peaks at wavelengths in the 200-350 nm range corresponding to UV absorption of SiO 2 [50]. The introduction of Cu, Zn, Fe and Cr in MCM-41 results in significant changes in the corresponding UV-Vis spectra ( Figure 2). The catalyst Cu/MCM-41 displays two clear bands, an intense band at 219 nm and a wide band between 300-800 nm that are mainly due to the presence of Cu 2+ in form of CuO NPs [51,52]. Zn/MCM-41 catalyst is characterized by two bands, the first is located at 257 nm and the second and wide band located at 305 nm, which can be assigned to encapsulated ZnO NPs [53,54]. In general, the band below 220 nm confirms the incorporation of Zn into the framework of MCM-41 and is associated with the charge transfer transitions of the zinc species with the oxygen of the framework [55]. The UV spectrum of the catalyst Cr/MCM-41 is very similar to that obtained in the literature in which it clearly shows the existence of Cr 2 O 3 [56]. The catalyst Fe/MCM-41 presents a strong band at 215 nm associated with another band at 268 nm, which is assigned to the charge transfer between the Fe 3+ and O 2− atoms (Fe-O-Si) in the framework of MCM-41 [57]. In addition to these bands, a shoulder appeared around 330 and 500 nm, indicating the presence of either extra-framework iron or iron oxide particles [58]. The investigation of the electronic states of MCM-41 doped by different metals was carried out by X-ray photoelectron spectroscopy (XPS). Figure 3 represents the Cr, Cu, Zn and Fe 2p XPS signal of the correspondent catalysts. XPS spectra of Cr 2p showed the Cr 2p1/2 and Cr 2p3/2 peaks on Cr/MCM-41 were located at 586.3 and 576.7. eV, which are characteristic of Cr2O3 species [59,60]. The O1s XPS peak ( Figure S5 of the Supplementary DATA) has a peak at 532 eV, which corresponds to the oxygen of the Cr2O3 network [53] and also to the Si-O bond and explains a close interaction between oxygen and silicon [61]. The XPS spectra of Cu 2p of the catalyst Cu/MCM-41 have two Cu peaks, belonging respectively to Cu 2p3/2 at around 933 eV and Cu 2p1/2 at 953 eV, suggesting the presence of Cu 2+ species in form of CuO [62,63]. The Zn 2p peaks for the catalyst Zn/MCM-41 show the Zn 2p1/2 and Zn 2p3/2, contributions, with peaks at 1045.8 eV and 1022.6 eV ascribed to Zn 2+ species [64]. The value of Zn 2p1/2 -Zn 2p3/2 is approximately 23.12 eV corresponding to the typical splitting value of ZnO [65,66]. The Fe 2p XPS spectra of Fe/MCM-41 exhibited a strong peak at 711.0 eV with a lower intensity satellite peak at 719.2 eV assigned to Fe 2p3/2 signal. The weak satellite peak of around 719.52 eV is characteristic of Fe 3+ in Fe2O3 [67]. The binding energy of Fe 2p1/2 is around 724.84 eV associated with other satellite peak at 732.01 eV [68]. The peaks at around 711.01 eV and 724.84 eV, indicating Fe components were mainly in trivalent status in Fe-MCM-41 [69]. The XPS O1s peak of all the catalysts is very similar (see Figure S5). A first peak, between 533.0-533.2 eV, corresponds to the oxygen of SiO2 [70]. The other peak, located at around 532.0-530.0 eV, is attributed to T-OH (T = Si, Al or Metal), T-O-Si (T = Metal) and T-O (T = Metal) [70][71][72]. These peaks were almost masked by the intense and wide oxygen signal from SiO2 [73]. The investigation of the electronic states of MCM-41 doped by different metals was carried out by X-ray photoelectron spectroscopy (XPS). Figure 3 represents the Cr, Cu, Zn and Fe 2p XPS signal of the correspondent catalysts. XPS spectra of Cr 2p showed the Cr 2p 1/2 and Cr 2p 3/2 peaks on Cr/MCM-41 were located at 586.3 and 576.7 eV, which are characteristic of Cr 2 O 3 species [59,60]. The O1s XPS peak ( Figure S5 of the Supplementary DATA) has a peak at 532 eV, which corresponds to the oxygen of the Cr 2 O 3 network [53] and also to the Si-O bond and explains a close interaction between oxygen and silicon [61]. The XPS spectra of Cu 2p of the catalyst Cu/MCM-41 have two Cu peaks, belonging respectively to Cu 2p 3/2 at around 933 eV and Cu 2p 1/2 at 953 eV, suggesting the presence of Cu 2+ species in form of CuO [62,63]. The Zn 2p peaks for the catalyst Zn/MCM-41 show the Zn 2p 1/2 and Zn 2p 3/2 , contributions, with peaks at 1045.8 eV and 1022.6 eV ascribed to Zn 2+ species [64]. The value of Zn 2p 1/2 -Zn 2p 3/2 is approximately 23.12 eV corresponding to the typical splitting value of ZnO [65,66]. The Fe 2p XPS spectra of Fe/MCM-41 exhibited a strong peak at 711.0 eV with a lower intensity satellite peak at 719.2 eV assigned to Fe 2p 3/2 signal. The weak satellite peak of around 719.52 eV is characteristic of Fe 3+ in Fe 2 O 3 [67]. The binding energy of Fe 2p 1/2 is around 724.84 eV associated with other satellite peak at 732.01 eV [68]. The peaks at around 711.01 eV and 724.84 eV, indicating Fe components were mainly in trivalent status in Fe-MCM-41 [69]. The XPS O1s peak of all the catalysts is very similar (see Figure S5). A first peak, between 533.0-533.2 eV, corresponds to the oxygen of SiO 2 [70]. The other peak, located at around 532.0-530.0 eV, is attributed to T-OH (T = Si, Al or Metal), T-O-Si (T = Metal) and T-O (T = Metal) [70][71][72]. These peaks were almost masked by the intense and wide oxygen signal from SiO 2 [73]. Figure 4a,b represent the ACE concentration and H2O2 conversion upon reaction time when using the different catalysts. It should be remarked that the ACE adsorption (see Figure S6) was negligible in all th siloxy e cases. Despite the well-developed surface area of the MCM-41 sample, it shows an almost negligible adsorption capacity of acetaminophen. This is mainly due to the hydrophilic nature of MCM-41, and it is in agreement with previous studies [74]. All the tests were performed at pH = 3, which is generally accepted as the optimal pH to perform CWPO reactions. At lower pH values the formation of hydrated metal complexes results in a lower production of hydroxyl radicals. Furthermore, at very acidic conditions a higher number of protons is free in the medium to react with hydroxyl radicals, which decreases the overall efficiency of the process. On the other hand, pH > 4 favors the formation of metal oxyhydroxides on the catalyst's surface, which inhibits both the production of hydroxyl radicals and the regeneration of the metal ions. ACE conversion varies in the following sequence Cr/MCM-41 >> Fe/MCM-41 > Cu/MCM-41 >> Zn/MCM-41. The ACE conversion was completed in less than 60 min using Cr/MCM-41 while the Fe/MCM-41 catalyst needs four times higher reaction time to achieve complete ACE conversion. The low initial acetaminophen concentration (5 mg·L −1 ) makes difficult the identification and accurate quantification of the reaction intermediates. No additional aromatic compounds were observed in HPLC chromatograms. However, short-chain organic acids, such as, oxalic, acetic and formic acids, were detected using ionic chromatography. The difference between the ACE and H2O2 final conversions (at 4 h) when using Fe/MCM-41 catalyst should be ascribed to the presence of intermediate oxidation byproducts, suggesting an incomplete mineralization of ACE. In contrast, Zn/MCM-41 catalysts showed very little ACE conversion, hardly 10% after 240 min of reaction. This activity can be linked to several factors such as the nature of the metal oxides, their crystallinity, their metal particle sizes and/or their specific surface [75,76]. The Figure 4a,b represent the ACE concentration and H 2 O 2 conversion upon reaction time when using the different catalysts. It should be remarked that the ACE adsorption (see Figure S6) was negligible in all th siloxy e cases. Despite the well-developed surface area of the MCM-41 sample, it shows an almost negligible adsorption capacity of acetaminophen. This is mainly due to the hydrophilic nature of MCM-41, and it is in agreement with previous studies [74]. All the tests were performed at pH = 3, which is generally accepted as the optimal pH to perform CWPO reactions. At lower pH values the formation of hydrated metal complexes results in a lower production of hydroxyl radicals. Furthermore, at very acidic conditions a higher number of protons is free in the medium to react with hydroxyl radicals, which decreases the overall efficiency of the process. On the other hand, pH > 4 favors the formation of metal oxyhydroxides on the catalyst's surface, which inhibits both the production of hydroxyl radicals and the regeneration of the metal ions. ACE conversion varies in the following sequence Cr/MCM-41 >> Fe/MCM-41 > Cu/MCM-41 >> Zn/MCM-41. The ACE conversion was completed in less than 60 min using Cr/MCM-41 while the Fe/MCM-41 catalyst needs four times higher reaction time to achieve complete ACE conversion. The low initial acetaminophen concentration (5 mg·L −1 ) makes difficult the identification and accurate quantification of the reaction intermediates. No additional aromatic compounds were observed in HPLC chromatograms. However, short-chain organic acids, such as, oxalic, acetic and formic acids, were detected using ionic chromatography. The difference between the ACE and H 2 O 2 final conversions (at 4 h) when using Fe/MCM-41 catalyst should be ascribed to the presence of intermediate oxidation byproducts, suggesting an incomplete mineralization of ACE. In contrast, Zn/MCM-41 catalysts showed very little ACE conversion, hardly 10% after 240 min of reaction. This activity can be linked to several factors such as the nature of the metal oxides, their crystallinity, their metal particle sizes and/or their specific surface [75,76]. The results illustrated in Figure 4 are not in agreement with the trend observed for the microporosity of the samples, suggesting that the catalytic activity of the catalysts is not strongly affected by the surface, as previously reported in other studies [20]. 021, 11, x FOR PEER REVIEW results illustrated in Figure 4 are not in agreement with the trend observed for croporosity of the samples, suggesting that the catalytic activity of the catalysts strongly affected by the surface, as previously reported in other studies [20]. (Table 1). Moreover, these samples have not been able to d pose H2O2, which justifies their lower activity in CWPO reaction. Fe/MCM-Cr/MCM-41 catalysts have higher metal contents. Several studies have shown t various oxide metals including magnetite [34,77,78] are effective heterogeneous catalysts which are in agreement with our results.
Catalytic Test
The best catalysts, Fe/MCM-41 and Cr/MCM-41, were used in the rest of the Figure 5 represents the evolution of ACE concentration and H2O2 conversion with r time when using different loads of Fe/MCM-41 and Cr/MCM-41 catalysts. The m catalyst was varied between 0.5 and 2 g/L while keeping the temperature (25 °C) a ≈ 3 of solution constant. The load of catalyst plays a very important role on the con of ACE that increases rapidly with increasing mass of the catalyst. A total conver ACE was obtained with Cr/MCM-41 catalyst at reaction time lower than 30 min using catalyst loads of 1 or 2 g/L. This is a consequence of the presence of more acti when using higher loads of catalyst. Another explanation also linked to the size chromium oxides which probably have a smaller size compared to the iron Cr/MCM-41 has a larger surface area than Fe/MCM-41, which may be due to the p of large particles of iron oxide inside the pores subsequently leading to a decrease number of sites, and thus a tortuous diffusion of reagents. Figure 5c,d shows H2O2 conversion using both catalysts. In the case of Cr/M higher conversions of H2O2 lead to higher and faster degradation of ACE. It sho noted that the reaction between H2O2 and ACE without catalyst does not lead to an radation which has been confirmed by Carrasco-Díaz et al. [20]. It has been show the total degradation of ACE requires approximately 45-55% of H2O2 conversion The low activity of Cu/MCM-41 and Zn/MCM-41 catalysts was probably linked to their lower metal content (Table 1). Moreover, these samples have not been able to decompose H 2 O 2 , which justifies their lower activity in CWPO reaction. Fe/MCM-41 and Cr/MCM-41 catalysts have higher metal contents. Several studies have shown that the various oxide metals including magnetite [34,77,78] are effective heterogeneous Fenton catalysts which are in agreement with our results.
The best catalysts, Fe/MCM-41 and Cr/MCM-41, were used in the rest of the work. Figure 5 represents the evolution of ACE concentration and H 2 O 2 conversion with reaction time when using different loads of Fe/MCM-41 and Cr/MCM-41 catalysts. The mass of catalyst was varied between 0.5 and 2 g/L while keeping the temperature (25 • C) and pH ≈ 3 of solution constant. The load of catalyst plays a very important role on the conversion of ACE that increases rapidly with increasing mass of the catalyst. A total conversion of ACE was obtained with Cr/MCM-41 catalyst at reaction time lower than 30 min when using catalyst loads of 1 or 2 g/L. This is a consequence of the presence of more active sites when using higher loads of catalyst. Another explanation also linked to the size of the chromium oxides which probably have a smaller size compared to the iron oxides, Cr/MCM-41 has a larger surface area than Fe/MCM-41, which may be due to the presence of large particles of iron oxide inside the pores subsequently leading to a decrease in the number of sites, and thus a tortuous diffusion of reagents. Figure 5c, [79], which are considered as Fenton-active sites for the degradation of ACE. The H 2 O 2 decomposition Catalysts 2021, 11, 219 9 of 17 mechanism has already been described in the literature confirming the intervention of these three species in the degradation of pollutants [78,79]. Higher reaction temperatures favor the reaction rate of ACE and the H2O2 decomposition. The decomposition of H2O2 increases significantly when the temperature increases from 25 to 55 °C, which can be explained by a higher conversion of ACE, as previously reported with other pollutants as phenol [80]. Concerning the Cr/MCM-41 catalyst, only a slight increase in the ACE removal was observed (Figure 6b) although the conversion of H2O2 was higher at higher reaction temperature (Figure 6d) due to its higher catalytic activity, with room temperature enough to achieve a total degradation of ACE. However, Fe/MCM-41 exhibited a lower catalytic activity and a higher temperature of 55 °C was necessary to reach similar results than those obtained with Cr/MCM-41. When using Fe/MCM-41 catalysts the reaction rate increases with the reaction temperature. Using the Arrhenius equation (Equation (1)) the activation energy for the acetaminophen degradation is 86 kJ·mol −1 (see Figure S7). In the case of Cr/MCM-41 the degradation is very fast at the reaction temperatures analyzed and no accurate value of the activation energy can be obtained. The Turnover Frequency (TOF) (Equation (2)) has been calculated as the mols of acetaminophen converted per mol of metal in the cata- increases significantly when the temperature increases from 25 to 55 • C, which can be explained by a higher conversion of ACE, as previously reported with other pollutants as phenol [80]. Concerning the Cr/MCM-41 catalyst, only a slight increase in the ACE removal was observed (Figure 6b) although the conversion of H 2 O 2 was higher at higher reaction temperature (Figure 6d) due to its higher catalytic activity, with room temperature enough to achieve a total degradation of ACE. However, Fe/MCM-41 exhibited a lower catalytic activity and a higher temperature of 55 • C was necessary to reach similar results than those obtained with Cr/MCM-41. When using Fe/MCM-41 catalysts the reaction rate increases with the reaction temperature. Using the Arrhenius equation (Equation (1)) the activation energy for the acetaminophen degradation is 86 kJ·mol −1 (see Figure S7). In the case of Cr/MCM-41 the degradation is very fast at the reaction temperatures analyzed and no accurate value of the activation energy can be obtained. The Turnover Frequency (TOF) (Equation (2) The stability of the catalysts was tested in continuous tests for seven days. Figure 7 shows the evolution of ACE concentration and H 2 O 2 conversion in those long-run tests. The two catalysts are very stable under the reaction conditions for seven consecutive days. The catalytic activity of each material was stable without losing their performance. The conversion of H 2 O 2 was constant during the seven days of treatment, which results in a total degradation of ACE. To completely assess that the catalyst does not suffer significant modification after long reaction times, additional characterization of the used catalyst is necessary. However, the results shown here allow us to state that the catalyst is capable to maintain the high acetaminophen conversion at those extremely long reaction times. Metal leaching has been studied in order to confirm the possible contribution of homogeneous Fenton to the degradation of ACE. These results confirm the stability of the Cr/MCM-41 and Fe/MCM-41 catalysts prepared by a simple process which consists in a partial exchange between metal and CTA + followed by calcination. It must be replaced by the next one where "H2O2" has been replaced by "H2O2" with subscripts in Y axys of figure 7b and in the legends of figures 7a and 7b. The mechanism of the oxidation of acetaminophen has been extensively studied in the literature (Figure 8). In the case of CWPO of acetaminophen, the reaction pathway begins with the formation of benzoquinone, which result upon further oxidation in the generation of benzaldehyde and benzoic acid [81][82][83]. These by-products further decompose in smaller aliphatic organic acids such as maleic, oxalic, acetic and formic acids, which are non-toxic products [82,83]. The last step involves the complete mineralization to CO 2 , H 2 O and NO 3 − . The mechanism of the oxidation of acetaminophen has been extensively studied in the literature (Figure 8). In the case of CWPO of acetaminophen, the reaction pathway begins with the formation of benzoquinone, which result upon further oxidation in the generation of benzaldehyde and benzoic acid [81][82][83]. These by-products further decompose in smaller aliphatic organic acids such as maleic, oxalic, acetic and formic acids, which are non-toxic products [82,83]. The last step involves the complete mineralization to CO2, H2O and NO3 -.
Catalyst Preparation
Mesoporous MCM-41 support was synthesized by a hydrothermal method as reported elsewhere [29,30,84]. The molar composition used in the formulation was Si/Al = 40, 0.12 CTAB, 0.25 NaOH, 1.5 EtOH and 100 H 2 O. Briefly, an aqueous solution with CTABr surfactant and ethanol was prepared at a temperature of 35 • C. After homogenization of the mixture, TEOS was added dropwise at the same time with a solution containing distilled water, sodium aluminate and NaOH in the solution containing the surfactant. The reaction mixture was placed under constant stirring for 3 h at 35 • C, after the product was hydrothermally treated at 100 • C for two days. The final product was filtered, washed several times with distilled water and then dried at 60 • C. Different transition metals (Cu, Cr, Fe and Zn) were exchanged on the MCM surface in order to obtain the CWPO catalysts. About 2 g of as-synthesized MCM-41 was dispersed in 100 mL of 0.1 M solutions of the different metal salts and stirred for 2 h at room temperature. Then the mixture was filtered, washed several times with distilled water and dried overnight at 60 • C. For the release of the porosity of the obtained materials through the removal of the organic template and the condensation of the silanol groups, the product was calcined for 6 h at 550 • C in an air atmosphere (to improve the formation of metal oxides in air oxygen). The catalysts were named as M/MCM-41 being M the metal exchanged (Cr, Fe, Cu and Zn) [49,85].
Catalysts Characterization
XRD powder diffraction patterns of calcined samples were obtained with a Bruker AXS D-8 diffractometer using Cu-Kα radiation at 2θ = 1-60 • . Nitrogen adsorption-desorption isotherms were obtained at −196 • C using ASAP 2020 Micromeritics apparatus. The samples were previously outgassed at 100 • C for 10 h prior to the sorption measurements. The thermal properties and mass losses were followed by means of a thermobalance (TGA DISCOVERY SDT-650) under nitrogen flow. The functional groups of the metal-modified MCM-41 before and after calcination were analyzed by Fourier transform infrared (FTIR) spectra in the range of 500-4000 cm −1 using a BRUKER ALPHA Platinum-ATR instrument. The metal coordination state with the mesoporous silica was analyzed by ultraviolet visible (UV-vis) absorbance spectra using SHIMADZU UV-2700 spectrometer. The amount of metal in each sample was determined by X-ray fluorescence using a XEPOS spectrometer (Spectro Ametek). The XPS measurements were carried out on a Kratos Axis Ultra using AlKα (1486.6 eV) radiation. High-resolution spectra were acquired at 20 eV pass energy with energy resolution of 0.9 eV, the C1s line of 284.5 eV was used as a reference to correct the binding energies for charge energy shift.
Adsorption Tests
The adsorption tests were performed in batch reactor at 25, 40 and 55 • C. Briefly, 1 g of catalyst was added to a 5 mg/L of ACE solution (1000 mL) at a fixed pH close to 3 using a 1 M HCl solution. The samples were taken at different times in maximum 2 h and they were analyzed by high-performance liquid chromatography (HPCL) in order to obtain the adsorption capacities of catalysts. The results show that adsorption equilibrium was obtained after 1 h with very low adsorption capacities.
Catalytic Tests
CWPO tests were carried out in batch reactor at controlled temperature. A catalyst concentration of 1 g/L was used while the ACE initial concentration was fixed at 5 mg/L. The pH was adjusted to 3 using a 1 M HCl solution. The mixture was stirred for 1 h in order to achieve adsorption equilibrium and then the stochiometric amount of H 2 O 2 was added to the solution to initiate the reaction. The conversion of ACE was followed by HPLC (Shimadzu Prominence-I LC-2030C with a diode array detector) at a wavelength of 246 nm using a reverse phase C18 column. A mixture of acetonitrile/acetic acid 0.1% v/v was used as mobile phase (0.7 mL/min). The effects of the metal active phase, catalyst mass (0.5, 1.0 and 2.0 g/L), and reaction temperature (25, 40 and 55 • C) were analyzed.
The stability of catalysts was investigated using a continuous reaction test for one week. An ACE solution was continuously fed to the reactor (0.243 mL/min) using the best catalysts (100 mg) at a reaction temperature of 25 • C. The same volume of solution was extracted and analyzed to follow the ACE conversion with time on stream. The H 2 O 2 concentration in the reaction medium was quantified by colorimetric methods with a Cary 60 UV-Vis (Agilent Technologies) spectrophotometer, using the titanium oxisulphate method [86].
Conclusions
The mesoporous material MCM-41 with a Si/Al ratio = 40 was prepared by a hydrothermal method. The as-synthesized MCM-41 was ionic exchanged with several transition metals such as Fe, Cr, Cu and Zn and then calcined at 550 • C. The results (XRD, XPS, UV-vis, FTIR) showed that Fe, Cr, Cu and Zn were incorporated into the MCM-41 framework, which was confirmed by the inflation of the mesh parameter (a 0 of the modified materials was higher compared to the parent material), and a part of the metals were transformed in their corresponding oxides form (Fe 2 O 3 , ZnO, CuO and Cr 2 O 3 ). The nitrogen adsorption at 77K showed that the surface area was reduced due to the pores blockage by metal oxides. However, different behavior was observed for the catalyst Cu/MCM-41 in which the formation of CuO thin layers slightly decreased the surface area.
Application of these solids in the oxidation of ACE showed that the ACE conversion varies in the following sequence Cr/MCM-41 > Fe/MCM-41 > Cu/MCM-41 > Zn/MCM-41. The effect of the initial concentration of ACE, reaction temperature and the catalyst mass were studied in which the Cr/MCM-41 and Fe/MCM-41 catalysts were judged as the best catalysts for this kind of reaction. The use of both these catalysts in long-time tests showed that ACE conversion was higher and stable, which confirms the high stability of the synthesized catalysts.
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2021-05-10T00:02:48.355Z
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2021-02-07T00:00:00.000
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13973155
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pes2o/s2orc
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v3-fos-license
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Aptamer-Drug Conjugates of Active Metabolites of Nucleoside Analogs and Cytotoxic Agents Inhibit Pancreatic Tumor Cell Growth
Aptamer-drug conjugates (ApDCs) have the potential to improve the therapeutic index of traditional chemotherapeutic agents due to their ability to deliver cytotoxic drugs specifically to cancer cells while sparing normal cells. This study reports on the conjugation of cytotoxic drugs to an aptamer previously described by our group, the pancreatic cancer RNA aptamer P19. To this end, P19 was incorporated with gemcitabine and 5-fluorouracil (5-FU), or conjugated to monomethyl auristatin E (MMAE) and derivative of maytansine 1 (DM1). The ApDCs P19-dFdCMP and P19-5FdUMP were shown to induce the phosphorylation of histone H2AX on Ser139 (γ-H2AX) and significantly inhibited cell proliferation by 51%–53% in PANC-1 and by 54%–34% in the gemcitabine-resistant pancreatic cancer cell line AsPC-1 (p ≤ 0.0001). P19-MMAE and P19-DM1 caused mitotic G2/M phase arrest and inhibited cell proliferation by up to 56% in a dose-dependent manner when compared to the control group (p ≤ 0.001). In addition, the cytotoxicity of P19-MMAE and P19-DM1 in normal cells and the control human breast cancer cell line MCF7 was minimal. These results suggest that this approach may be useful in decreasing cytotoxic side effects in non-tumoral tissue.
INTRODUCTION
Pancreatic ductal adenocarcinoma (PDAC) is the 12 th most common cancer worldwide and the 7 th most common cause of cancer-related death. 1 In contrast to other cancer types, the mortality rate of PDAC is increasing, and it has been predicted that PDAC will become the second leading cause of cancer-related mortality by 2020. 2 Despite efforts to improve the treatment and outcome of patients with PDAC, limited progress has been made. 2,3 The survival rate remains less than 5% at 5 years taking into account all stages of the disease. 4 Pancreatic resection remains the only curative option for patients with localized tumors; however, only 15%-20% of patients have resectable disease without metastatic spread at the time of presentation. 5 For patients with locally advanced or metastatic disease, gemcitabine, a nucleoside analog with a structure similar to cytarabine, has been the standard treatment for more than 10 years. 6 Gemcitabine, however, only increases the 1-year survival rate from 16% to 19%. Despite the introduction of new chemotherapy regimens, the median survival of PDAC patients remains less than 12 months. 6,7 Nevertheless, as systemic chemotherapy provides significant survival benefits and improves the quality of life of patients in comparison with best supportive care alone, it is still recommended by current guidelines. [8][9][10] One of the current limitations of aggressive chemotherapy in PDAC is treatment-related toxicity.
Although combination therapy has been shown to be associated with higher response rates compared to single-agent regimens, 7 multiagent regimens are associated with more severe cytotoxic side effects, such as neutropenia, sensory neuropathy, vomiting, and diarrhea, all which have a negative impact on quality of life. 7 In view of these problems, improvements in selective drug delivery are imperative for newer cytotoxic drugs.
The relatively new agents monomethyl auristatin E (MMAE), a synthetic analog of the natural product dolastatin, 11 and the maytansinoid DM1, a derivative of maytansine, have both been investigated in several preclinical studies. 12,13 Both MMAE and DM1 are highly potent antimitotic drugs that bind to microtubules. 14 Due to their high toxicity, however, both MMAE and DM1 cannot be used as cytotoxic drugs in their own right. Instead, both MMAE and DM1 have been used in the construction of antibody-drug conjugates (ADCs). ADCs are composed of a cytotoxic drug linked to a monoclonal antibody (mAb), which targets a tumor-associated antigen, thus delivering the cytotoxic drug directly to the cancer cell and so reducing toxicity in non-tumoral cells. 15 ADCs can, however, induce an immune response driven by different parts of the conjugates thus compromising their safety and efficacy. 16 This study aimed to improve current systemic chemotherapy for PDAC by constructing aptamer-drug conjugates (ApDCs). Aptamers are small structured single-stranded RNA chains capable of recognizing cell-specific receptor targets.
Aptamer-Drug Conjugation: Nucleoside-Analog-Linked ApDCs
The construction of ApDCs involved conjugating the pancreatic-cancer-specific RNA aptamer P19 17 to well-known cancer drugs. These included gemcitabine triphosphate (dFdCTP) and 5-fluorouracil (5-FU) triphosphate (5FdUTP). The active metabolites of the drugs, dFdCMP and 5FdUMP ( Figure 1A), were incorporated enzymatically into the P19 RNA aptamer. The structure of P19 conjugated with dFdCMP and 5FdUMP is depicted in Figure 1B, where the inclusion of these drugs only marginally increased the molecular weight of P19-dFdCMP (to 1,862 g/mol) relative to P19 due to excess fluorine residues, while P19-5FdUMP remained unchanged ( Figure S1A). The sequences of P19-dFdCMP and P19-5FdUMP are shown in Table S1.
To determine the binding affinity of the ApDCs, we performed fluorescent binding assays with PANC-1 human pancreatic cancer cells to calculate the apparent dissociation constant (K D ). Treatment with P19 aptamer alone in PANC-1 was previously measured at a K D of 13.07 nM. 17 Both P19-dFdCMP (K D = 7.76 nM) and P19-5FdUMP (K D = 6.9 nM) ( Figure 1C, left and right panels) showed slightly increased binding affinity when compared to P19 alone. To determine whether the nucleoside analog ApDC was effectively internalized, we labeled P19-dFdCMP and P19-5FdUMP with Cy3 fluorescent dye and incubated them with our panel of cell types. P19-dFdCMP and P19-5FdUMP showed specific internalization into pancreatic cancer cell line PANC-l cells, while breast cancer cells (MCF7) did not show internalization ( Figure 1D). From this, we concluded that both P19-dFdCMP and P19-5FdUMP maintained the same epitope specificity to PDAC cells as our original P19 aptamer. 17 P19-dFdCMP and P19-5FdUMP Induce DNA Double-Strand Breaks and Inhibit Cell Proliferation in Pancreatic Cancer Cells The most well-known mechanism of action for nucleoside analogs is inhibition of DNA synthesis and DNA damage. 18 To characterize the level of DNA damage induced by P19-dFdCMP and P19-5FdUMP in PANC-1 cells, we used an indirect immunofluorescence assay (IFA) to measure the appearance of the phosphorylated form of histone H2AX Ser 139 (g-H2AX), a specific biomarker of DNA double-strand breaks (DSBs). 19 P19-dFdCMP and P19-5FdUMP significantly increased the incidence of nuclear DSBs, as indicated by increased g-H2AX in ApDC-treated cells relative to negative control or P19-only-treated cells (Figure 2A). Quantification of g-H2AX relative to control demonstrated a significant 16-fold increase in DSBs for P19-dFdCMP and a 12-fold increase for P19-5FdUMP (p % 0.0001, one-way ANOVA) ( Figure 2B; Table S2).
Aptamer-Drug Conjugation: Cytotoxic-Drug-Linked ApDCs
We constructed antimitotic ApDCs by chemical conjugation of a truncated form of the P19 aptamer to MMAE and DM1. First, in order to facilitate increased binding affinity and allow large-scale chemical synthesis, the full length of P19 was truncated to a smaller 27-mer unit (tP19) ( Table S1). The binding affinity of tP19 was confirmed at a K D of 8.7 nM, ( Figure 3A), and this was also comparable to full-length P19. Next, to prevent structural hindrance of tP19, its 5 0 end was attached to either MMAE (Figure 3B) or DM1 ( Figure 3C) via a sticky sequence (SE). After chemical conjugation, each compound was high performance liquid chromatography (HPLC) purified. Then, to assemble the ApDCs, tP19-SE linked to MMAE-SE or DM1-SE was annealed in folding buffer to create functional tP19-MMAE and tP19-DM1 complexes ( Figure S1B).
To confirm whether this newly truncated tP19 complex maintained the same epitope specificity to PDAC cells as our previously reported full-length P19, 17 a cell internalization assay was performed using PANC-1 and a panel of non-pancreatic cancer cells, including Huh7, HepG2, and MCF7. tP19 maintained binding specificity and internalization to PANC-1 cells only ( Figure 3D). The ApDCs linked to the truncated aptamers were also screened for target cell specificity. Both tP19-MMAE and tP19-DM1 were tested against PANC-1 and MCF7 cells, but only PANC-1 cells showed internalization of tP19-MMAE ( Figure 3E) and tP19-DM1 ( Figure 3F). Since MMAE and DM1 are strong anti-mitotic drugs inhibiting microtubule polymerization, to characterize cell-cycle perturbation induced by tP19-MMAE and tP19-DM1, we used flow cytometry to measure the effect on cell-cycle progression. PANC-1 cells were treated with 500 nM tP19, tP19-MMAE, or tP19-DM1 for 72 hr. tP19 treatment did not disrupt any phase of the cell cycle ( Figures 4A and 4B). tP19-MMAE and tp19-DM1 treatment caused a significant increase in cells entering the G2/M phase ( Figures 4A and 4B). To confirm the antiproliferative effects of both ApDCs and their cell selectivity, an MTT cell proliferation assay was carried out in PANC-1, BJ, MCF7, U251-MG, and HCT116 cells treated with tP19-MMAE ( Figure 4C) and tP19-DM1 ( Figure 4D). tP19-MMAE and tP19-DM1, while showing no response in BJ and MCF7 cells, induced significant inhibition in proliferation of PANC-1 at 72 hr dose dependently. Since PANC-1 represents cells of an undifferentiated and aggressive metastatic phenotype, we tested both ApDCs on other cells bearing a similar phenotypic trait. U251-MG glioblastoma cells and HCT116 colorectal carcinoma cells were treated with tP19-MMAE and tP19-DM1. Both treatments cause significant inhibition in cell proliferation (Figures 4C and 4D). Interestingly, our original full-length P19 aptamer also showed suppression in proliferation of U251-MG and HCT116 similar to that observed with tP19 (Figure S2). When compared side by side, it was noted that both tP19-MMAE and tP19-DM1 caused stronger suppression in proliferation of U251-MG and HCT116 when compared to PANC-1 cells ( Figures 4C and 4D). This observation suggests that P19/tP19 may also be a relevant vehicle for cells bearing an aggressive metastatic phenotype; however, this is an area that has yet to be investigated.
DISCUSSION
To improve drug-specific delivery to cancer cells, aptamers have been at the forefront of development in nanomedicine for targeted delivery of chemotherapeutic agents. These aptamers offer significant advantages over antibodies, including better stability, lower toxicity, lower immunogenicity, and a better safety profile. 20,21 RNA aptamers can be synthesized chemically or enzymatically, thus making it possible to incorporate nucleoside analogs for anti-cancer drugs into aptamers, such as gemcitabine and 5-FU.
In this study, ApDCs consisting of chimeric aptamers conjugated to the cytotoxic active metabolites of the nucleoside analogs gemcitabine and 5-FU and to the cytotoxins MMAE and DM1 were used for targeted delivery to PANC-1, a cell line representing PDAC (Figure 5). Successful construction and biological use of these ApDCs were demonstrated using a range of assays, including fluorescent internalization imaging and a MTT cell proliferation assay. In this setting, we demonstrated specific internalization of ApDCs into cancer cells bearing an aggressive metastatic phenotype, directly delivering active anti-metabolites and cytotoxins, and inducing selective and potent proliferative activity while sparing normal cells. To incorporate the nucleoside analogs gemcitabine and 5-FU in our previously reported PDAC-specific aptamer, P19, three-phosphate fluoropyrimidines of dFdCTP and 5FdUTP were used. During the conjugation process, two phosphates were removed and the final outputs were active monophosphate forms of the fluoropyrimidines (dFdCMP and 5FdUMP). Once internalized, dFdCMP and 5FdUMP would subsequently be phosphorylated to diphosphate or triphosphate fluorodeoxycytidine (dFdCDP and dFdCTP). 18 Since fluorines are electronegativity charged, they strongly bind to other molecules and prevent DNA chain elongation and subsequently DNA synthesis. 22 Their effects have been principally ascribed to misincorporation of fluoronucleotides into DNA, RNA and inhibition of thymidylate synthase (TS). [23][24][25] From our study, we confirmed that ApDCs were internalized into cells ( Figures 1D and 3D-3F) and induced DNA damage (Figures 2A and 2B).
In vitro cell proliferation assays demonstrated significant cell growth inhibition in PANC-1 and even in the gemcitabine-resistant cell AsPC-1 ( Figure 2C). Since low cell penetrance is the most likely paradigm for gemcitabine resistance, our data suggest that ApDCs may help ablate chemoresistance in pancreatic cancer by allowing internalization and delivery of the drug to otherwise inaccessible cells. 26 MMAE and DM1 are potent anti-mitotic agents. However, because of their high toxicity, they cannot be administered on their own. Most experimental evidence for the efficacy and safety of MMAE is derived from in vivo studies and xenograft models with ADCs. 27 Up to now, there are few published studies regarding the use of MMAE with ADCs in pancreatic cancer. In a phase 1 study of patients with platinum-resistant ovarian cancer or unresectable PDAC, MMAE conjugated with a monoclonal antibody against mesothelin had antitumor activity in both types of cancer, with acceptable dose-limiting toxicity. 28 In another phase 1 study of patients with gastric or pancreatic cancer, MMAE targeting SLC44A4 was generally well tolerated but had limited antitumor activity. 29 Another promising cytotoxic drug is DM1, which is a potent microtubule-targeted compound, but in clinical trials, it showed lack of tumor specificity and unacceptable systemic toxicity. When combined with ADC, DM1 inhibited growth of grafted PDAC tumor nodules in preclinical models. 13 However, ADCs have potential pitfalls that can affect their application in clinical practice, including instability, poor pharmacokinetic properties, and immunogenicity. 30 In the field of targeted cancer therapy, ApDCs and ADCs are used similarly. However, ApDCs display different advantages over ADCs, as they do not aggregate, are more stable, have less toxicity, have greater specificity, and are easier and more economical to produce. [31][32][33][34] We previously demonstrated that aptamers could be used as potential drug-delivery vehicles with customized specificity to cell-surface receptors followed by efficient internalization to these cells. 17 In this study, an ApDC was constructed to deliver chemotherapeutic agents specifically to PDAC cells. To construct ApDCs with MMAE and DM1, our previously reported P19 was truncated in order to increase binding affinity and allow large-scale chemical synthesis. This modified aptamer was termed tP19. An in vitro cell proliferation (MTT) assay demonstrated that tP19-MMAE and tP19-DM1 significantly inhibited growth in PANC-1 cells. We also observed cross anti-proliferative effects of tP19-MMAE and tP19-DM1 in the undifferentiated and highly proliferative U251-MG, glioblastoma (GBM) cell lines and HCT116, colorectal carcinoma cell lines. Interestingly, our original full-length P19 aptamer also showed cross-reactivity, with these two cells bearing an aggressive tumorigenic phenotype ( Figure S2). This observation implies that the P19 aptamer or the truncated tP19 aptamers might be candidate vehicles to deliver chemotherapeutic agents to cells with an aggressive metastatic profile, such as glioblastomas and colon cancer cells.
As the technology to synthesize these oligonucleotides becomes more amenable to large-scale production under good manufacturing practice (GMP) conditions, ApDCs present as an attractive therapeutic moiety for safer cell-specific delivery of anti-cancer drugs.
Cell Lines
The following cell lines were purchased from the American Type RNA was labeled with Cy3 fluorescent dye using the Cy3 Silencer siRNA labeling kit (Thermo Fisher Scientific). Cy3-labeled aptamers were added to the cells at 200 nM or 500 nM and incubated for 2 hr. Live-cell confocal imaging was performed with a Zeiss LSM 510 Meta inverted two-photon confocal microscope system using a C-Apo 40Â/1.2 numerical aperture (NA) water-immersion objective and AIM 4.2 software (Carl Zeiss).
Flow-Cytometry-Based Binding Assays
To determine the K D of truncated P19 interactions with PANC-1 cells, aptamer binding was assessed by flow cytometry. PANC-1 cells were detached using Accutase (Sigma-Aldrich), washed with PBS, and suspended in binding buffer (PBS solution [DPBS without Ca 2+ and Mg 2+ , Corning] and 5 mM MgCl 2 ). Next, Cy3-labeled aptamers were added and incubated with PANC-1 cells for 30 min at room temperature. Cells were washed with binding buffer and immediately analyzed by Fortessa flow cytometry (BD Biosciences). DAPI (1 mg/mL) was used to identify and exclude dead cells. Data were analyzed with FlowJo software. The mean fluorescence intensity (MFI) was calculated for each aptamer concentration and for the unselected library controls. Control values were considered to be background fluorescence and were subtracted from the aptamer values, as previously described by Sefah et al. 35 K D was calculated using a onesite binding model. Non-linear regression was performed using GraphPad Prism (GraphPad Software).
MMAE and DM1 Aptamer Conjugation with a "Sticky Bridge Sequence"
MMAE and DM1 were chemically attached to the 5 0 end of a sticky bridge sequence to maintain aptamer structure. The same concentration of tP19-SE and MMAE-SE or DM1-SE was annealed in binding buffer at 95 C for 5 min and slowly cooled down and incubated at 37 C for 20 min more to make the ApDCs.
g-H2AX Evaluation
To investigate whether 5FdUMP and dFdCMP induced doublestrand breaks in nuclear DNA, g-H2AX evaluation was performed by IFA. 5 Â 10 4 PANC-1 cells were seeded in four-chamber slides and grown in appropriate media for 24 hr. P19, P19-5FdUMP, and P19-dFdCMP were added to cells at 500 nM and incubated for 48 hr. The cells were fixed in 3% paraformaldehyde solution, permeabilized in 0.1% Triton X-100, and stained with anti-g-H2AX (Cell Signaling, 1:2,000), followed by Alexa-488-labeled secondary antibody. Nuclear dots, indicative of nuclear DSBs, were counted using Image-Pro software (Media Cybernetics). The images were analyzed using Image Pro Premier, where the nuclear specks were counted using smart segmentation under count/size. The setting of smart segmentation was saved and then applied to subsequent images so that all images were analyzed in an identical manner. The representative images were depicted in Figure S3.
Cell-Cycle Analysis
For cell-cycle evaluation, 5 Â 10 5 PANC-1 cells were treated with tP19, tP19-MMAE, or tP19-DM1 at 500 nM. After 4-hr incubation, cells were washed and new media was added. Cells were harvested after 72 hr, fixed in 70% ethanol overnight, and washed with PBS. Cells were centrifuged for 10 min at 300 Â g and resuspended in 500 mL propidium iodide (PI)/Triton X-100 staining solution (0.40 mL of 500 mg/mL PI, 0.1% [v/v] Triton X-100, and 2 mg DNase-free RNase A, to 10 mL) for 15 min at 37 C. Fluorescence of the PI-stained cells was measured with flow cytometry and analyzed with ModFit deconvolution software (Verity Software House).
Cell Proliferation Assay
To determine the inhibition of cell proliferation, PANC-1 and AsPC-1 cells were seeded at 5 Â 10 3 cells per well in 96-well plates and grown in appropriate media for 24 hr. P19, P19-5FdUMP, and P19-dFdCMP were added to cells at 1 mM and changed the media to remove the remaining ApDCs. Inhibition of cell proliferation was measured by MTT assay after 72-hr incubation.
PANC-1, MCF7, U251-MG, HCT116, and BJ cells were seeded at 5 Â 10 3 cells per well in 96-well plates and grown in appropriate media for 24 hr. tP19-MMAE and tP19-DM1 were added to cells at various concentrations (from 1 mM to 0.45 mM, 3-fold dilutions) and incubated for 4 hr. Cells were washed and new media was added after 4-hr incubation to remove remaining ApDCs. After 72 hr, the inhibition of cell proliferation was measured by MTT assay (Promega).
Statistical Analysis
Statistically significant differences were determined by Student's t test and ANOVA using GraphPad Prism software (GraphPad Software).
SUPPLEMENTAL INFORMATION
Supplemental Information includes three figures and two tables and can be found with this article online at http://dx.doi.org/10.1016/j. omtn.2016.11.008.
ACKNOWLEDGMENTS
We would like to thank the light microscopy digital imaging core facility of the Beckman Research Institute at the City of Hope for www.moleculartherapy.org technical assistance. Research reported in this publication also included work performed in the Analytical Cytometry and Drug Discovery and Structural Biology Cores supported by the National Cancer Institute of the NIH under award number P30CA033572. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We also like to thank Sarah Wilkinson, scientific writer, for language editing. The authors would like to acknowledge support from Apterna Ltd. and NIAID R01 AI029329. T.M.P. and Y.W. are supported by the Intramural Research Program of the NIH, National Library of Medicine.
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2018-04-03T06:25:00.465Z
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2016-12-10T00:00:00.000
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The burden of comorbidities in pulmonary arterial hypertension
Abstract Patients with comorbidities are often excluded from clinical trials, limiting the evidence base for pulmonary arterial hypertension (PAH)-specific therapies. This review aims to discuss the effect of comorbidities on the diagnosis and management of PAH. The comorbidities discussed in this review (systemic hypertension, obesity, sleep apnoea, clinical depression, obstructive airway disease, thyroid disease, diabetes, and ischaemic cardiovascular event) were chosen based on their prevalence in patients with idiopathic PAH in the REVEAL registry (Registry to EValuate Early and Long-term PAH disease management). Comorbidities can mask the symptoms of PAH, leading to delays in diagnosis and also difficulty evaluating disease progression and treatment effects. Due to the multifactorial pathophysiology of pulmonary hypertension (PH), the presence of comorbidities can lead to difficulties in distinguishing between Group 1 PH (PAH) and the other group classifications of PH. Many comorbidities contribute to the progression of PAH through increased pulmonary artery pressures and cardiac output, therefore treatment of the comorbidity may also reduce the severity of PAH. Similarly, the development of one comorbidity can be a risk factor for the development of other comorbidities. The management of comorbidities requires consideration of drug interactions, polypharmacy, adherence and evidence-based strategies. A multidisciplinary team should be involved in the management of patients with PAH and comorbidities, with appropriate referral to supportive services when necessary. The treatment goals and expectations of patients must be managed in the context of comorbidities.
Introduction
The definition of comorbidity was first proposed by Feinstein in 1970, as the presence of 'any distinct additional clinical entity that has existed or that may occur during the clinical course of a patient who has the index disease under study'. 1 The development of comorbidities can be driven by genetics, lifestyle and societal factors, and/or the interaction of both. 2 Comorbidities in pulmonary arterial hypertension (PAH) may be present at the time of PAH diagnosis or may develop during the course of treatment for PAH. The concurrence of PAH and comorbidities increases the complexity of disease management for patients, who may require multiple pharmacological interventions to treat both PAH and the comorbidity.
At the current population level, approximately one in four adults lives with two or more chronic conditions. 3 In PAH, approximately three quarters of patients have at least one comorbidity, 4 with patients aged 65 years and over having a greater number of comorbidities. 4,5 Current research suggests that the presence of comorbid conditions in patients with PAH negatively affects outcomes. 6,7 To optimize patient care, it is important that the effects of comorbidities on PAH are well understood. The 2015 European Society of Cardiology (ESC)/European Respiratory Society (ERS) guidelines recommend that assessments of patients with PAH should provide information on comorbidities. 8 Physicians should assess patients on a regular basis to identify clinically relevant comorbidities. 8 The aim of this review is to explore how comorbidities affect the diagnosis and management of PAH. There are multiple causes of PAH; however, for the purpose of this review, known associations of PAH with connective tissue disease (CTD), HIV infection, portal hypertension, congenital heart disease, and schistosomiasis, 8 which can be classified as PAH sub-entities, are not considered comorbidities of PAH. Similarly, combinations of Group 1 pulmonary hypertension (PH) with Groups 2-5 PH are not discussed in detail, given these are classified as multiple causes of PH. Furthermore, pregnancy as an important transient comorbidity is not addressed here but is discussed elsewhere. 9 Comorbidities discussed in this review were selected based on those with the greatest (>10%) prevalence in idiopathic PAH from the REVEAL Registry (Registry to EValuate Early and Long-term PAH disease management), 10 which included patients from over 50 centres in the USA. These comorbidities were: systemic hypertension, obesity, sleep apnoea, clinical depression, obstructive airway disease, thyroid disease, diabetes mellitus, and ischaemic cardiovascular event. 10 This is not an exhaustive list and there are many other comorbidities associated with PAH that have been discussed elsewhere, such as anaemia, 11 chronic kidney disease, 12 chronic liver disease, 13 chronic pain, 14,15 chronic muscle disease, 16,17 frailty, peripheral vascular disease, cancer, and dementia. All comorbidities add a significant burden to PAH patients' lives, as well as their caregivers, and presents a challenge to the treating physician. It is also important to note that, across all PH diagnoses, PAH (Group 1) patients typically present with the fewest comorbidities at the time of diagnosis ( Figure 1). Summarizing the latest guidance on all of the above-listed comorbidities for all the PH groups is beyond the scope of this review, and condition-specific considerations are therefore limited to those with the greatest (>10%) prevalence in idiopathic PAH from the REVEAL Registry.
The impact of comorbidities on diagnosing pulmonary arterial hypertension Comorbidities in pulmonary arterial hypertension The concurrence of PAH and comorbidities ( Figure 2) increases the complexity of disease identification and management. Patients may require multiple pharmacological therapies or medical interventions for PAH and comorbidities, which can affect the choice of PAH-specific therapies due to drug interactions, dosing regimens, or contraindications. Similarly, patients may require care services to be co-ordinated and a multidisciplinary team to manage their conditions. 22 However, patients with comorbidities are frequently excluded from clinical trials, and as such there is a paucity of data on the effectiveness of PAH-specific therapies in patients with multiple comorbid conditions.
In populations of patients with three or more of the following comorbidities: body mass index (BMI) 30 kg/m 2 ; hypertension; diabetes; and coronary disease, patients experienced a similar treatment effect compared with those with two or fewer comorbidities. A post hoc analysis of the GRIPHON study revealed that selexipag was as effective in patients with three or more comorbidities (n ¼ 99) as those with two or fewer (n ¼ 653), and safety profiles for both subgroups were similar and consistent with the known profile of selexipag. 23 In the AMBITION study, patients with three or more comorbidities (n ¼ 105) experienced a Figure 1 The burden of comorbidities observed across pulmonary hypertension groups at the time of diagnosis in authors' experience. 18-21 a Comorbidities that are typically associated with pulmonary hypertension Group 5, but may affect all groups. b Comorbidities that are typically associated with IPAH, but may affect all groups. This schematic is used to illustrate the typical scenarios the authors observe in clinic; it is not drawn to scale and is largely based on authors own experience rather than a robust body of evidence in the literature. The blue triangle and the rectangular 'Group' boxes represent the burden of comorbidity and thus, the placement of the pulmonary hypertension group boxes within the triangle, as well as their size, represent the relative prevalence of comorbidities in each group (with the lowest and largest group having the greatest prevalence). Group 5 pulmonary hypertension is shown outside the triangle because published data and the authors' findings from the clinic are not sufficient to draw conclusions from; there is no consensus on the prevalence of comorbidities in Group 5 pulmonary hypertension at the time of diagnosis. COPD, chronic obstructive pulmonary disease; IPAH, idiopathic pulmonary arterial hypertension; PH, pulmonary hypertension.
K22
I.M. Lang and M. Palazzini similar treatment effect to first line combination therapy of ambrisentan and tadalafil compared with patients who had two or fewer comorbidities (n ¼ 500), with good tolerability. 24 Similar responses to treatment (World Health Organization functional class; exercise capacity and natriuretic peptide levels) were also observed after 12 months between patients (with 3 comorbidities, n ¼ 139; with 2 comorbidities, n ¼ 421) in an analysis of the COMPERA registry, who were receiving either an endothelin receptor antagonist (ERA), a phosphodiesterase-5 inhibitor (PDE-5), or a prostacyclin analogue or combination therapy. 25
Differentiation of pulmonary hypertension groups
Pulmonary hypertension is a complex disease with several possible causes, which are not always mutually exclusive and management is made more difficult in cases where a patient's diagnosis includes multiple PH groups. For example, there is high prevalence of left heart disease and/or interstitial lung disease in patients with CTD, thus it can be difficult to distinguish whether Group 1 (PAH associated with CTD), Group 2 (PH due to left heart disease), or Group 3 (PH due to lung diseases and/or hypoxia) is the leading or primary condition. 8,26 It is important to identify the primary condition driving PH progression to ensure appropriate treatment. 8 For example, the use of PAH-specific therapies is not recommended in patients with Groups 2 and 3 PH and there is a paucity of evidence regarding their efficacy in these patients. 8 Common comorbidities such as ischaemic left heart disease, diabetes, or aortic valve stenosis increase pulmonary artery wedge pressure, which can lead to diagnostic uncertainty in the presence of precapillary disease. For example, severe aortic stenosis in a case of precapillary PH (such as Eisenmenger syndrome) can lead to combined pre-and post-capillary disease, for which the outcomes with PAHapproved therapies are uncertain. 8
Confounding the diagnosis of pulmonary arterial hypertension
The presence of comorbidities can lead to delayed diagnosis by masking the symptoms of PAH, 27 particularly due to difficultly identifying and differentiating between non-specific symptoms of PAH such as dyspnoea, fatigue, syncope and chest pain, and symptoms of common comorbidities. Research has shown that patients may experience symptoms for a number of years before receiving a diagnosis of PAH. 28,29 Given the progressive nature of PAH, a delayed diagnosis and subsequent delay to treatment initiation can have severe consequences. The importance of screening patients for PAH to allow treatment to be started in a timely manner is discussed in more detail in a review focused on screening, also in this supplement. 30 The presence of comorbidities, such as systemic hypertension, obstructive airway diseases, and obesity, can also confound the interpretation of prognostic tests for PAH such as the 6-min walk test, contributing to a delay in diagnosis. The diagnostic PAH sign of interventricular septum flattening may be mitigated by concurrent increase in left ventricular pressures, such as in the course of cardiomyopathy. It is therefore important that results are interpreted in the context of known comorbidities. 6 One example of a population with a high prevalence of comorbidities is in the elderly. Among patients with idiopathic PAH, approximately a quarter of older patients (65 years) were found to have at least four of the following comorbidities: systemic hypertension, diabetes, ischaemic stroke, ischaemic heart disease, atrial fibrillation, obesity, or kidney dysfunction, compared with less than 7% of those aged under 65 years. 4 Patients aged over 50 years with PAH were diagnosed with more advanced, severe disease compared with patients younger than 50 years in a review of the UK registry of patients with idiopathic, heritable, or anorexigeninduced PAH. 5 Management considerations for an older population with PAH are discussed in more depth in a separate review in this supplement. 31
Management considerations with comorbidities and pulmonary arterial hypertension
Patients with PAH and comorbidities are likely to be on complex pharmacological treatment regimens that need close monitoring and regular adjustment, with an overview by physicians from different specialties. A multidisciplinary team and network are therefore important when patients with PAH are also being treated for comorbidities. Patients with PAH and comorbidities may require different aspects of care including cardiac exercise rehabilitation (for peripheral artery disease, heart failure, myocardial infarction, and atrial fibrillation) and surgical interventions, in addition to multiple pharmacological therapies. [32][33][34][35][36][37] Patients may also require additional support in their daily lives from social workers, occupational therapists, and psychotherapists. Treatment interventions, and expectations must be managed in the context of comorbidities, 8 and the goals of patients with PAH may need to be reassessed following, or in preparation for, major surgical procedures required to manage a comorbidity.
The ESC/ERS treatment algorithm for treating patients with PAH 8 was developed for patients with no significant cardiovascular comorbidities. 38 An amended treatment algorithm that was endorsed by the Cologne Consensus Conference in 2018, takes into account patients with cardiopulmonary comorbidities, and recommended that oral monotherapy should be the first initiated treatment for PAH, regardless of risk category. 38 Treatment should then be escalated in the case of an inadequate clinical response. 38 This recommendation was made based on the lack of efficacy data in the population of patients with cardiovascular comorbidities, in addition to safety concerns arising from a high incidence of drug discontinuations attributed to adverse events. 38
Condition-specific considerations
Systemic hypertension and ischaemic cardiovascular events Systemic hypertension increases the risk of cardiovascular morbidity/mortality, including coronary events and stroke. 39 Up to three-quarters of patients who experience a first stroke or myocardial infarction have concomitant systemic hypertension. 40 Lifestyle modifications should be used to decrease the required dose levels of anti-hypertensive drugs, where appropriate. 39 The use of beta-blockers, angiotensinconverting enzyme inhibitors, angiotensin receptor blockers, diuretics, and calcium channel blockers have all been found to cause a similar reduction in cardiovascular events on the basis of decreased blood pressure. 39 Care must be taken when PAH-specific therapies are used concomitantly with anti-hypertensive medications due to the risk of excessive systemic hypotension. 8 The use of beta-blockers as a first line anti-hypertensive drug should be avoided in PAH patients.
Calcium channel blockers can also be used in the treatment of PAH, and relatively high doses are needed to provide a benefit in idiopathic PAH following vasoreactivity testing. 8 Patients with PAH who are treated with calcium channel blockers should be monitored closely, with complete reassessment (including right heart catheterization) after 3-4 months of therapy. 8 Similarly, patients who have had an ischaemic stroke are likely to be taking anti-coagulants, 41 such as warfarin. The potential benefits of anticoagulation therapy in idiopathic PAH are unclear due to contrasting evidence. Analyses of the REVEAL Registry and GRIPHON study did not demonstrate a survival benefit for patients with PAH taking anticoagulants, compared with those who were not. 42,43 However, analysis of the COMPERA registry showed that during the 3-year followup, there was a significantly lower mortality rate in patients receiving anticoagulants compared with the nonanticoagulated group. 44 Careful monitoring of patients using bosentan and warfarin is required, as bosentan increases warfarin metabolism and subsequent dose adjustments may be necessary. 8 Obesity The prevalence of obesity in patients with PAH is between 30% and 40%. 10,45 Increased mortality has been observed in patients with BMI 40 kg/m 2 with PAH aged under 65 years (hazard ratio ¼ 3.01), 45 and a higher BMI (30 kg/m 2 ) has been associated with worse functional class. 6 Not surprisingly, patients with obesity and PAH also had significantly lower 6-min walk distances than patients without obesity. 46 These findings indicate that weight management is an important consideration in patients with PAH and who have obesity. 45 The presence of obesity must be considered when using the 6-min walk test as a prognostic tool for PAH, as it may act as a confounding factor but may also contribute to PAH severity. 47 Bariatric surgery for patients with obesity can positively affect PH with lower right ventricular systolic pressure (RVSP) 48 and pulmonary artery pressure. 49 In patients with PH and a pre-operative RVSP 35 mmHg who underwent bariatric surgery there was no mortality in the first 30 days following surgery, indicating that bariatric surgery can be performed safely in this population without the need for bridging therapies to improve PH. 48 Obesity is linked to a number of other conditions such as sleep apnoea, insulin resistance, and obesity hypoventilation syndrome, all of which have been linked to the development of PH. 50 Thus, reductions in body weight can lead to improvement in or resolution of these associated conditions as well as an improvement of PH.
Sleep apnoea and obstructive airway disease
In the REVEAL Registry, obstructive airway disease was defined as obstructive lung disease (including asthma and bronchiectasis), reactive airways disease, and chronic obstructive pulmonary disease (COPD). 10 Most of these conditions, in addition to sleep apnoea, which was considered a separate comorbidity, are classified as a cause of Group 3 PH. 8 Due to this, there is a paucity of research and evidence into the management of these comorbidities and PAH, despite their presence in approximately one-quarter of patients with PAH. 10 When sleep apnoea is present in combination with comorbidities that also cause hypoxaemia, patients commonly present with more severe PH. 50 Sleep apnoea is associated with increased pulmonary artery pressure, which can be reduced by treatment with continuous positive airway pressure. 51 During the daytime, patients with PAH and sleep apnoea display lower arterial oxygen and higher arterial carbon dioxide tension compared with patients without PAH, 52 which may necessitate supplemental K24 I.M. Lang and M. Palazzini oxygen therapy. There is limited evidence of a benefit of PAH-specific therapies in Group 3 PH (due to lung diseases and/or hypoxaemia), 8 therefore, in our opinion, when patients with PAH present with or develop conditions such as sleep apnoea and COPD physicians should carefully monitor for the absence or loss of efficacy of PAH-specific therapies. The potential effects of PAH-specific therapies on the symptoms and progression of the lung disease must also be considered. 8
Clinical depression
Clinical depression was observed in over 25% of patients with idiopathic PAH in the REVEAL Registry. 10 However, there is some concern that the diagnosis of depression may be missed due to the overlapping symptoms of fatigue and apathy for PAH and depression. 53 Furthermore, less than 25% of PH patients with a psychiatric diagnosis go on to receive psychiatric treatment. 54 Given the progressive and debilitating nature of PAH, paired with the changes in lifestyle such as job loss and financial concerns, screening, identification, diagnosis and treatment of depression in this population should be prominent in PAH management. 53 Despite the prevalence of depression in patients with PAH, optimal pharmacological approaches are not well defined. 55 In the general population, psychotherapy is recommended in mild depression, with the addition of pharmacological therapies for moderate-to-severe depression. Selective serotonin reuptake inhibitors (SSRIs) have been associated with higher risk of mortality and clinical worsening in patients with PAH, compared with patients not taking SSRIs. 56 Patients with PAH may also consider joining patient support groups to help them cope with the uncertainty associated with the disease. 55,57 Thyroid disease Thyroid disorders are classified as mechanisms for the development of Group 5 PH 8 ; however, thyroid disease is also commonly observed concomitantly in patients with PAH (either present during the initial assessments or developing during the course of PAH). 10,58 In both hypothyroidism and hyperthyroidism, cardiac output and pulmonary vascular resistance (PVR) are increased, which in turn can act as a driver for PH. 59,60 As such, tests of thyroid function should be conducted as part of the investigation into PAH, and in established PAH, thyroid function tests should be conducted at least once a year or in cases of rapid deterioration. 8,60 Early and aggressive treatment of patients with PAH and hyperthyroidism is critical, due to potentially fatal complications of thyroid disease. 61 The increases in cardiac output and PVR can be reversed with beta-blockers and antithyroid medications such as propylthiouracil and saturated solutions of potassium iodide, and can return the patient to a euthyroid state. 59,61 Beta-blockers are not ordinarily recommended for patients with PAH but can be used when necessitated by the presence of comorbidities. 8 For example, there is the potential for a drug-drug interaction between sildenafil, which is metabolized by cytochrome (CYP)-3A4, and those beta-blockers that are CYP3A4 substrates. 8 The concomitant use of PAH-specific therapies and beta-blockers should also be carefully managed to avoid excessive systemic hypotension. 8 Diabetes Glucose intolerance and insulin resistance are increasingly thought to influence both the pathogenesis and prognosis of PAH, 62 and diabetes as a comorbid condition to idiopathic or heritable PAH reduces right ventricular function. 63 Patients with PAH and diabetes have been shown to have significantly lower 10-year survival compared with PAH patients without diabetes. 63 Identification and mitigation of modifiable risk factors for diabetes, and targeted treatment of diabetes if it develops, may delay PAH progression and improve patient quality of life. 62 Patients should be educated about the risks of diabetes and PAH, and there should be regular testing for diabetes in these patients. 62
Considerations regarding polypharmacy
Polypharmacy has been associated with poor treatment outcomes in the general population, which may be due to the adverse side effects of drugs, drug interactions, and/or non-adherence to the treatment regimen, 64 and the impact of multiple medical conditions. 65 In a study of 174 patients with PAH, the median number of drugs per patient was nine, and over 80% were taking five drugs or more. 66 The side-effect profiles of PAH-specific therapies are well documented when used as monotherapy or in combination; however, new side effects may be observed when concomitantly administered with therapies for other conditions. Drug-drug interactions must also be considered ( Table 1). For instance, PDE-5 inhibitors for PAH, such as sildenafil, cannot be concomitantly used with nitrates, which are used to treat a number of coronary conditions, 70 and there are known interactions between anticoagulants and both prostacyclin analogues and ERAs, which contraindicate their concomitant use. 71 Adherence may reduce as the number of medications increases, and patients may not notice the short-term effects of occasional missed doses 72 ; however, given the progressive nature of PAH, the loss of steady-state from repeated missed doses may have negative consequences for longer-term disease progression. Therefore, patient education through appropriate communication channels is important to maintain adherence. 72 The dosing regimen of PAH-specific therapies should also be considered in patients who are taking multiple medications. 73 For instance, ambrisentan, macitentan, and tadalafil are taken once per day, bosentan and selexipag are taken twice per day, and sildenafil and riociguat are taken three times daily. The pill burden of each therapy should be considered when deciding on medications for monotherapy or combination therapy, in addition to treatment regimens for comorbidities. Older adults with PAH are the most likely to have multiple comorbidities, and the administration of complex therapy regimens in these patients is more challenging. 72 This is discussed in more detail in a specific review on management of PAH in older patients, which is also in this supplement. 31 The burden of comorbidities in PAH K25
Conclusions
Comorbidities can mask the symptoms of PAH, which can lead to a delayed diagnosis and deleterious consequences for disease progression and survival. Similarly, the presence of comorbidities increases the difficulty of evaluating disease progression and treatment effects by confounding prognostic assessments. The management of comorbidities in addition to PAH should consider drug interactions, polypharmacy, adherence and evidence-based strategies. Thus, it is important that any and all comorbidities are identified and diagnosed so that each patient receives the optimal treatment regimen. A multidisciplinary team approach is essential in the management of patients with PAH and comorbidities. In addition to management by physicians of different specialties, patients may require social, financial, and psychological support. Furthermore, healthcare professionals must manage each patient's treatment goals and expectations in the context of comorbidities.
Funding
Medical writing and editorial support were provided by Victoria Atess, Zoe Schafer and Richard McDonald of Watermeadow Medical, an Ashfield Company, funded by Actelion Pharmaceuticals Ltd (Allschwil, Switzerland).
Conflict of interest: I.M.L. has received grants, personal fees and non-financial support from Actelion Pharmaceuticals Ltd, and grants and personal fees from AOP Orphan Pharmaceuticals. M.P. has received grants, personal fees and non-financial support from Actelion Pharmaceuticals Ltd.
|
2019-12-19T09:18:58.831Z
|
2019-12-01T00:00:00.000
|
{
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205244987
|
pes2o/s2orc
|
v3-fos-license
|
LigParGen web server: an automatic OPLS-AA parameter generator for organic ligands
Abstract The accurate calculation of protein/nucleic acid–ligand interactions or condensed phase properties by force field-based methods require a precise description of the energetics of intermolecular interactions. Despite the progress made in force fields, small molecule parameterization remains an open problem due to the magnitude of the chemical space; the most critical issue is the estimation of a balanced set of atomic charges with the ability to reproduce experimental properties. The LigParGen web server provides an intuitive interface for generating OPLS-AA/1.14*CM1A(-LBCC) force field parameters for organic ligands, in the formats of commonly used molecular dynamics and Monte Carlo simulation packages. This server has high value for researchers interested in studying any phenomena based on intermolecular interactions with ligands via molecular mechanics simulations. It is free and open to all at jorgensenresearch.com/ligpargen, and has no login requirements.
INTRODUCTION
Recent advances in computer hardware, algorithm development and force field parameterization have contributed greatly to increased use of molecular simulations in many aspects of biomolecular studies (1,2). Nowadays, computational approaches have become widely popular to study the structure, mechanism of action and biomolecule-ligand interactions of proteins and nucleic acids. These methods, mostly based in molecular mechanics (MM), require force fields (FF) that provide an accurate description of the interactions to provide valid predictions. For instance, a 1.4 kcal/mol difference in binding free energy translates to an order of magnitude difference in binding. Most of the common MM force fields, such as CHARMM (3), OPLS-AA (4) or AMBER (5) provide complete parameters for proteins and nucleic acids, but the parameterization of the ligands remains an open problem due to the vast chemical space found in combinatorial and medicinal chemistry. For-tunately, bonded and van der Waals parameters can generally be ported from existing analogous atom types. Thus, the most critical issue in ligand parameterization is the estimation of a balanced set of partial atomic charges effective at reproducing experimental properties such as hydration free energies (HFE) or free energies of binding ( G bind ).
The OPLS-AA force field was initially developed and parameterized to reproduce experimental heats of vaporization and densities of small organic molecules and was later extended to include proteins (6,7) and nucleic acids (8). As individual parameterization of sets of partial atomic charges for the countless possible organic ligands is not practical, several different quantum mechanics (QM) based charge models have been proposed. In particular, the CMx models developed by Cramer et al. (9)(10)(11)(12)(13)(14) have been tested extensively in condensed phase simulations in combination with the OPLS-AA force field, usually by comparison between calculated and experimental hydration free energies, heats of vaporization and densities. The most accurate charge models to reproduce experimental HFEs with OPLS-AA have been found to be 1.20*CM5 (15) and 1.14*CM1A (16) charges. The accuracy of 1.14*CM1A charges is further improved by the use of localized bond charge corrections (LBCC), making 1.14*CM1A-LBCC (17) the best among existing QM charge models at the same computational cost of CM1A charges.
Once an appropriate set of parameters is obtained the problem is reduced to transferring them into the desired software. Each simulation program requires the specification of geometries, topologies and parameters in one or more files in specific formats. Although most of the packages provide utilities to prepare these files using their builtin parameter set and charge model, it is far from trivial to use a different set of parameters in any given program. The LigParGen web server was developed to address this problem by providing a simple, automatic procedure to assign the parameters and generate the files needed to do calculations in some of the most commonly used simulation packages.
There are other servers that provide force field parameters for MD simulations. Among them the CGenFF (18,19), MATCH (20), CHARMM-GUI (21) and CHAR-MMing (22) servers provide files formatted for CHARMM W332 Nucleic Acids Research, 2017, Vol. 45, Web Server issue with parameters from the CHARMM36/CGenFF force field and charges assigned by analogy. The SwissParam (23) server produces CHARMM input files combining bonded parameters from MMFF and nonbonded ones from CHARMM22. Two servers that generate Amberformatted files are H++ (24), which uses the ambertools (25) package to assign GAFF parameters with AM1-BCC charges, and R.E.D (26) that provides RESP and/or ESP charges fitted from an ab initio calculation. It must be noted that some of the simulation packages provide utilities to read files formatted for other packages. Lastly, the PRO-DRG (27) server provides GROMOS FF parameters and charges in a GROMACS formatted file.
The LigParGen server was designed to provide OPLS-AA parameters for neutral organic molecules using two CM1A charge models, 1.14*CM1A and 1.14*CM1A-LBCC, while unscaled CM1A charges are provided for charged molecules. The output files are formatted to be used directly in the popular molecular dynamics MD programs CHARMM (28), NAMD (29), GROMACS (30) and OpenMM (31) and the Monte Carlo (MC) software BOSS and MCPRO (32). The server can also generate a single file in PQR format, which are used for Poisson-Boltzmann calculations or docking. In addition, the input is very flexible. The input molecule can be specified with 3-dimensional coordinates in a MOL or PDB file, or a simplified 2D representation can be entered as a SMILES string, or the structure may be drawn on the web page through the use of the JSME (33) plugin, or it may be copied directly from a drawing program such as ChemDraw (34).
MATERIALS AND METHODS
The core of the LigParGen server is the internal use of the BOSS (32) software to assign the bonded and van der Waals parameters by analogy to the existing atom types in the latest OPLS-AA force field (4). Subsequently a semiempirical AM1 (9) calculation is performed to calculate and assign the charges. The server can, as directed by the user, utilize one of two CM1A-derived charge models as described briefly below. For further information about technical details and comparisons, please read the original papers (9,16).
In general, quantum mechanics population analysis methods distribute the total electron density of a molecule into partial charges centered on each atom of the molecule. As partial charges are not observables, there are different ways to partition the electron density. The CM1A method uses the Mulliken population analysis from the electron density obtained by the AM1 method from the ligand geometry. Mulliken charges for an atom A are computed using the following equation: where q A is the partial Mulliken charge, Z A is the nuclear charge of the atom A and N A is the electron density assigned to atom A as described by the equation: where N is the total number of electrons in the molecule, C n,i is the molecular orbital coefficient for the atomic orbital χ n and S nk is the QM overlap integral. This electron density definition is based on the linear combination of atomic orbital-molecular orbital (LCAO-MO) method where the molecular electronic distribution per each molecular orbital is defined each as a linear combination of atomic orbitals (n). The CM1A charges are then computed using a multilinear transformation of the Mulliken charges based in the computed bond orders to improve the molecular dipole moment using empirical parameters. Then, for neutral molecules, the 1.14*CM1A model scales the charges by a factor 1.14, which was fitted to improve the agreement of the HFEs to the experimental values (16). If the total charge of the molecule is not zero, partial charges are not scaled. It should be noted that, as in all quantum mechanics based charges, the CM1A charges can have some variations due to the molecular geometry. The typical variations observed in our tests are in the 0.03-0.05 e range, with a few cases involving intramolecular hydrogen bonds reaching 0.1e.
A later evaluation of HFEs for a set of 426 organic molecules showed that some moieties such as phenyl rings, aldehydes or ketones are not well parameterized by the 1.14*CM1A charge model, leading to a mean unsigned error (MUE) of 1.5 kcal/mol with respect to experimental HFE data. The performance of CM1A charges was improved by adding Localized Bond Charge Corrections (LBCC), by which small charge adjustments are made to the partial charges for atoms in problematic bond types such as, CT-OH in aliphatic alcohols. Only 19 LBCCs were enough to reduce the errors with the 1.14*CM1A charges for the 426 HFE values to only 0.61 kcal/mol. These adjustments give rise to the 1.14*CM1A-LBCC charge method which can also be provided by the LigParGen server.
Overall description
The LigParGen web server is a free, robust and multiplatform tool to generate OPLS-AA parameters for organic molecules using the 1.14*CM1A and 1.14*CM1A-LBCC charge models. It has been implemented in CGI-python using the Apache HTTP server and the Bootstrap framework to allow access from any internet device (computers, tablets, cell phones, etc.) while keeping an optimal appearance. In order to facilitate its use, a sample button uses the benzene molecule to provide a quick exploration of the server features for novice users. It is important to mention that there is no login required for job submission (see Figure 1A). The server includes in its home web page the Clustrmap tracking system to analyze user relevant information such as location, operating system or internet browser for statistical purposes, future improvements, or for debugging in case specific failures are detected.
Additionally, a section of tutorials by example is included in the LigParGen web page to illustrate the preparation of ligand-water boxes or protein-ligand systems for molecular dynamics simulations using the output templates gen- erated by the server. This tutorial section includes detailed explanations, comments, and snippets to understand and perform the equilibration and production phases of MD in GROMACS, NAMD and openMM. Furthermore, LigPargen server aims to be a dynamic tool with constant improvement in response to users' feedback. For this reason, a contact section requests details for suggestions.
Input
The server currently accepts three different standard input formats for molecular structures: SMILES codes, PDB and MOL files. As the request will be processed by the BOSS package, which is used to calculate the CM1A charges, the maximum number of atoms is currently 200. A singlepoint calculation is done by default on the input structure, but users can request a structure optimization using the assigned OPLS-AA parameters and 1.14*CM1A or 1.14*CM1A-LBCC by checking the 'Optimize Molecule' radio button (see Figure 1A and B). The structure optimization is carried out by BOSS using the Broyden-Fletcher-Goldfarb-Shanno variable metric algorithm (35)(36)(37)(38). The flow of the different operations in the server includes a robust error management system to detect possible problems in the submission process. For instance, if the proposed total charge of the ligand is not realistic an error page will be shown with an explanation (see Figure 1D).
Furthermore, the web server offers the possibility to interactively draw a 2D structure of the desired molecule using the JSME javascript plugin. JSME allows actions such as edit the molecule, export it in different formats or search in molecular databases, and it is compatible with iPhone, Android smartphones, iPad, and Android tablets. This option is located in the 'Draw Molecule' section and contains the same submission options than the default input section. Prior to the submission, any 2D structures generated by JSME, can be converted into SMILES code using the 'make SMILES' button, and the SMILES code can be copied for future submissions (See Figure 1B).
Output and representation of the results
The input molecule provided by the user in 2D (SMILES) or 3D (MOL or PDB) format is converted in several stages to a BOSS Z-matrix for processing by the BOSS program. If needed, the SMILES string is first transformed to a 3D MOL file using the RDKit chemoinformatics package. If no errors are detected in the input, the BOSS utility autozmat is used to create a Z-matrix. The latter is used by LigParGen to call the BOSS program to calculate the CM1A charges and assign parameters for bonded and van der Waals interactions. At this point the sum of the CM1A charges and the user defined charge are compared. If the two total charges match, the parameter and coordinate files are generated in all the different formats and displayed in the LigParGen output page (see Figure 1C).
The output page of LigParGen provides a publication quality image generated using Pymol (Schrodinger, LLC), as well as an interactive 3D visualization panel for the submitted molecule powered by the JSMOL (39) JavaScript application (see Figure 1C). The latter can be useful to identify possible mistakes in the submitted structures, particularly when the structure is generated from a SMILES code. The user can download each output template file needed to run an MD or MC simulation for CHARMM/NAMD, GRO-MACS, OpenMM, BOSS/MCPRO by clicking directly on the individual download buttons or a compressed zip file with all of the files can be downloaded by clicking the 'ALL' button. In addition to the MD/MC files, a PQR file format is generated with radii based on the OPLS-AA σ parameters and the selected charge model to perform Poisson-Boltzmann or docking calculations. It should be noted that all the files generated during a run are tagged by including in the name a unique six-character alphanumeric code used to manage different concurrent jobs and at the same time acting as a queue system. Also, it avoids vulnerabilities in the submissions. As an additional security measure, the server does not store any information about the users and the submitted structures, and all the job-related files are removed after an hour.
Processing time
The time required for processing depends on the ligand size and the number of optimization steps needed. The calculations of the charges are the most expensive compared to other parameters and are computed on the fly. Since these are based on semiempirical QM calculations, the total computational time for small or medium molecules is negligible, and output generation takes <5 s without optimization. If ligand optimization is requested in the submission, the running time can be increased up to 30-45 s depending on the system size and quality of the input structure. For SMILES input, the optimization time is decreased because structures are generated close to the equilibrium conformation.
RESULTS
The server capabilities and robustness are illustrated in two different sets of calculations. The first set present a direct comparison between single-point energy evaluations in vacuum from a representative test set of molecules to demonstrate the coherence between the different template outputs. The second case shows a comparison between experimental and computed HFE results, demonstrating a practical application of the output files generated by the server.
Comparison of gas phase energetics
Fifty two molecules from the SAMPL4 (40) dataset server were chosen to verify the coherence of parameters, topology and structure between the BOSS, OpenMM, NAMD and GROMACS programs using SMILES codes as input. BOSS single-point energies in vacuum were used as reference values to compute the mean unsigned error (MUE) for each energy term and package as shown in Table 1. A more detailed comparison of the energetics for all ligands can be found in the SI table. As seen in Table 1, the total single-point energies from GROMACS, NAMD and OpenMM are in very good agreement with the BOSS energies, showing a MUE of 0.03-0.02 kcal/mol for the entire test set. In most cases, the bond energy term produces the highest MUE, due to the sensitivity of the large stretching force constants to the loss of accuracy in the input coordinates (a BOSS Z-matrix uses five decimal figures while PDB/GRO uses just 3). On the other hand, the difference in non-bonded energies is due to the different values for the vacuum permittivity constants used by NAMD, OpenMM and GROMACS. Overall, the differences seen in the total energy is in the order of 10-20 cal/mol, and are not expected to have a significant effect on the outcome of MD simulations.
Evaluation of hydration free energies
Calculating HFE is an integral part of the estimation of binding free energy. To demonstrate the applicability of parameters and topology generated by the LigPar-Gen server, MD/FEP calculations of the absolute HFE for 10 molecules which produce accurate results from the SAMPL4 data set were performed. The simulations were done both in water and gas phase using the NAMD package to calculate HFE. Water molecules were represented us- ing the TIP3P water model while the ligands were represented using the OPLS-AA/1.14*CM1A-LBCC force field parameters generated by the server. Each ligand was solvated in a cubic box with a 13Å padding, and a cutoff of 10 A was used for calculating non-bonding interactions with PME for long range electrostatics. The Lennard-Jones interactions were smoothed off over last 0.5Å and long-range corrections were included. All simulations were run at constant pressure and temperature, 1 atm and 300 K. A detailed description of the methodology is presented elsewhere (41). In brief, each ligand was annihilated in water and in the gas phase over 36 windows. Each window was equilibrated for 1 ns and the averaging was done over the next 1 ns. Following a decoupling scheme, the electrostatic interactions were switched off first, followed by LJ interactions. Soft-core potentials were used to avoid any end-point catastrophes. Both forward and backward simulations were performed to improve the accuracy of FEP calculations. The Bennett Acceptance Ratio estimator implemented in VMD was used to obtain the free energy changes (42). The free energies of hydration were obtained from the difference between the aqueous-phase and gas-phase free energies of annihilation, and are summarized in Table 2. From the comparison with the experimental data given in Table 2, it can be seen that the OPLS-AA/1.14*CM1A-BCC parameters produced by the LigParGen server perform notably well for hydration free energies with MUE and Mean Signed Error (MSE) of 0.6 kcal/mol and 0.0 kcal/mol, respectively.
CONCLUSIONS AND FUTURE DEVELOPMENT
Here, we present the first server to generate, in an automated manner, OPLS-AA/CM1A parameter files for organic ligands starting from 3D MOL or PDB files, or even 2D chemical drawings or SMILES strings. The ligand parameters can be generated for common simulation software packages such as NAMD, GROMACS, OpenMM, BOSS and MCPRO. These capabilities allow the users to obtain highquality parameters for MM simulations without extensive knowledge about MM force fields or QM methods. This server will be useful for any group interested in the study of biomolecule-ligand interactions, with direct applications to computational drug design, or condensed-phase ligand properties via MD or MC methods. The LigParGen server is an ongoing project, and future improvements will reflect the feedback provided by the users. In the next update, we are planning to generate parameter files for the AMBER software.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
|
2017-09-16T03:03:59.158Z
|
2017-04-21T00:00:00.000
|
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|
14389060
|
pes2o/s2orc
|
v3-fos-license
|
Laparoscopic removal of gastrointestinal stromal tumors of uncinate process of pancreas
The pancreas is an extremely rare location for gastrointestinal stromal tumors (GIST). We present a case of a patient with a GIST located in the uncinate process of the pancreas that was treated successfully with a laparoscopic technique. Computed tomography, magnetic resonance imaging and scintigraphy suggested a neuroendocrine tumor. Due to the fact that the image suggested a neuroendocrine tumor with a diameter below 2 cm, the patient was qualified for a laparoscopic procedure of tumor enucleation. Postoperative care proceeded in accordance with the principles of the ERAS concept. The postoperative course was uncomplicated. He was discharged home on the second postoperative day. In the obtained histopathology result a GIST was found. During a 6-month observation, including control computed tomography examination, no signs of tumor progression were found. Despite the fact that stromal tumors of the gastrointestinal tract localized in the pancreas are very rare, they should be considered in the differential diagnosis of tumors of this organ.
Introduction
Gastrointestinal stromal tumors (GIST) are rare lesions comprising about 1% of neoplasms of the gastrointestinal tract [1]. They are usually localized in the stomach (60%), but they can involve the small intestine (30%), large intestine (4%), or esophagus (< 1%) [2,3]. Even rarer cases of GIST developing outside of the GI tract (extragastrointestinal stromal tumors -EGISTs), in organs such as the pancreas, gallbladder, urinary bladder, or omentum, are described [4]. The mainstay of treatment of GIST is surgery based on resection of the lesion with a healthy tissue margin [5].
Below we present a case of a patient with GIST located in the uncinate process of the pancreas that was treated successfully with a laparoscopic technique.
Case report
A 55-year-old man came to our clinic because of a 1.5 cm pancreatic tumor that was accidentally found during a computed tomography examination (Photo 1). At that time the patient was completely asymptomatic. In order to widen the diagnostics he was referred for magnetic resonance imaging study of the abdominal cavity. In that examination a lesion sized 20 × 17 × 16 located in the uncinate process of the pancreas was found. It intensified significantly after intravenous contrast administration (Photo 2). The image primarily suggested a neuroendocrine tumor. To confirm the diagnosis, scintigraphy was performed using a marked somatostatin analogue (99MTC). The examination confirmed the presence of an area of higher expression of somatostatin receptors located in the uncinate process of the pancreas
A b s t r a c t
The pancreas is an extremely rare location for gastrointestinal stromal tumors (GIST). We present a case of a patient with a GIST located in the uncinate process of the pancreas that was treated successfully with a laparoscopic technique. Computed tomography, magnetic resonance imaging and scintigraphy suggested a neuroendocrine tumor. Due to the fact that the image suggested a neuroendocrine tumor with a diameter below 2 cm, the patient was qualified for a laparoscopic procedure of tumor enucleation. Postoperative care proceeded in accordance with the principles of the ERAS concept. The postoperative course was uncomplicated. He was discharged home on the second postoperative day. In the obtained histopathology result a GIST was found. During a 6-month observation, including control computed tomography examination, no signs of tumor progression were found. Despite the fact that stromal tumors of the gastrointestinal tract localized in the pancreas are very rare, they should be considered in the differential diagnosis of tumors of this organ.
(Photo 3). In the imaging studies no distal lesions were found within the abdominal cavity. No cancer markers were elevated either. Due to the fact that the image suggested a neuroendocrine tumor with a diameter below 2 cm, the patient was qualified for a laparoscopic procedure of tumor enucleation. The procedure was performed using 5 ports ( Figure 1). Intraoperative ultrasound was performed, visualizing a 15 mm hypoechogenic structure within the uncinate process of the pancreas, which was then enucleated completely and sent for histopathologic verification (Photos 4 and 5). No drain was left in the abdominal cavity. Postoperative care proceeded in accordance with the principles of the ERAS concept [6]. A few hours after the procedure the patient started to walk around. During the first 24 h drinks and a fluid diet were administered. The further postoperative course was uncomplicated. The patient (Table I). The risk of tumor progression assessed according to the Joensuu criteria was defined as low. The histopathologic examination confirmed an R0 resection. Because of this, it was decided not to widen the margin of excision, even though the resection margin was smaller than 10 mm. During 6 months of observation, including control computed tomography examination, no signs of tumor progression were found.
Discussion
The GIST are rare lesions, and their occurrence outside of the gastrointestinal tract (EGIST) is sporad- ic. These neoplasms may also be located in the urinary bladder, omentum, gallbladder, or retroperitoneal space and pancreas, among other locations [4,7]. Most pancreatic tumors are lesions of epithelial origin. Neuroendocrine tumors comprise a small percentage of lesions of this organ [8]. The pancreas is an extremely rare location for EGIST. So far about 20 cases of neoplasms of this type localized in the pancreas have been described [9].
The mainstay of GIST treatment is surgical treatment based on a radical excision of the lesion with a macroscopically healthy tissue border with a 1-2 cm margin. Unlike malignant tumors of epithelial origin, removal of the regional lymphatic system seems unnecessary, because neoplasms of the GIST type very rarely metastasize to lymph nodes [10]. Such characteristics encourage the use of minimally invasive techniques in treatment of these neoplasms [11]. Most cases of pancreatic EGIST described in the literature were large lesions [9]. In more than a half of them the tumor diameter was over 10 cm, and the lesion itself often extended beyond the pancreas. Because of that, the surgical treatment involved extensive resection procedures with the classical method. However, in the case described by us the pancreatic tumor was small. We decided on enucleation of the lesion because the preoperative tests suggested presence of a NET type tumor with a diameter below 2 cm. The small size of the lesion and lack of infiltration of neighboring tissues allowed the use of laparoscopy, even though the tumor was located in the uncinate process of the pancreas, which from the anatomical point of view seems to be a place that is hard to reach in surgery. The lesion was removed with a small healthy tissue margin, because its widening would be connected with the need of pancreaticoduodenectomy. According to our knowledge the procedure performed by us is the first surgery of that kind performed within the pancreas due to GIST.
Due to a small number of pancreatic GIST cases described in the literature we only have rudimentary information concerning the results of treatment of these tumors. However, existing scientific papers comparing laparoscopic treatment of stomach GIST with the procedures performed using the classic method suggest that the minimally invasive treatment allows similar results to be obtained in terms of oncology, and at the same time, it is linked with a smaller perioperative injury, less blood loss, and a quicker recovery of proper function of the gastrointestinal tract. It shortens the time of hospitalization and reduces the risk of complications [12]. Due to the very rare occurrence in the literature there is a lack of scientific research documenting the advantage of one method over the other in the case of GIST localized in the pancreas.
In the case described by us, perioperative procedures according to the ERAS concept were implemented [6]. Postoperative care based on the principles of, among others, early mobilization of the patient, early administration of per os feeding and optimal analgesia, allowed the patient to be discharged home within 48 h of the procedure.
Clinically, stromal tumors of the gastrointestinal tract are characterized by a high variability. They range from small lesions with a slow, almost stationary course, recognized often accidentally during endoscopic examinations or surgical procedures, to tumors with a very aggressive clinical picture with multiple metastases at the time of diagnosis [13]. Basic criteria allowing the determination of the degree of GIST malignancy include characteristics such as tumor size, value of the mitotic index, and lesion location [1,14]. Available literature suggests that GIST located in the stomach are characterized by a better prognosis than GIST in other locations. The limited quantity of data does not allow us to clearly determine the prognosis in the case of pancreatic GIST. However, in accordance with the above information it can be suspected that GIST of the pancreas have a worse prognosis than lesions located in the stomach. This necessitates special oncological caution in future follow-ups. In the case presented by us, the tumor was characterized by a low degree of malignancy and was radically removed. However, the short follow-up period does not allow the assessment of long-term treatment.
Conclusions
Despite the fact that stromal tumors of the gastrointestinal tract localized in the pancreas are very rare, they should be considered in the differential diagnosis of tumors of this organ. The case described by us is an example showing that minimally invasive procedures can be used in the treatment of this type of tumor within the pancreas. Nevertheless, it is necessary for the operator to have significant experience and proficiency in surgical procedures due to oncological indications. Also, patients should remain under constant postoperative care, irrespective of the degree of tumor malignancy.
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2016-05-12T22:15:10.714Z
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2015-06-10T00:00:00.000
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High-intensity exercise prescription guided by heart rate variability in breast cancer patients: a study protocol for a randomized controlled trial
Background Breast cancer is a chronic disease with a large growth in its treatments, prognosis, improvements, side effects and rehabilitation therapies research. These advances have also highlighted the need to use physical exercise as a countermeasure to reduce the cardiotoxicity of pharmacological treatments, increase patients' strength and quality of life and improve body composition, physical condition and mental health. However, new investigations show the need for a closed exercise individualisation to produce higher physiological, physical and psychological benefits in remote exercise programs. To this end, the present study will use, in a novel way in this population, heart rate variability (HRV) as a measure for prescribing high-intensity training. Thus, the primary objective of this randomised clinical trial is to analyse the effects of a high-intensity exercise program daily guided by HRV, a preplanned moderate to high-intensity exercise intervention and a usual care group, in breast cancer patients after chemotherapy and radiotherapy treatments. Methods For this purpose, a 16-week intervention will be carried out with 90 breast cancer patients distributed in 3 groups (a control group, a moderate to high-intensity preplanned exercise group and a high-intensity exercise group guided by HRV). Both physical exercise interventions will be developed remotely and supervised including strength and cardiovascular exercises. Physiological variables, such as cardiotoxicity, biomarkers, lipid profile, glucose, heart rate and blood pressure; physical measures like cardiorespiratory capacity, strength, flexibility, agility, balance and body composition; and psychosocial variables, as health-related quality of life, fatigue, functionality, self-esteem, movement fear, physical exercise level, anxiety and depression will be measure before, after the intervention and 3 and 6 months follow up. Discussion Personalized high-intensity exercise could be a promising exercise intervention in contrast to moderate-intensity or usual care in breast cancer patients to reach higher clinical, physical and mental effects. In addition, the novelty of controlling HRV measures daily may reflect exercise effects and patients' adaptation in the preplanned exercise group and a new opportunity to adjust intensity. Moreover, findings may support the effectiveness and security of physical exercise remotely supervised, although with high-intensity exercise, to reach cardiotoxicity improvements and increase physical and psychosocial variables after breast cancer treatments. Trial registration ClinicalTrials.gov nº NCT05040867 (https://clinicaltrials.gov/ct2/show/record/NCT05040867). Supplementary Information The online version contains supplementary material available at 10.1186/s13102-023-00634-2.
Background
Worldwide cancer incidence is growing each year reaching 19.3 million new cancer cases in 2020. Whereas it is expected to worsen to 28.4 million cases in 2040, an increase of 47%. Concretely, female breast cancer has surpassed lung cancer as the most diagnosed type, with an estimated 2.3 million new cases every year (11.7%), accounting for 1 in 4 cancer cases and being the fifth leading cause of cancer mortality worldwide, with 685,000 yearly deaths. However, although new advances and treatments are increasing cancer-related survivorship, estimated to be 29.1% higher in 2029 [1], breast cancer survivors had an almost twofold higher risk of dying compared with cancer-free women [2]. An aspect to consider given that by 2040 it is expected that 73% of survivors will be over 65 years of age [1].
One of the principal causes of these differences leads to cancer and treatments side-effects in breast cancer survivors increasing their comorbidity rates and decreasing patients' health-related quality of life (HRQoL) and survivorship [3]. Regarding long-term side effects, breast cancer patients may suffer, even several years after the diagnosis, cardiotoxicity [4], autonomic dysfunction [5], sarcopenia [6,7], joint pain and cancer-related fatigue [8], among others, are quite common and severe. These interrelated consequences, caused among other aspects by pharmacological toxicity [6,9] and physical inactivity, are associated with mortality [10][11][12], worse prognosis and cancer evolution [13,14] and morbidity [15]. Cardiotoxicity, the toxic effect produced in patients' cardiovascular system by some anticancer drugs, could cause the appearance of several cardiovascular abnormalities such as hypotension or hypertension, cardiorespiratory capacity decline, arrhythmias, myocardial infarction, thromboembolism, myocarditis, greater arterial rigidity, over-activation of the sympathetic nervous system and a parasympathetic decline -measured by heart rate variability (HRV)- [16][17][18]. Such is its gravity, that breast cancer survivors are at high risk of heart failure [19], autonomic dysfunction [20] and cardiovascular disease [19] up to the point of being the second most common cause of death among breast cancer survivors [21].
However, physical exercise programs, as recent literature is showing, could be an effective tool to reduce these severe side effects [22]. Although the investigations performed in breast cancer patients, not murine models, are limited it seems that exercise may increase left ventricular ejection fraction and cardiorespiratory capacity [23], decrease biomarkers such as Brain Natriuretic Peptide (BNP), high-sensitivity cardiac troponin and c-reactive protein [22,24,25] and blood pressure, and improve the sympathovagal imbalance increasing baroreflex sensibility and reducing resting heart rate [26,27]. These physiological changes have been proved to be produced specially by endurance training due to DOX-induced increases in oxidative stress and apoptosis, decreases chronic inflammation, angiotensin II, renin and atherosclerosis and increases the patient's vasodilatation [25]. To reach the mentioned effects it seems that exercise programs focused on cardiotoxicity need to involve at least 36 sessions [24]. The exercise intensity carried out in the interventions is quite heterogeneous involving most of them from moderate to high intensity [22,24,25]. Whereas, more current research, but not focused on cardiotoxicity, has found also positive psychosocial, physical and physiological effects with high-intensity exercise interventions [23,26] and it seems to be a potential tool to provide a higher glycolytic metabolism [28], induce a decrease of intratumorally lactate concentration [29] and moderate the overexpression of reactive oxygen species limiting the tumor growth or cancer recurrence and inflammation [30].
Nevertheless, most of the interventions focused their exercise programs only on aerobic training forgetting the role of muscle mass and function to reduce the toxicity produced by treatments and tumor prognosis [6], even though sarcopenia is associated with an increased risk of overall mortality in breast cancer survivors and breastcancer-specific mortality [11,31,32]. In this regard, resistance training interventions have been stated to be crucial to recovering the loss of muscle mass and muscle function caused by chemotherapy [33,34], reducing myomatosis and chronic inflammation [35], reducing oxidative free radicals and oxidative stress [35], improving body composition [36], reducing pain join and cancer rated fatigue [37], increasing cardiorespiratory fitness [23,38] and HRQoL [37], reducing mortality from cardiovascular disease [39] and all causes of death [40]. Commonly, resistance programs, performed alone or like concurrent training, lasted at least 12 weeks, utilized a moderate intensity, between 50 and 80% of 1 repetition maximum (RM) or high intensity < 85% of 1 RM [36,37].
The debate and the controversial results about optimal exercise intensity and type for breast cancer survivors could be produced due to cancer investigation and clinical practice, especially in group-based programs, all the participants from the same group train at the same corresponding intensity. In this regard, programs are not daily personalized without considering participants' daily physiological and psychological requirements. So that, those participants who do not respond correctly to highintensity programs could be because they do not tolerate the pre-planned intensity equally every day. Innovative investigations in sports sciences are starting to prescribe daily training intensity by utilizing participants' matutine heart rate variability [39] a new useful tool not employed before, as far as we are concerned, in breast cancer survivors. HRV provides a multidimensional registration of autonomic modulation [40] presenting the physiological stress and the sympathovagal imbalance, so common in breast cancer survivors [5]. Besides, some investigations have stated the relation between pain perception, so common in breast cancer patients, and HRV scorings, showing that people with high pain have lower parasympathetic parameters [41]. For these reasons, the current study will aim to prescribe high-intensity exercise involving cardiovascular and resistance training, guided daily by HRV trying to individualize exercise to patients' physiological stress.
Moreover, concerning the new habits and the current risk due to the coronavirus pandemic (COVID- 19) there is a need for novel intervention in a supervised and remote-controlled context. In this sense, it could be also one of the first exercise programs where the physical specialist control and monitor each exercise session in realtime in contrast to most remote interventions based on self-delivery exercise, physical exercise recommendations or mobile apps [42].
Aim and research questions
Therefore, given the potential benefits of physical exercise and the possibilities of optimizing how it is prescribed for cancer patients, the current randomized controlled trial aims to analyze the effectiveness of a high-intensity remote exercise program guided daily by HRV compared to a pre-planned remote moderate to high-intensity exercise program in breast cancer survivors. For this purpose, the effects of both physical exercise programs on physiological (cardiotoxicity, inflammatory factors, cardiovascular variables, body composition and anthropometric parameters), physical (low and upper body strength, cardiorespiratory fitness, low and upper body flexibility, agility and balance) and psychological health (HRQoL, fatigue, life satisfaction, self-esteem, anxiety and depression, shoulder disability perception, physical activity, kinesophobia and exercise motivation) measurements of breast cancer patients will be assessed and compared an inactive control group.
Study design
The research will be conducted following the Helsinki declaration [43] and the American Society of Clinical Oncology policy statement [44]. Patients will be randomly assigned to three groups where two of them will participate in a physical exercise intervention and the left group will continue with usual care for 16 weeks (see Fig. 1). Moreover, the study methods described below follow the Standard Protocol Items Recommendations for Interventional Trials (SPIRIT) of 2013 [45] (SPIRT schedule and checklist are shown in the Additional file 1). After the positive response from the ethics committees, the trial was registered at ClinicalTrials.gov, recognized by the World Health Organization and the International Committee of Medical Journal Editors, under the identification number of NCT05040867.
Recruitment and eligibility
A total of 90 patients are being recruited from the Oncology Service of Cantabria, concretely, they are being enrolled in the University Hospital of Marqués de Valdecilla. The enrolment consists of two phases. First, an oncologist and a radiologist select patients who fulfill the following inclusion criteria: (a) women aged between 18 and 65 years, (b) having luminal or triple-negative breast cancer, (c) having received and finished chemotherapy and radiotherapy treatment. In addition, patients are excluded if: (a) they have been scheduled for surgery during the study (b) they have HER2 + breast cancer, (c) they have severe heart disease before chemotherapy, (d) they have finished the cancer treatment more than 5 months ago, (e) they have metastatic cancer, (f ) they have any injury which prevents them to perform the intervention or the evaluation correctly, (g) they have severe psychiatric illnesses.
In the second phase, patients are called to the hospital by the oncologist and the physical exercise professional to explain in detail the study and each intervention group requirement. After answering patients' questions and asking for some technical needs for mobile phone application licenses, a complete description of the study is given to the participants.
Randomization and allocation
The study is a blinded, randomized, controlled trial. Before baseline assessments, participants are randomly assigned to one of the three groups: a group that performs the planned physical exercise program based on daily heart rate variability (HRVG), a group that performs the conventional pre-planned physical exercise program (PEG) and a control group (CG).
Ethical approval and registration
The design of this study complies with the Helsinki Declaration of 2014. Moreover, the ethical standards of the study have been approved by the ethical committee of the Rey Juan Carlos University (approval number: 1901202103121) and the ethical committee of the University Hospital of Marqués de Valdecilla, named Valdecilla Health Research Institute (IDIVAL) (register identification number: 42//2021). Both, the study registration and the positive ethics committee approval were carried out before the recruitment. Besides, all the participants are informed about the relevant aspects of this study before starting their program including the potential benefits and risks of practicing exercise. Any change in the protocol will be expressed on the clinical registration website of ClinicalTrials.gov.
Sample size
Given that, to our knowledge, no previous studies have examined the effects of an exercise program guided by daily HRV in breast cancer patients and its comparison to a moderate to high-intensity exercise group and a control group, the sample size was calculated by considering the exercise intensity differences between groups. In this regard, we have taken as reference values interventions carried out with three groups, a high-intensity group, a moderate-intensity group and a control group. Moreover, the oxygen consumption peak (VO 2peak ) has been selected to make the calculations due to its importance in cardiovascular and physical health. The chosen tool to analyze sample size has been using G-Power 3.1 and the calculation were done basing us on the data from the investigations carried out by Martin, Battaglini [46] and Northey, Pumpa [47] two different studies. In both cases, between-group significant differences were reported. A total of 18 patients, (11 allocated to the experimental and 11 allocated to the control group) would be needed in the worst of the cases to achieve a 99% statistical power for VO 2peak . Moreover, regarding the low number of participants obtain, the calculations concerning the effects of exercise in Global Longitudinal Strain (GLS) compared to a control group were also done. In this case, the data employed for the calculation was from Costello, Roberts [48] where significant differences within and between groups were achieved. So, in the prior calculations performed, we would need a total of 50 patients [25 in each group] to achieve a 99% statistical power (two tails). Therefore, assuming 20% of dropouts, 90 patients will be recruited and allocated to one group or the other. Due to the large number of patients that are estimable to be enrolled in the study, the recruitment and intervention will be carried out progressively over 24 months depending on the number of patients to be recruited.
Physical exercise programs
First, the common aspects between the two exercise groups are going to be mentioned, to explain afterwards the peculiarities of each physical exercise program. The programs of both experimental groups, PEG and HRVG, will last 14 weeks, and patients will train three times per week. All sessions will be supervised and remote. So that, each patient will connect with the physical exercise professional and with the other 4 participants by videocall. Moreover, the practitioner will monitor their heart rate in real-time because each patient will be given a MyZone heart rate (HR) control and platform access to MZ-Remote. In this way the professional will be able to adjust their workout intensity according to their HR, in the cardiovascular exercises and the weightlifting load in the resistance exercises; and to control and correct, participants' posture and technique. Moreover, these measures will be determinant to asses participants' exercise adherence. During the sessions, participants will be able to see their HR and HR of the rest of their training group. In addition, to promote social interaction an extra link will be given to those patients which want to see each other while practicing exercise. The necessary material to perform the exercise programs will be given to each participant in the before-intervention evaluation. This training kit will include a barbell of 2 kg, 2 weight discs of 5 kg, 2 weight discs of 2.5 kg, 2 weight discs of 1.25 kg and a Myzone's MZ-3 Belt chest strap.
Each session will last approximately 60 min and will include a warm-up, a main part composed of cardiovascular resistance exercises and a cooldown. The warmup, lasting from 5 to 10 min, will include upper, lower body and truck mobility, and core muscular activation. The cooldown part will last about 5 min and will combine stretching of the muscles involved during the corresponding session. Moreover, during the stretches, breathing feedback will be given to fully relax and enhance elongation during the exhalation phase.
As for the main part of the sessions, lasting from 45 to 50 min approximately, the training schedule will be divided into three mesocycles. The first, with a duration of 4 weeks, will be focused on neuromuscular adaptation, learning exercise techniques and improving participants' aerobic capacity and HR self-control. In this regard, the exercises proposed in the period will be of low complexity and will be performed with the lowest program intensity. In the 5th week will begin the second mesocycle until week 10th when the intensity will increase progressively including exercises with greater technical complexity. In the last mesocycle from weeks 11th to 16th participants will experiment with the highest level of technical complexity and intensity archiving the highest weight load and cardiovascular intensity. In this way, the sessions, and their progression, will be designed according to the principles of training to achieve the greatest possible adaptations [49]. Concretely, during the first mesocycle, the participants will alternate two training circuits and during the second and third mesocycle patients will combine 3 different circuits, one each training day of the week. Part of the exercises has a cardiovascular component through intervallic training. These exercises will be combined with resistance training by performing them either at the beginning of the circuit, or between the strength exercises, or at the end of the strength circuit.
The cardiovascular component of the exercise program will be performed with different exercises such as: walking with knees up, jogging in place, skipping, sideways running by changing direction, jumping jacks, etc. The duration of the cardiovascular and its intervallic duration will vary in each mesocycle. In the first one, participants will perform 2 sets composed of 4 repetitions of 1 min with 45 s of rest between intervals and 2 min between sets. In the second and third mesocycles, the cardiovascular sets will be conducted between each resistance exercise. In this way, each circuit repetition will include 5 sets composed of 3 intervals of 30 s with 10 s of resting between repetitions in circuits 3, 4, 6 and 7. Whereas in circuits 5 and 8, which will be carried out on the third day of the training week of the second and third mesocycle, the intervals will be developed at the end of all the resistance exercises. Furthermore, every 6 weeks, on the third training day, the cardiovascular interval part will be done with dancing choreographies with the songs chosen by participants. Furthermore, every 5 weeks, on the third training day, the cardiovascular interval part will be done inside dancing choreographies with songs chosen by participants.
Regarding the resistance exercise of the training program, exercises will combine lower body strength with a predominance of both knees (e.g. squat or stride) and hip (e.g. deadlift or hip thrust) and upper body strength exercises of pushing (e.g. chest press, shoulder press or arm-triceps extension) and gripping (e.g. rowing or biceps curl). As mentioned, participants will progress in the complexity of the exercises, for example, from a squat starting from a chair to a barbell squat, or from a gluteal bridge to hip thrust. Table 1 shows a programming example that can serve as a reference for a better understanding of what the intervention will entail.
Pre-planned remote moderate to high-intensity physical exercise program The current exercise group will perform exercise following the recommendations of several and important literature such as the American College of Sports Medicine guidelines carrying the exercise with an intensity going from moderate to high intensity. Concretely in the cardiovascular exercise, the participant will start with an intensity of 65% of their reserve HR, in the first mesocycle; and will end with an intensity of 80% of their reserve HR. In the resistance exercise, they will commence lifting weights corresponding to their 55% RM and finish in the last mesocycle lifting loads matching their 70% RM.
High-intensity remote physical exercise program guided daily by HRV Although all participants will daily measure their HRV, only in this intervention group each day's results will influence their exercise intensity. From the outcomes reported with smartphone photoplethysmography, explained the measurement process in the outcomes part, the root means squared differences of successive RR intervals (rMSSD) will be chosen as a reflection of vagal activity to prescribe the workout intensity [50]. In this regard, the natural logarithm of rMSSD will be calculated to make parametric statistical comparisons assuming a normal distribution. For establishing the training intensity and load, a 7-day rolling average measure (LrRMSS- D7day-roll-avg) will be utilized. Subsequently, the scoring obtained will be contrasted in the smallest worthwhile change (SWC) to analyze if the daily result is inside SWC upper and lower limits, calculated as LnrMSSD reference week mean ± 0.5 × SD [51,52]. The reference week of the first 4 training weeks will take the limits created in the baseline week, and in the rest of the program, an updated measure will be carried out every 4 weeks with the SWC for the past 4 weeks, because of the influence of exercise in participants cardiac autonomic modulation [53].
In this regard, when the daily LnrMSSD fell inside the limits created by the SWC patients will perform highintensity training characterized by a cardiovascular intensity from 80 to 95% of their reserve HR (increasing a 5% every four weeks) and a weighting load from 70 to 85% of their RM (increasing the load 5% ever mesocycle, four weeks). Whereas if any day patients' scoring fell out of the SWC, they will train at the same intensity as the PEG (Fig. 2).
Usual care control group
Participants who will be allocated to the CG will participate in the before and after intervention assessments will measure daily their HRV. They will be advised to continue with their regular activities of daily living without any exercise restrictions.
Outcomes
The following outcomes will be assessed in person by the oncology group at the hospital (Marqués de Valdecilla Hospital) or by a physical exercise professional at the sports center (GOfit) when it corresponds. The evaluations will be carried out before, after the intervention and 3 and 6 months after the end of the intervention to all the participants (HRVG, PEG and CG). To ensure CG measurements and follow up evaluations the principal investigator will call or write participants each month.
Physiological outcomes
Cardiotoxicity measurements Cardiotoxicity will be assessed at the hospital to have a more exhaustive control and safety of the participants and at the end of the program. On the one hand, ç hemograms will be carried out to assess of High-Sensitivity Cardiac Troponin, Troponin I, N-terminal portion of B-type natriuretic pro-peptide (NT-proBNP), Troponin T due to their predictive values of cardiac damage in patients under the effects of chemotherapy [54,55]. Although these variables were primarily planned in the trial registration, the hospital will be only able to assess High-Sensitive Cardiac Troponin and NT-proBNP.
In addition, an echocardiogram will be performed to measure the left ventricular ejection fraction and GLS due to its modification caused by cardiotoxicity [56][57][58] together with mean and maximum aortic valve gradient, ventricles' diameters and thicknesses and valve velocities because their possible aortic stenosis risk [59]. Moreover, a resting electrocardiogram will also be performed on each patient to obtain values for heart rate, heart rhythm, heart rate variability, I-axis and aVF, Q-T interval, QRS complex, S-T segment and T-wave [60].
Measurement of biomarkers
On the one hand, the measurement of the following biomarkers will be included: Tumor Necrosis Factor, interleukins IL-6, IL-8 and IL-1b, C-reactive protein, creatine kinase, global lactate dehydrogenase, alkaline phosphatase, bilirubin levels, monocyte chemotactic protein and vitamin D. On the other hand, the anti-inflammatory cytokines IL-1ra and IL-10 [61]. As occurred in the cardiotoxicity variables, the interleukins will not be able to be measured at the hospital. Lipid profile and glucose measurement Apart from the mentioned outcomes, other variables from the patients' blood tests related to cancer prognosis and cardiovascular risk will be taken. Triglycerides, low-density lipoproteins and high-density lipoproteins will be collected due to their association with cancer prognosis and mortality [62]. Relatedly, fasting glucose will be analyzed because of its relation to metabolic syndrome in breast cancer survivors [63]. Other serum and hematological analyses will be done to control exhaustively each of the patients.
Blood pressure and resting heart rate measurement Blood pressure measurements will be taken using a validated oscillometer Omron Healthcare Oscillometer [64]. The participants will remain at rest for five minutes before the assessment and will be lying in a supine position with the legs and arms completed. They will place their left or right arm, depending on the affected breast, straight, so that the cuff is at the level of the heart, 2 cm from the elbow. In addition, any clothing that may alter the results will be removed. Once in this position, the air tube of the cuff will be placed on the front of the arm aligned with the middle finger and with the blue date on the cuff. Systolic and diastolic blood pressure data will be taken; and, subsequently, the mean arterial pressure will be calculated by the following formula [(systolic blood pressure + (2 × diastolic blood pressure))/3] [65]. The resting heart rate will be measured for 5 min. The participants will lie relaxed in the supine position on a stretcher isolated from the floor. This data will be essential for the correct prescription of the intensity.
Heart rate variability and heart rate in rest measurements The assessment of heart rate in rest and HRV will be performed by the Polar H10 chest strap and by photoplethysmography (PPG) with the validated mobile app of HRV4training [50,66]. The participants will remain in the supine position for 8 min to obtain 5 min of a stable signal. After removal of artifacts, by employing the Kubios ® clinical software the temporal variables of standard deviation time domains of all RR intervals (SDNN), rMSSD, Average of all NN intervals (AVNN) and percentage of differences between adjacent NN intervals that are greater than 50 ms (pNN50) will be calculated [67]. Moreover, by also utilizing Kubios software the frequency measurements of low frequency (LF, 0.04-0.15 Hz), high frequency (HF, 0.15-0.4 Hz) and the LF/HF ratio will be obtained [67]. The HRV4Training app directly calculates the HRV outcomes.
On the other hand, daily heart rate variability (HRV) is measured using the HRV4Training application, a validated mobile application [50,66] that allows HRV values to be obtained by PPG (rMSSD, LF, HF, SDNN, SDNN, AVNN, pNN50 heart rate and recovery points). The morning measurement will be performed every day, in the supine decubitus position upon awakening, for 1 min. During the basal week before starting the intervention, participants will measure their HRV, to establish the reference values for each participant of the GHRV group. In the intervention weeks, the participants will measure their HRV every morning, therefore, collecting morning HRV data (rMSSD, heart rate and recovery points). The value that will be taken into consideration to establish the GHRV training intensity will be the rMSSD as it represents the parasympathetic activation of the central nervous system. The detailed information of its utilization to exercise prescription is explained in the exercise program intervention part. To eliminate the placebo effect of the use of the application, the participants of the three physical exercise groups will perform this protocol. In this way, the HRV data of all the participants will be collected to analyze their evolution with the different interventions. To reduce measurement error, the application functioning is explained to all participants, mentioned to repeat the measurement if a message saying that the sign was not optimal or poor the need to repeat it. Moreover, the exercise professional will check the quality of the sign before its analysis.
Body composition measurement
The assessment of the percentage of fat and the visceral level of the participants are variables to be taken into consideration, due to the existing risk in women with overweight and obesity, of a greater relapse in breast cancer [68] and the possible effective role of high-intensity exercise [69]. Therefore, by utilizing the impedance Inbody analyzer [70] the body fat mass [68], body fat percentage, visceral fat level, musculoskeletal mass, body water, the bone mineral content, segmental fat mass (trunk, right and left arm and right and left leg) and segmental lean mass (trunk, right and left arm and right and left leg) of the patients will be evaluated [71]. In addition, all participants will be weighed and measured to obtain their Body Mass Index (BMI) by the formula of [weight/height 2 ].
Measurement of anthropometric parameters
Patients will use a non-elastic tape measure for the evaluation of waist circumference, hip circumference, chest circumference, arm circumference, leg circumference following the guidelines set by the American College of Sports Medicine [72].
Physical exercise evaluations
Cardiorespiratory fitness measurement To record the cardiorespiratory capacity, maximum heart rate and the patients' perception of effort, the Bruce incremental sub- maximal test (modified) [73] will be performed on a treadmill. During the test, the patients will wear a Polar H10 chest strap to monitor their heart rate. Thus, following the protocol created by Bruce, the treadmill will start with a 0% incline and a speed of 1.7 m/h which will be maintained during the first 3 min. Every 3 min both the speed and the incline will be increased until the participants decide that their fatigue is too high to continue. In addition to constant heart rate monitoring to record the maximum heart rate, the patient will be asked about her perception of exertion every minute. By acquiring the maximum heart rate we can then prescribe the target intensity from the reserve heart rate using Karvonen's formula [74]. At the end of the test, the recovery heart rate will be recorded 1 min after stopping the test in order to assess the recovery rate. Subsequently, the maximum oxygen consumption of each patient (VO 2max = 2.282* (time) + 8.545) will be calculated from the formula proposed by the creators. This submaximal test has been commonly used in cancer patients in different exercise interventions [75].
Lower and upper body strength measurement Lower body strength resistance. For the measurement of the lower body strength resistance, the 30 s chair stand test will be used. The patients will use a chair without armrests, with the back against the wall so that it does not move during the test [76]. The evaluation will begin with the participant seated in the middle of the chair with arms flexed at the chest and hands resting on the opposite shoulder of the chair. The patient will stand up, positioning herself fully stretched, and will sit the maximum number of repetitions possible in 30 s. During the development, the patient will be encouraged and reminded of the need to sit down completely for each repetition. The number of repetitions achieved is recorded. This test has been performed previously in breast cancer survivors [37]. Upper body strength resistance. As for upper body strength-endurance, the patients will be measured by the 30-s arm curl test of the Senior fitness Manual [76]: a 2.5 kg weight will be used according to the Senior fitness Manual. The participants will be seated in a chair and with the dominant arm, they will have to perform elbow flexion extensions starting in full extension. The test will report the maximum number of repetitions in 30 s [76]. This assessment has been used previously in the breast cancer population [77,78].
Lower and upper maximum force, velocity, and power. The evaluation of the maximum force will be carried out by using a linear encoder for the correct calculation of the maximum weight that the patient can lift in one repetition (RM) from the speed and power generated when moving the weight vertically. For the easy and correct performance, the different tests will be carried out on a multipower machine with a counterweight mechanism which will guide the vertical movement in a rectilinear way without the influence of the barbell weight.
The participants will begin to perform each of the exercises using the barbell as the only weight for 3 repetitions as a warm-up. After, at least three incremental weight discs will be added depending on the exercise and the patient's perception of effort and the speed of execution decline. At the end of each repetition, patients will be asked about their perception of perceived exertion using the modified Borg scale (Rate of Perceived Exertion, RPE) [79] and when this value has dropped to 2/10, the next weight will be added.
Diverse variables will be analyzed in each of the repetitions by employing the Chronojump by Boscosystem linear encoder, the MyLift mobile app [80] or the kinovea software [81] See supplementary table. So that, each participant's repetition will be recorded by an iPhone camera of 240 frames per second. This protocol will be repeated for each of the following exercises: • Barbell Squat (Back Squat Test). Participants will hold the bar behind the back of their neck with the weight resting on their shoulders. If they have difficulty maintaining this position, the evaluation will be done by holding the bar in front of them under their neck with their arms crossed. Start with your knees fully extended and your feet shoulder-width apart. You will progressively bend your knees, without exceeding the line of the toes, until you reach 90° of flexion. Once you have reached this point, you will recover the initial position by extending your knees. • Deadlift Test. Participants will begin the movement with feet hip-width apart, hands in the centre of the bar (placed on the floor) and shoulder-width apart, the bar will be placed close to the shins and the knees bent until the hands can grasp the bar. The chest should remain upright and the back straight throughout the exercise. The hips should be above the knee so that the shoulders are positioned in front of the bar. The movement starts with a slight knee extension, followed by a hip extension and ends with a simultaneous knee and hip extension until the shoulders are positioned behind the bar, thus aligning the whole body [82]. • Lunge Test. Participants will start the movement by placing their feet shoulder-width apart, holding the bar behind the back of their neck, leaving the weight of the bar on their shoulders. They will take one step forward. The distance of this will correspond to the distance from the superior iliac spine to the medial malleolus of the tibia. Once this distance is reached, bend the knees, touching the floor with the back knee and reaching 90° of flexion in the front knee. Subsequently, they will recover the initial position by fully extending the front leg and progressively transferring the weight of the body to the back leg [83]. • Chest press (Chess press). Participants lie on the bench with their legs bent to the sides of the bench at slightly more than 90°. They will grasp the bar with their hands wider than shoulder-width apart. The movement will begin with the elbows fully extended and with the bar at throat level. Progressively lower the bar by bending the shoulders, followed by bending the elbows until the bar reaches one centimeter from the chest. Once the maximum flexion is achieved, they will extend the elbows and shoulders until they return to the initial position [37,84]. • Shoulder press. Participants sit on the bench with their backs against the backrest. Start the movement by grasping the bar with elbows fully extended with hands 5 cm wider than shoulder-width apart. Lower the bar continuously until it comes into contact with the upper chest. Then start the concentric phase by extending the elbows completely to the starting position [85]. • Bent over row. Participants perform the exercise with their backs bent at 45°. They begin the movement by holding the bar in front of their shoulders with their hands shoulder-width apart. Proceed to bend the elbows, bringing them behind the body until the barbell touches the middle of the abdomen [86]. • Biceps curl with barbell. Participants will stand in an anatomical position with knees slightly bent and feet apart. They will hold the bar with a supine grip, arms parallel to the sides of the body. Begin the movement with elbows fully extended and progressively bend the elbows to their maximum. Subsequently, they will extend their elbows to the starting position [87].
Finally, the strength-power will be measured by employing a countermovement jump test (CMJ) and from squat (SJ) to evaluate the explosiveness of the patient, as has been done in previous studies on patients with breast cancer [88]. it was decided to eliminate the assessment of hip trust and triceps barbell strength, included in the clinical registration because performing all the tests would take too long for the patients.
Vertical applied force. The force-power will be measured utilizing a CMJ and from SJ as a reflection of the application of force in the unit of time, inter and intramuscular coordination and the use of the elastic energy of those actions in which the stretch-shortening cycle of the lower limbs predominates, which is present in the majority of actions in daily life, as has been carried out in previous studies in patients with breast cancer [88]. In the evaluations after the intervention, those patients in whom it is possible to evaluate the jump with progressive loads, the vertical force-velocity profile will be performed, which will give us information on how the subject is currently producing power and how it should (based on their characteristics) in order to be able to evaluate the explosiveness of the patients [89]. To obtain it, together with the SJ and CMJ jumps without load, the patients will perform 2-3 more attempts with progressive loads, determining the increase in load according to the height of the jump without weight [90]. In addition, for the corresponding calculations, two body measurements will be taken to determine the push-off distance (pod). For the evaluation of the different variables indicated, different technologies will be used such as the Chronojump's contact platform and the recording with an IOS camera of 240 f.p.s of each of the jumps for subsequent analysis with the validated application of My Jump [91] and Kinovea [92].
Horizontal applied force. For the evaluation of this manifestation, of force, an acceleration action will be recorded (the only requirement is to start from speed 0 and to cover a distance of 30 m in the shortest possible time, the time used not being a limitation for the performance of the test). This recording will be used to calculate the horizontal force-velocity, which will give a mechanical description of how the patient has applied forces to move horizontally. For the complete assessment of the horizontal profile in the post-intervention evaluation, patients will perform 3 progressive 30 m sprints by running or walking (at 50%, 70% and 90% of the participants' maximum self-perceived speed) [93]. For this, an IOS device with 240 frames per second will be used and subsequently analysed with the validated App for IOS devices (My Sprint) [94]. The device, iPhone will be mounted to a tripod (in the frontal plane) to film the sprint from the side, at the 15 m marker and at 18 m from the track, to register the entire sprint. Since the iPhone 6 was in a fixed position, video parallax was corrected to ensure 5 m, 10 m-, 15 m-, 20 m-, 25 m and 30 m split times were measured properly.
Measurement of agility and balance
The Timed up and go test will be employed to assess patients' agility as it has been done before in breast cancer patients [95]. For its performance, a chair will be placed against the wall and a cone will be placed in 3 m from the front edge of the chair to the back of the cone. The patient will sit in the middle of the chair keeping her back straight, her hands on her thighs and both feet on the floor. At the "go" signal, the participants will get up from the chair and walk as fast as possible to the cone, and around the cone, back to the chair. The time taken to complete the test will be recorded. The test will be repeated twice, and the best score achieved. The One-leg stand test belonging to the ALPHA battery in adults [96] will be used to evaluate static balance. To do this, the patient will be placed in monopodal support, placing the sole on the inside of the knee. The test will be performed first with the right feet on the ground and then with the left. Patients will be able to use their arms to balance only when necessary.
Lower and upper body flexibility measurement Hamstring flexibility will be measured using the V Sit and Reach test [8] where the participant will be seated on the floor with knees fully extended and feet 30 cm apart. Place a tape measure on the floor, equidistant between the feet, placing the 0 cm in line with the heels. Putting the palms of the hands together and keeping the elbows fully extended, the participants will bend their trunks as much as possible on the tape measure. The missing centimetres or the centimeters that are passed from their maximum bending point and the 0 cm will be noted.
Upper body flexibility will be evaluated by utilizing the Back scratch test, previously used in breast cancer patients [95] and belonging to the Senior Fitness Test battery [76]. During the test, the participants will be placed in a standing position, having to try to reach above their head, with their arm in flexion and external rotation, to touch their other hand, which will be placed on their back in supine position with the arm flexed [95]. The test will be performed first with the arm through which the chemotherapy was administered and then repeated with the other arm.).
Psychosocial and physical exercise outcomes
Psychological variables will be assessed utilizing selfadministered questionnaires specifically validated for cancer patients.
Health-related quality of life measurement To assess HRQoL patients will complete the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) [97] which includes functional and cancer-specific symptom assessment and the results will be analyzed following the instructions given in its manual [98]. Concretely, the QLQ-C30 is composed of both multi-item scales to assess functional, symptoms and global health status and six single-item measures to score the left symptoms. Inside the functional outcomes, 6 items correspond to physical function, 2 to functional role, 4 to emotional function, 2 to cognitive function and 2 to a social function. Moreover, the assessed symptoms will be fatigue (3 items), nausea and vomiting (2 items), pain (2 items), dyspnoea (1 item) insomnia (1 item), appetite loss (1 item), constipation (1 item), diarrhea (1 item) and financial difficulties (1 item). All the scales and single-item measures range in score from 0 to 100. A high scale score represents a higher response level. Thus, a high score for a functional scale represents a high/healthy level of functioning, a high score for the global health status / QoL represents a high QoL, but a high score for a symptom scale/item represents a high level of symptomatology/problems [23].
Cancer-related fatigue measurement For the assessment of cancer-related fatigue, the fatigue subscale of Functional Assessment of Chronic Illness Therapy-Fatigue questionnaire [99] will also be used and analyzed according to the instructions of its creators [100]. The questionnaire is composed of 13 items where participants would answer a Likert scale of 4 points being 1 "Not at all" and 4 "Very much". The questions are asked about perceived fatigue in the last 7 days. The score could range from 0 to 52, so that, higher scorings represent high fatigue symptoms.
Life satisfaction, self-esteem, depression and anxiety measurements Life satisfaction will be assessed using the Satisfaction With Life Scale questionnaire [101], validated in Spanish breast cancer patients [102]. The scale is composed of 5 items to answer with a Likert scale from 1 to 5 being 1 "strongly disagree" and 5 "strongly agree". Higher scores mean higher satisfaction with life [101]. In addition, the Rosenberg Self-Esteem Scale [103] employed before in cancer patients [104] will be utilized for analyzing Spanish breast cancer patients' self-esteem. The scale, validated in Spanish [105], comprises ten items with a Likert scale of four options. According to the sum of their items, the scale is divided into three ranges: normal or high selfesteem (between 30 and 40 points), medium self-esteem (26-29 points), and low self-esteem (< 26 points). It has a Cronbach's alpha of 0.87 and a reliability of 0.74 [105].
In addition, the Hospital Anxiety and Depression (HAD) Scale will be used and analyzed according to the authors' indications [106]. This questionnaire was validated in Spanish oncological patients to obtain results regarding anxiety and depression [107]. The HAD scale consists of 14 items referring to participants' perceptions regarding the last week. The odd-numbered items make up the anxiety subscale and their response scale are scored from 3 to 0. The even-numbered items make up the subscale of depression and are scored from 0 to 3. The total score in each subscale is obtained by adding the scores of the corresponding items, with a range each from 0 to 21. In both cases, the higher the score, the higher the level of anxiety or depression [106]. Shoulder mobility disability and kinesiophobia measurements For the assessment of their perception of disability due to shoulder mobility so common after breast cancer treatment, the Quick Disabilities of the Arm, Shoulder and Hand questionnaire will be used [108,109]. The scale is composed of 11 items graded from 0 to 100 asking patients to rate the level of difficulty and pain in performing several tasks over the past week. Patients will answer each question scoring from 1 to 5 selecting 1 when the activities are performed with no difficulty and 5 for those activities unable to perform with "extreme difficulty". Relatedly, fear of movement will be measured using the 11-items Tampa Kinesiophobia Scale [110,111]. The scale has two subscales, one focused on activity avoidances (reflecting the belief that activity may result in injury or increased pain) and the other concerning somatic focus (indicating the belief in underlying and serious medical problems). The total score ranges from 11-44 points with higher scores indicating greater fear of pain, movement, and injury [111]. Items on the TSK-11 are scored from 1 (strongly disagree) to 4 (strongly agree) [111].
Physical activity and motivation for exercising measurements Finally, the International Physical Activity Questionnaire be administered to evaluate the level of physical activity of the patients in the assessment periods [112]. The 8 questions refer to the time patients spent being active in the last 7 days to analyze the time spent in making low-intensity activities, moderate-intensity activities, high-intensity activities and sedentary activities [112]. In addition, the existence or not of behavioral change/motivation towards exercise will be assessed using the Behavioural Regulation of Exercise Behaviour Scale-2 [113]. This questionnaire is composed of a 19-item scale with five factors (amotivation, external, introjected, identified and intrinsic motivation) [114]. Patients will reflect their opinion by a Likert scale from 1 to 5 corresponding 1 to "strongly disagree" and 5 to "strongly agree".
Statistical analysis
All the outcome results will be included in an anonymous database to conduct the statistical analyses. A descriptive, quantitative, and graphics analysis will be performed of all the included variables. The statistic software employed will be the IBM Statistical Package for the Social Sciences (version 25.0; SPSS, Inc., Chicago, IL, USA) [115]. The number of statistical analyses will be high and diverse depending on the nature of the results. In general, we will proceed with the usual steps to check the normality and homogeneity of variables. First, the Kolmogorov-Smirnov and Shapiro Wilk tests will be used to determine whether parametric or non-parametric statistical analyses should be applied to each of the variables analyzed. In this regard, normal distribution will be assumed when the p-value is greater than 0.05 indicating that the variable tested was not significantly different from a normal distribution. Descriptive results will be presented as mean and SD or median (range) according to the results of the normality tests. Subsequently, to evaluate the effects of the program, a repeated-measures ANOVA or Kruskal-Wallis, depending on the normal distribution, will be used. The interaction factors of the ANOVA, or Kruskal-Wallis, will be the group (HRVG, PEG or CG) and the timing of the measure (before, after the intervention and follow-up). Besides, a Bonferroni analysis will be performed to adjust the results. Results shall include the Cohen's d effect size (95% confidence interval) and statistical significance for each dependent variable concerning time and its interaction effects (group × time). Moreover, to analyze the differences between the group independently in each of the variables, the T-test for Independent Samples and the T-test for related samples, or a Mann-Whitney U-test or Wilcoxon in case of not fulfilling the principle of normality, will be employed.
Concurrently, a correlation analysis is planned to be done to establish novel relations between some of the measures performed. For instance, a Pearson o Spearman analysis (depending on the results of the normal distribution analysis) will be calculated between the change in strength, the body composition, cardiotoxicity variables and the psychological variables. Moreover, a correlation between the results obtained in the different questionnaires will be calculated.
Discussion
The present study will be the first, as far as we are concerned, on evaluating the physiological, physical and psychosocial effects of high-intensity exercise guided by HRV in contrast to pre-planned exercise and usual care. In addition, it will also distinguish because of its novelty in analyzing cardiotoxicity effects, together with a detailed physiological, physical and psychosocial study, in high-intensity exercise versus moderate to high intensity and in performing a remote, but supervised in real-time, intervention.
First, as has been mentioned in the article, all the patients will register daily their HRV measurements. Some studies have been carried out before analyzing the effects of exercise in autonomic modulation variables in breast cancer patients but none of them have registered daily participants' measurements [116]. In a meta-analysis recently published, the data obtained showed significant differences between the exercise and the control groups in all the HRV variables analyzed (SDNN, rMSSD, LF, HF, LF/HF ratio) although it could be noticed that interventions involving higher intensities reached greater effects [116]. In this regard, as in the current study patients in the HRVG will perform high-intensity training, we expect to get at least the same HRV positive modifications or better due to the higher intensity reached by the meta-analysis participants was 60% VO 2max [117,118] or 13-14 RPE [119,120]. Moreover, as it has been developed before in athletes [39], but not in clinical populations, the intensity of HRVG participants will be adjusted according to rMSSD values, as a reflection of the parasympathetic activity. rMSSD suffers normally a decrease during and after cancer treatments [121,122], which exercise may regain by a decrease of renin and angiotensin, affecting the renin-angiotensin-aldosterone system; a decline of oxidative stress and inflammation caused by the increase in catecholamines impacting, therefore, in tumour cell microenvironment by an exponential decrease of reactive oxidative species and an increment in antioxidants [61,[123][124][125][126]. In this way, to get an rMSSD increase and adapt to their training load to their physiological stress, if patients' rMSSD goes beyond their individual limits, they will not perform in those days high-intensity and will follow PEG intensity until their values return to their corresponding normality [127]. This novelty way to prescribe and adapt high-intensity exercise to patients daily could provide a new tool to reach higher benefits. Two recent meta-analyses have stated that high-intensity exercise does not produce higher positive changes in the VO 2max [23] and HRQoL [26] of cancer patients than moderate intensity. In this regard, we hypothesize that maybe not all patients reacted equally to high-intensity, and they need more individualization concerning their disease's long-term side effects, especially the related cardiovascular abnormalities.
Relatedly, the second aspect to highlight is the novelty of the exercise interventions compared to ones perform so far analyzing cardiotoxicity variables. To date, from our perspective, is the first study that will apply high and moderate to high-intensity cardiovascular and resistance training in breast cancer patients to analyze the cardiotoxicity effects, among others. Furthermore, except for one study protocol recently published [128] and a few studies in rats [129,130], it would be also one of the first of including resistance training with so high weight and volume loads. Moreover, regarding exercise timing, the exercise programs focused on the improvement of cardiotoxicity variables such as LVEF, GLS, or troponin, most of the studies are carried out during breast cancer patients' treatments [131] and not after them as our study will perform. However, cardiac abnormalities in breast cancer patients may appear within the first year after the end of chemotherapy, in case of early cardiotoxicity, or after several years following the completion of treatment (median of 7 years) [4]. Accordingly, to improve patients' cardiorespiratory fitness, troponin and GLS values and maintain the levels reached by acquiring physical exercise habits, in the current study patients have recently finished chemotherapy and radiotherapy to prevent the potential early and late cardiotoxicity. We will try to teach patients how to train during the 16 weeks of the exercise programs and to generate positive and self-determination experiences through exercise [132]. So that, we will promote exercise adherence to continue exercising even after its end knowing their cardiovascular and resistance levels at the end of the programs. Moreover, maybe if exercise programs are developed uniquely during chemotherapy or before it, patients can associate exercise to unpleasant cancer treatments or phases being unlikely to maintain exercising after treatments.
The final aspect of the study to be highlighted, but not less important, is the novelty of the exercise programs modalities. Common exercise interventions are carried out supervised by in-person training or unsupervised by home-based exercise, but none have involved remotely supervised exercise programs. The COVID-19 situation has forced physical professionals to adapt exercise programs to online supervision, whereas in breast cancer patients, only a case study has reported its development during the pandemic where, by video calls, two patients made cardiovascular and resistance training with the intensity controlled by HR monitors controlled by patients [133]. In this line, another intervention, involving aerobic exercises, was carried out in-person at the begging, but the covid situation restrictions obliged them to transform the rest of the intervention supervised remotely by video calls [134]. In contrast, our exercise interventions are supervised from the beginning and HR is monitored in real-time by the physical professional. However, the rest of the home-based investigations, to the best of our knowledge, are not supervised by video calls, so real-time corrections and HR control are not commonly done. In these cases, interventions are usually based on physical activity recommendations or aerobic exercise such as walking [135] and very few include resistance training [136,137] where counseling is performed by weekly calls or diaries, exercise is controlled by RPE or pedometer [137] and exercises intensity is commonly prescribed from low to moderate intensity. In this regard, the only home-based high-intensity intervention, carried out unsupervised-unlike ours-, was developed by Ochi, Tsuji [138] where patients trained for 10 min 3 times per week including bodyweight exercises in a smartphone application.
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2023-03-08T15:19:31.226Z
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2023-03-08T00:00:00.000
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238583718
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pes2o/s2orc
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Unsupervised Neural Machine Translation with Generative Language Models Only
We show how to derive state-of-the-art unsupervised neural machine translation systems from generatively pre-trained language models. Our method consists of three steps: few-shot amplification, distillation, and backtranslation. We first use the zero-shot translation ability of large pre-trained language models to generate translations for a small set of unlabeled sentences. We then amplify these zero-shot translations by using them as few-shot demonstrations for sampling a larger synthetic dataset. This dataset is distilled by discarding the few-shot demonstrations and then fine-tuning. During backtranslation, we repeatedly generate translations for a set of inputs and then fine-tune a single language model on both directions of the translation task at once, ensuring cycle-consistency by swapping the roles of gold monotext and generated translations when fine-tuning. By using our method to leverage GPT-3's zero-shot translation capability, we achieve a new state-of-the-art in unsupervised translation on the WMT14 English-French benchmark, attaining a BLEU score of 42.1.
INTRODUCTION
Recent work on generative pre-training has shown that with sufficient data and scale , large language models (LMs) can learn a diverse suite of tasks without explicit supervision (Radford et al., 2019), and that even stronger performance on these tasks can be elicited using few-shot demonstrations . While few-shot prompting is flexible and enables strong performance on a diverse suite of NLP tasks to be coaxed out of generatively pre-trained LMs without further fine-tuning, its benefits are most pronounced with larger models, with commensurate training, inference, compute, and data costs. Furthermore, the very generality of the pre-training objective which enables multi-task learning can produce LMs with more knowledge than is immediately apparent, requiring carefully designed prompts to bring out fully. The desire to unlock and amplify these latent abilities while also reducing the cost of few-shot prompting motivates our present work, which allows us to continue fine-tuning our models, obtaining more performance from smaller models and pushing our larger models even further, without resorting to few-shot prompting at test time or any additional supervision at train time.
We target the domain of unsupervised neural machine translation (NMT), which typically involves bootstrapping a weak translation model before amplifying its translation ability via backtranslation. Recent work in unsupervised NMT has been dominated by large encoder-decoder architectures where the bootstrap is implemented by denoising/autoencoding tasks (e.g., multilingual Cloze (Devlin et al., 2019;Conneau & Lample, 2019), masked-span prediction (Raffel et al., 2020;Xue et al., 2021), reconstruction from corrupted inputs (Wang et al., 2019;) intended to produce strong encoders and aligned multilingual representations for decoding. In our present work, we show that generative language modeling alone can implement the entire unsupervised NMT pipeline, and derive state-of-the-art unsupervised NMT systems using only generatively pre-trained language models. We implement the bootstrap by first sampling a small number of zero-shot translations from GPT-3. These are then used as few-shot prompts to sample a larger dataset of synthetic translations. The few-shot prompts are then discarded and the generated samples are distilled by fine-tuning the model on these synthetic data in the zero-shot format. This produces a language model aligned to our translation format and amenable to large-scale backtranslation. By using our method to leverage GPT-3's zero-shot translation capability, we achieve a new state-of-the-art in unsupervised translation on the WMT14 English-French benchmark, attaining a BLEU score of 42.1.
At the same time, recent work on large-scale generative pre-training has demonstrated that with sufficient data and model scale , transformer language models begin learning a variety of tasks without explicit supervision (Radford et al., 2019) and that even stronger performance can be coaxed from them using few-shot prompts . Our present work unifies these lines of research by using generative language modeling to simplify unsupervised NMT even further: we show how with sufficient scale, pre-training, and clever prompting, a single generative language model can implement the entire unsupervised neural machine translation pipeline, avoiding optimizations such as denoising autoencoding, auxiliary / adversarial losses in latent space, or ad-hoc bilingual dictionaries.
Our reliance on large-scale generative pre-training is similar to prior work in unsupervised NMT which uses large-scale language modeling tasks on internet data as part of the bootstrap (Conneau & Lample, 2019;. The role of few-shot prompting and distillation in our method is related to recent work on unsupervised data augmentation using language models (Anaby-Tavor et al., 2020;Kumar et al., 2020;Papanikolaou & Pierleoni, 2020; and is also in the same spirit as recent work on selftraining and noisy-student training (Mi et al., 2021;Vu et al., 2021;Xie et al., 2020). Recent work on scaling laws for neural machine translation has shown that transformer decoders exhibit more favorable scaling than encoders (Ghorbani et al., 2021). The few-shot distillation component of our method bears some resemblance to contemporaneous work by Wang et al. (2021b) which uses fewshot prompting for unsupervised data augmentation, though they focus only on inference for text classification rather than generation for sequence-to-sequence tasks like machine translation and do not study the phenomena of self-amplification nor few-shot data efficiency (Section 6) as we do.
BACKTRANSLATION VIA LANGUAGE MODELING
Backtranslation was first introduced in the context of machine translation as a method for data augmentation using target-side monolingual data by sampling synthetic source-to-target data from another target-to-source translation model (Bojar & Tamchyna, 2011;Sennrich et al., 2016;Poncelas et al., 2018). In our present work, we cast machine translation as a language modeling task and jointly train and sample generations from a single language model for both source-to-target and target-to-source translation.
Algorithm 1 Iterated backtranslation using a single generative language model Input: Source monotext M S ; target monotext M T ; number of iterations I; number of samples per iteration J; monotext formatter f (·); bitext formatter g(·, ·); parameters θ of language model p θ (·) trained to complete outputs of f to outputs of g. Output: Final model parameters θ.
1: for i = 1 to I do 2: estimate θ by maximizing log p θ of g(x, y) for x, y ∈ B back Given bitext 〈seq1, seq2〉 in languages L 1 and L 2 , we format the translation task as follows: [L2] and we parse a candidate translation <sampledSeq> from the sampled completion. Backtranslation is implemented by reversing the roles of seq and sampledSeq and fine-tuning on the bitext 〈sampledSeq, seq〉.
We remark that in contrast to the interpretation of backtranslation as a wake-sleep algorithm (Cotterell & Kreutzer, 2018), where the forwards and backwards translators are trained alternately, we use a single language model for both forwards and backwards translation and train on both directions jointly at every iteration. There are various ways to train a model using backtranslation, e.g., completely online (interleaving minibatch gradient updates and sampling) versus offline (backtranslating the entire training dataset at each epoch; potentially re-training the model from scratch after sampling new backtranslations). In practice, we find that data scaling of a model's optimal test loss and BLEU score quickly saturates on backtranslations from previous versions of the model, and opt for a semi-online setup where we synchronously sample a relatively small number of L 1 -L 2 and L 2 -L 1 pairs before resuming training for a single epoch on the newly sampled data. We refer to this as a single iteration of backtranslation.
Formally, Algorithm 1 describes our implemention of backtranslation using a single generative language model p θ (·). We assume that p θ (·) has already been trained to complete formatted monotext (
THE BOOTSTRAP: GENERATIVE PRE-TRAINING, FEW-SHOT AMPLIFICATION, AND DISTILLATION
The modern approach to unsupervised NMT is parametrized by a choice of initialization or bootstrap. The bootstrap has typically relied on some form of unsupervised cross-lingual representation learning, e.g., bilingual dictionaries initialized from unsupervised cross-lingual word embeddings (Lample et al., 2018;Artetxe et al., 2018) or multilingual masked language modeling followed by denoising autoencoding with a shared encoder and decoder (Conneau & Lample, 2019).
In Section 3, we formulated iterative backtranslation in terms of language modeling, assuming a language model which has already been trained to follow a particular instruction format for translation. To complete our procedure, we must supply such a language model. Unlike previous work on unsupervised NMT, we use language models from the GPT-3 family which have been generatively pre-trained on a large corpus of Internet data. A key observation from the body of work around GPT-3 is that generative pre-training at scale induces strong in-context metalearning abilities, two special cases of which are (1) instruction following and (2) few-shot prompting: a sufficiently trained large language model benefits from both detailed natural language descriptions of tasks and, when given in-context examples, can achieve strong performance on a diverse suite of [en] [fr]
[[TRANSLATE]]
Figure 1: Illustration of our bootstrap procedure, which we call few-shot distillation. We use fewshot prompts sampled from GPT-3 to generate an initial dataset of synthetic translations from a generatively pre-trained language model (left). The few-shot examples are then discarded and the synthetic bitext reformatted for fine-tuning on the autoregressive language modeling objective (right).
tasks (e.g., question-answering, natural language inference, translation.) We implement the bootstrap by exploiting both of these abilities, by using natural language instruction to produce zero-shot translations and few-shot prompting during amplification.
FEW-SHOT AMPLIFICATION AND DISTILLATION
It thus remains to adapt our generatively pre-trained models' few-shot translation ability to the zero-shot format specified in Section 3. We do this in a two-stage process. We first sample a small number of zero-shot translations from GPT-3. Given bitext 〈srcSeq, tgtSeq〉 in srcLang and tgtLang, and a stop-sequence <sep>, we use the following format for zero-shot prompting: <sep> Given the following passage in <srcLang>: <sep> <srcSeq> <sep> a good <tgtLang> translation is: <sep> <tgtSeq> <sep>.
At test-time, we sample a completion until the stop-sequence <sep> is detected; throughout we set <sep> to be \n---\n.
We amplify these zero-shot translations by using them as few-shot prompts to sample a much larger synthetic dataset from a smaller model. We then distill this dataset by discarding the few-shot prompts and fine-tuning on formatted bitext, producing a language model aligned with our task format and amenable to backtranslation. In detail, we implement the bootstrap as follows: 1. Generatively pre-train a language model p θ (·) on a large corpus of Internet data. 2. Sample a pool of N S synthetic target-side translations and N S target-side translations zeroshot from another language model q(·) for few-shot prompting. Using k few-shot examples randomly drawn from N S (resp. N T ), sample C S synthetic target-side translations (resp. C T synthetic source-side translations) from p θ (·), using the monolingual source-side corpus M S (resp. target-side corpus M T ). 3. Discard the few-shot prompts, reformat the (gold prompt, sampled translation) data as specified in Section 3, and fine-tune the language model p θ (·) on these data. 4. Reverse all data and continue fine-tuning the language model p θ (·) on the backtranslations (sampled translation, gold prompt).
Why amplify and distill? While few-shot prompting is flexible and enables strong performance on a diverse suite of NLP tasks to be coaxed out of generatively pre-trained LMs, its benefits are most pronounced with larger models, with commensurate training, inference, compute, and data costs. It is also unclear how to iteratively fine-tune a language model in a way that preserves its few-shot ability while remaining aligned with a zero-shot format like in Section 3. Few-shot amplification allows us to generate data for the bootstrap in an unsupervised fashion, possibly avoiding the overhead of few-shot sampling from GPT-3 itself by few-shot prompting a smaller model p θ (·), while distillation enables iterative backtranslation.
RESULTS
Experimental setup For our experiments, we focus on the well-studied WMT14 English-French benchmark. In the notation of Algorithm 1, we obtain source and target monotext M S and M T by splitting the WMT14 English-French training set in half, each with approximately twenty million examples, and use only the English text from one half and only French text from the other to avoid implicit sentence-level alignment between source and target monotext. At each iteration of backtranslation, we sample one million translations in either direction, i.e,. J = 2e6, and train for one epoch on the newly sampled data. For all of our results, unless otherwise specified, we run 40 iterations of backtranslation after the bootstrap and report BLEU using the final model checkpoint.
To implement the bootstrap, we additionally set aside 2048 training examples, and sample N S = 1024 English-French (resp. N T = 1024 French-English) translations zero-shot from GPT-3 to use as few-shot prompts. During few-shot amplification, we sample four million initial target-and source-side translations respectively using few-shot prompts, i.e., C S = C T = 4e6 in the notation of Section 4.1, drawing monolingual prompts from as M S and M T defined above. We fine-tune for two epochs in the forwards direction (distillation) and for another two epochs in the backwards direction (initial backtranslation). For few-shot prompting, we use k = 3 in-context examples. In Section 6.3.1 we will see that we can minimize the number of few-shot examples to N S = N T = 3 with little effect on evaluation BLEU score after iterative backtranslation.
We use the same training setup and BPE tokenizer as GPT-3. During fine-tuning, we use a constant learning rate of 0.05 · , where is the pre-training learning rate, a weight decay of 0.1, and residual dropout 0.1. When sampling during the bootstrap or during backtranslation, we default to using temperature τ = 0.3. We ablate other values of τ in Section 6.1. We also filter all fine-tuning bitext by length, discarding pairs with a source/target length ratio exceeding 2.0.
We report BLEU score on the official WMT14 English-French test set with greedy (argmax) sampling and sacreBLEU 1 (Post, 2018). In Table 3 we give a comparison to previous work on unsupervised NMT using multi-bleu.perl and the XLM (Conneau & Lample, 2019) tokenizer.
FEW-SHOT SELF-DISTILLATION AND BACKTRANSLATION
We first report results using self-distillation, i.e., where during the bootstrap (Section 4) we sample from a single model which is then trained to imitate and then backtranslate its own few-shot prompted generations; for these experiments, the few-shot demonstrations themselves are generated zero-shot by GPT-3. This is then followed by the iterative backtranslation procedure described in Section 3. We apply this methodology to the small, medium, large, and xl models from the GPT-3 family , with 125M, 350M, 760M, and 1.3B parameters respectively. Table 1 displays test BLEU throughout our procedure for all model sizes. We see that translation out of English benefits significantly from the backtranslation part of the bootstrap alone. We also see that our models are much stronger at the translation task compared to few-shot prompting after only self-distillation. Finally, all models benefit significantly from iterative backtranslation, with English-French BLEU always converging to a slightly higher value than the reverse direction.
DISTILLING SELF-AMPLIFIED GPT-3 INTO SMALLER MODELS
Although we do not apply our full methodology to the 175B parameter GPT-3 model due to compute constraints, we observe that for few-shot distillation, instead of training a model on few-shot samples from itself, we can just as well distill on few-shot samples from a much larger model instead-in this case, the full-size 175B parameter GPT-3 model (henceforth just "GPT-3"). That is, we use GPT-3 to self-amplify its own zero-shot translations to produce an initial dataset for distillation. Table 2: English-French (top) and French-English (bottom) test BLEU throughout the bootstrap and after iterative backtranslation, this time using generations from self-amplified GPT-3 for the bootstrap. We observe the best performance by mixing in monotext from the English and French components of the CC100 dataset during backtranslation.
We now proceed to apply the same method as in Section 5.1 to all model sizes, but this time using few-shot samples from GPT-3 for the bootstrap. We display the evaluation BLEU scores throughout the bootstrap and after iterative backtranslation in Table 2. Interestingly, the higher-quality samples from GPT-3 appear to saturate the smaller models and they improve very little. Motivated by the possibility that our models are beginning to overfit to the WMT14 English-French training data, we attempt another experiment where 50% of the monotext for backtranslation is sampled from the English and French components of the CC100 dataset . The extra monolingual data significantly benefits all model scales, improving English-French BLEU by approximately 3 points compared to iterative backtranslation on WMT data alone. With this setup, the xl attains a new unsupervised state-of-art of 42.1 BLEU on the WMT14 English-French benchmark.
DISCUSSION AND FURTHER ABLATIONS
Bias towards English generation Previous work has shown that after generative pre-training on a corpus of English-dominated Internet text, GPT-3 models are far more capable of translating into English than translating out of English. This is reflected by the disparity between English-French and French-English BLEU scores immediately after few-shot distillation and before backtranslation on the few-shot prompted data. Interestingly, after only two epochs of backtranslation on the relatively scarce few-shot prompted data, this gap is reversed, with all models achieving significantly higher English-French BLEU than French-English BLEU. The data efficiency of the bootstrap suggests that coming out of pre-training, the models are merely misaligned rather than deficient in knowledge about French, and that their latent knowledge about translation out of English can be surfaced using backtranslation. Relatedly, high-quality samples in one language in the previous round of backtranslation lead to higher-quality synthetic bitext for training the reverse direction in the next. This turns the asymmetry towards English generation into an advantage during backtranslation. However, if the initial disparity between the quality of the translation directions is extreme (as with the self-distilled small, which after distillation achieves < 1 BLEU for English-French versus ≈5 BLEU for French-English), then we see that the evaluation BLEU scores for either direction are unstable and oscillates between iterations, though they eventually converge upwards as backtranslation continues.
Comparison to previous work In Table 3, we compare the BLEU scores attained by our best model (an xl distilled on self-amplified GPT-3 followed by 40 rounds of backtranslation) to prior work in unsupervised neural machine translation on the WMT14 English-French benchmark. To ensure comparability to prior work, we report tokenized BLEU using multi-bleu.perl and the XLM tokenizer. This was used to report the few-and zero-shot performance of GPT-3 in , which we also include in Table 3 for completeness. We emphasize the improvement of our model compared to zero-shot GPT-3, which was used to initialize the bootstrap.
Potential data contamination from pre-training For high-resource language pairs such as English-French, naturally occuring demonstrations of translation are virtually guaranteed to appear in Common Crawl-like datasets; indeed, Radford et al. (2019) provide examples of English-French parallel text embedded in the WebText corpus. Train/test contamination is also a growing area of concern when training large language models on internet-derived data. The data contamination study conducted by found virtually no test set contamination for the WMT translation datasets they considered, including the WMT14 English-French dataset. We emphasize that throughout our entire procedure, no explicit supervision is given for the translation task during pre-training, distillation, or backtranslation. Table 4: English-French (top) and French-English (bottom) test BLEU using few-shot prompted samples generated with temperatures τ = 0.0, 0.3, 1.0 throughout the bootstrap. We see that the temperature used for sampling has little effect on evaluation BLEU after few-shot distillation, while high-temperature samples are harmful during the backtranslation part of the bootstrap.
It was shown by Edunov et al. (2018) that backtranslation is more effective when the translations are slightly noisy, i.e., sampled with nonzero temperature or via a noised beam search. This motivated our use of the temperature τ = 0.3 throughout. We ablate this choice of temperature when sampling data for few-shot distillation, and study the effect of using τ = 0.0 and τ = 1.0 during the bootstrap using a large model. We display the results in Table 4. We see that lower temperatures lead to marginally higher test BLEU scores during distillation while τ = 1.0 results in lower test loss and no overfitting after two epochs of training. However, regardless of the temperature of samples used for self-distillation, the differences in both test BLEU and test loss almost vanish after the backtranslation part of the bootstrap when training to backtranslate low temperature samples (τ = 0.0 or τ = 0.3).
FEW-SHOT SELF-AMPLIFICATION
We observed that few-shot prompting GPT-3 with its own zero-shot translations produced better translations than zero-shot prompting alone. We investigate this further by comparing the BLEU scores of zero-shot translations (sampled using the same prompt described in Section 4) to the BLEU scores of self-amplified few-shot prompted translations (i.e., where the few-shot demonstrations are the zero-shot translations sampled from the same model) for all the model sizes studied in this paper. Our results are displayed in Table 5. We see that self-amplification improves translation quality at all model scales. Table 5: Zero-shot versus few-shot self-amplified test BLEU for all model sizes studied in this paper. For zero-shot generation we use the same prompt format described in Section 4. For self-amplified generation, we use the model's own zero-shot generations as in-context few-shot examples.
USING REAL FEW-SHOT EXAMPLES
So far our results have been completely unsupervised, but few-shot learning is typically studied in the context of semi-supervised learning (Wang et al., 2020), where the few-shot demonstrations are real training data. In this section, we ablate the usage of synthetic few-shot translations in our methodology and reproduce our experiments from Section 5 using real few-shot demonstrations. We observe virtually no difference in BLEU score after iterative backtranslation.
We modify the few-shot prompting described in Section 5 as follows. Rather than sampling zeroshot translations for each half of our held-out pool of N=2048 training examples, we sample from these examples directly during few-shot prompting. Table 6 displays test BLEU throughout the bootstrap and after iterative backtranslation for the same model sizes studied in Section 5.1. We see that our models converge to the same test BLEU (c.f. Section 5.1). Table 7 displays analogous results when distilling samples from GPT-3 with the small and large models, this time few-shot prompted using real examples. We again see that using real rather than synthetic few-shot demonstrations to sample the initial bootstrap data from GPT-3 has no effect on final BLEU score after iterative backtranslation. We see that N has minimal impact on the BLEU score of the sampled translations. Moreover, the difference in BLEU between the models bootstrapped using N = 3 versus N = 2048 disappears after iterative backtranslation.
of N, the number of available few-shot examples. Remarkably, even when N is decreased to 3, there is only a slight negative impact on the BLEU score of the few-shot sampled translations. We do not ablate lower values of N in order to maintain the assumption of k=3 distinct in-context examples for few-shot prompting. We then run our entire procedure with a large model, using N=3 real few-shot demonstrations for the bootstrap followed by iterative backtranslation. We observe a final English-French BLEU of 38.0 and French-English BLEU of 34.2, on par with the final BLEU scores reported in Table 6.
CONCLUSION AND FUTURE DIRECTIONS
We remark that backtranslation, like reinforcement learning, is simply a way of exchanging compute for data. Instead of grounding the model with a reward signal from an environment, however, backtranslation exploits the symmetry of the translation task to ground the model by training it to cross-lingually denoise its own samples. Our present work can be viewed as part of a recent trend towards data-driven architecture engineering Rozière et al., 2021), where task-specific inductive biases, if any, are engineered into and learned from the training data instead of being hardcoded into the model architecture. In formulating the translation task in terms of language modeling, we see that the input-output inductive bias imposed by an encoderdecoder architecture can be simulated with prompt formatting. Similarly, we see that generative language modeling at sufficient scale combined with clever prompting for automated data generation can attain state-of-the-art results in unsupervised translation, rendering methods intended to produce strong encoders and aligned multilingual representations unnecessary.
Although we have focused solely on the domain of machine translation in this work, our methodology is applicable to any sequence-to-sequence task whose forwards and inverse directions are (1) jointly learnable by an autoregressive decoder-only transformer and (2) amenable to few-shot prompting after large-scale generative pre-training. Backtranslation is simply reverse self-training (Bojar & Tamchyna, 2011) and is fundamentally untied to the translation domain; we invite the research community at large to further explore this technique, moving beyond translation and towards applications reflecting the full generality of the transformer architecture.
|
2021-10-12T01:34:16.652Z
|
2021-10-11T00:00:00.000
|
{
"year": 2021,
"sha1": "55d1133ec3bd8851dc0172cf7454063872a11898",
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|
245018767
|
pes2o/s2orc
|
v3-fos-license
|
Big Data Technology in Intelligent Management and Control System of Drainage Pipe Network
With the rapid development of urban construction, smart city has become an important trend in the future, which is inseparable from the intellectualization of drainage pipe network. At the same time, with the increasing frequency of urban flood disasters, the old drainage pipe network system will cause significant economic losses, which requires modern cities to establish intelligent management and control of drainage pipe network. However, the situation of drainage facilities in most cities in China is complex, which requires strengthening modern means such as monitoring, operation management and planning services. Therefore, the research on intelligent management and control system of drainage pipe network based on big data technology is of great significance. Firstly, this paper analyzes the importance of intelligent management and control system. Then, this paper puts forward the main algorithms for big data processing. Finally, this paper constructs the intelligent management and control system of drainage pipe network.
Introduction
With the rapid development of modern science and technology, the monitoring of drainage pipe network system has been difficult to meet the needs of the information age, which needs to study the requirements of big data in the intelligent management and control of drainage pipe network [1]. Through the real-time wireless automatic monitoring system, the intelligent city can improve the monitoring capacity of drainage network management, pump stations, valves and other facilities and equipment, which will realize the functions of intelligent discharge and pump gate linkage [2][3]. Therefore, big data technology can provide scientific basis for flood prevention command [4]. In recent years, sudden rainstorms have occurred in many cities in China, which has caused major problems of waterlogging and water environment pollution. Urban waterlogging has exposed many problems existing in the management of urban drainage pipe network, such as unclear drainage facilities, unclear operation status of facilities, low efficiency of manual management, slow response to accident treatment, etc., which needs to strengthen the management means of drainage system [5]. Through intelligent management and control, we can improve the drainage capacity of the city, which will improve the current situation of urban drainage problems [6].
Requirements for smart city construction
Smart water is an important part of smart city construction, which is an important support and guarantee to ensure people's livelihood and public services. Smart water is an important part of smart city construction, which needs to be built with the development and practice of new technologies as the core. Therefore, modern cities need to establish intelligent drainage pipe network system, which is an essential part of urban modernization. Water conservancy modernization is an important link for China to realize national modernization [7]. The intelligent management and control system of drainage pipe network is an efficient and low consumption operation management system, which has highly shared data information and systematic problem solutions. Therefore, the drainage pipe network system can better meet the requirements of modern water conservancy information construction, which is the inevitable choice of water conservancy modernization [8].
Strong guarantee for water resources management
At present, there are many problems in urban water resources, such as huge rigid demand, serious waste of water resources, serious water pollution exceeding the standard, etc. it is urgent to establish an intelligent, integrated and automatic monitoring equipment [9]. Through the intelligent management and control system, we can monitor and supervise water affairs, which can ensure a more comprehensive perception index. Chinese cities must better protect national resources, which can implement the strictest water resources management system by quantitative means [10].
An important starting point for the construction of ecological civilization
Water resources management is an important link in the construction of ecological civilization. Water quality and quantity are the key to the success of the construction of ecological civilization. At the same time, the construction of ecological civilization is not a leap, which dynamically forms multiple departments into a large comprehensive system. Through inter departmental communication, cooperation and operation, we can promote the construction of ecological civilization in all directions, all fields and the whole society [11].
Adaptive needs of information technology development
After entering twenty-first Century, the information age is coming, including Internet plus, Internet of things, BIM, etc. Among them, China's State Grid and transportation network have been upgraded with the help of information means. Therefore, as an important part of urban infrastructure, urban drainage system urgently needs to build a system suitable for information technology [12]. Through the intelligent management and control system of drainage pipe network, modern cities can build an intelligent management and control system, which will improve the overall service capacity of the city and promote the construction of urban informatization [13].
Reliability Analysis
Reliability is the abbreviation of reliability, which mainly reflects the internal stability and consistency. There are many methods for reliability analysis. At present, the most commonly used is Cronbach reliability coefficient.
Correlation analysis
Correlation analysis is an analysis method that reflects the attachment relationship between random variables. Pearson correlation coefficient method is commonly used, as shown in formula 2.
(2) The comprehensive index formula of intelligent management and control system construction maturity is shown in Formula 3.
Among them, are infrastructure construction index, security system development index, operation system construction index and service construction index respectively.
System function construction
Through accurate early warning and forecasting, we can effectively deal with the rainstorm problem, which will reduce economic losses. However, some managers are difficult to grasp the accurate operation of drainage facilities in time, which will be difficult to make accurate judgment and scientific dispatching. Through the intelligent management and control system of drainage pipe network, we can provide real-time online monitoring capability of drainage and waterlogging prevention. Through video, optical fiber broadband and Internet of things technologies, the system can monitor the working conditions of sluice, pump station, pipe network and other facilities in real time, which will realize the management of urban waterlogging prone points. By deploying liquid level meter, liquid level gauge and HD camera, the system can focus on monitoring the ponding at urban waterlogging prone points, which will further realize the functions of intelligent discharge and pump gate linkage. Through big data analysis, the system can better detect and control. This paper constructs the functions of the system, as shown in Figure 1.
Overall architecture design
The core data of drainage pipe network system is the attribute data of drainage pipe network facility space and business expansion. The main functions of the intelligent management and control system of drainage pipe network are dynamic update of drainage pipe network data, query and statistics of pipe network facilities, application analysis, drawing and printing, etc. Based on the data characteristics of drainage network, the system can be implemented with C / S structure, which can greatly reduce the calculation pressure of the server. Based on the characteristics of system data management, we can get the system logic structure diagram, as shown in Figure 2.
Conclusion
Through intelligent management and control, cities can use traditional artificial drainage pipe network management methods, which will improve the digital and information management ability of urban drainage facilities. Through big data technology, computer technology, GIS technology, GPS technology and sensor communication, modern cities can establish grid and fine management means, which will improve the scientific and effective inspection mechanism of drainage facilities. Through the intelligent management and control system, modern cities can make real-time early warning and prediction, which can realize the real-time monitoring and control of the operation status of drainage facilities.
|
2021-12-11T20:07:12.139Z
|
2021-12-01T00:00:00.000
|
{
"year": 2021,
"sha1": "3855a7f46f9ee639477eca3604c5aa614911687a",
"oa_license": null,
"oa_url": "https://doi.org/10.1088/1742-6596/2143/1/012023",
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|
119002040
|
pes2o/s2orc
|
v3-fos-license
|
Massive charged Dirac fields around Reissner-Nordstr\"om black holes: quasibound states and long-lived modes
The behavior of a massive charged test Dirac field in the background of a Reissner-Nordstr\"om black hole is investigated. Especially, we obtain the frequencies of quasibound states by solving the Dirac equation numerically both in time and frequency domain. Our results suggest that although the absence of superradiance excludes the existence of stationary solutions for massive Dirac fields, it is still possible to find arbitrarily long-lived solutions.
I. INTRODUCTION
In classical general relativity, according to the no-hair conjecture [1,2], all asymptotically flat stationary black hole (BH) solutions can be completely characterized by only three parameters: mass, electric charge, and angular momentum. A perturbed BH will return to a stationary state and all other external fields will inevitably decay with time.
Although the no-hair conjecture forbids any nontrivial field distribution in BH spacetimes, it does not rule out the existence of dynamical solutions that decay very slowly [3]. In Refs. [3,4], Barranco et al. studied the evolution of massive scalar fields around a Schwarzschild BH and found configurations that can survive for a very long time, even for cosmological time scales. Zhou et al. further showed that such long-lived nontrivial distributions can be extended to Dirac fields [5]. These dynamical resonance solutions are basically described by the so-called quasibound states of massive fields in the BH spacetimes, which have attracted much attention in recent years [6][7][8][9][10][11][12][13][14][15][16][17][18][19]. Recent studies have shown that these resonances will dominate the dynamics of the massive fields in BH spacetimes, and could affect the late time waveforms of the gravitational radiation [20][21][22].
A common feature of the results in Refs. [3][4][5] is that the long-lived modes are obtained in the limit of smallmass coupling M µ ≪ 1, where M and µ are the masses of the BH and field, respectively. There is a simple physical explanation for this: smaller value of M µ corresponds to weaker gravitational attraction between the external field and BH, which implies that it takes a longer time for the BH to absorb the external fields around it. However, the small mass coupling requirement for long-lived field configurations is no longer necessary in at least two scenarios.
Firstly, massive bosonic fields in rotating BH spacetimes can form stationary clouds, which correspond to Secondly, for a massive charged scalar field on the Reissner-Nordström (RN) background, the decay rate of the field configuration could be extremely slow when M µ qQ [31,32], where q and Q are the charges of the field and BH, respectively. Although scalar fields on the RN background are subject to amplification by a charged version of superradiance, no stationary scalar modes exist, because the superradiance and quasibound state conditions are incompatible with each other in this case [33][34][35].
The study of a Dirac test field in a black hole background is a subject of long-standing interest and the Dirac-Maxwell-(Einstein) system has been considered in a series of paper by Finster et. al., see e.g. [36,37]. Among other things, these papers establish the absence of normalizable, time-periodic solutions in a RN background, together with the existence of solitonic solutions sustained by gravity effects.
In this paper, we shall consider a classical massive charged Dirac field propagating on the RN background and intend to work out whether long-lived Dirac configurations surrounding a RN BH exist. The organization of this paper is as follows. In Sec.II, the separation of variables procedure for massive Dirac fields around RN BHs is reviewed. Then, in Sec.III and Sec.IV, we compute the quasibound state solutions by solving the Dirac equation on the RN background in frequency and time domains, respectively. Finally, the paper ends up with a discussion and conclusion in Sec.V. Throughout the paper, we use natural units in which G = c = = 1. We start by the Dirac equation in RN BH spacetime. The RN metric, in Boyer-Lindquist coordinates, is given by where ∆ = r 2 −2M r+Q 2 . The electromagnetic potential of the charged black hole reads A µ = (−Q/r, 0, 0, 0). The RN BH has two horizons, r ± = M ± M 2 − Q 2 . When Q = 0, the potential A µ and the inner horizon vanish, and the BH reduces to a Schwarzschild one.
Massive charged Dirac field in curved spacetime is described by [38] ( where µ is the mass of the field, Ψ is the Dirac four-spinor and γ µ are coordinate-dependent Dirac four-matrices, whose components are the functions of spacetime coordinates. The spinor covariant derivatives in Eq.(2) are given by where q is the charge of the Dirac field, and Γ ν are the spinor connection matrices. The separation of variables of the Dirac equation (2) could be done through several equivalent approaches [39][40][41][42]. Here, following [8,43], we employ the canonical orthonormal (symmetric) tetrad for the RN spacetime, and decompose the Dirac four-spinor as where (5) Substituting the ansatz (4) and the background metric (1) into the Dirac equation (2) yields the following coupled first-order differential equations and where K = ωr 2 − qQr and λ is the separation constant. Solutions of the angular Eqs. (8) and (9) are spinweighted spherical harmonics and the eigenvalues are given by (See [44] for details) where j is a positive half-integer, i.e. j = 1/2, 3/2, · · · , which denotes the total angular momentum. The orbital angular momentum is characterized by the non-negative integer ℓ = 0, 1, 2, · · · . By the way, the relation between λ and j is given by It should be noted that Eqs. (6) and (7) can be written as the following second-order form 1 where the form of a Schrödinger-like wave equation where the new radial function ψ is defined by and the effective potential is given by Here, the auxiliary function F (r) is defined by
B. Quasibound states
As is known, quasibound state solutions are ingoing at the horizon, and exponentially decaying at infinity. To obtain such solutions, we solve the radial equations (6) and (7) under appropriate boundary conditions. By doing this, we can pick out a discrete set of complex frequencies, which correspond to the quasibound states of the Dirac field. Here, we express a complex frequency by ω = ω R + iω I . In the limit of small coupling parameters, the massive charged Dirac field in the RN BH spacetime has the following spectrum [45] 1 where the principal quantum number and the excitation number n = 0, 1, 2, · · · . From Eq. (19), the binding energy ω R − µ tends to zero in the limit of M µ → qQ. One may also find that Eq. (20) can be written in a simpler form In next section, we shall use the continued fraction method [46,47] to calculate the quasibound state frequencies beyond the small parameter regime (18), where the analytic formula (19) is no longer valid. It is worth noting that, due to the absence of superradiance, no growing modes could be found for Dirac fields around a RN BH.
III. FREQUENCY DOMAIN ANALYSIS
The main problem for solving the coupled equations (6) and (7) via the continued fraction method is that the asymptotic solutions of R 1 and R 2 are different at r + and infinity. Accordingly, we define two new radial functions as then, the radial equations (6) and (7) become and (24) Close to r + , the "ingoing" solutions behave as At infinity, the asymptotic solutions of Eqs. (23) and (24) are where and Here, we are only interested in quasibound states which decay exponentially at infinity. Thus, we choose Re(k) < 0. Based on the discussions above, solutions of Eqs. (23) and (24) can be expanded as where U n are two dimensional vectorial coefficients. Substituting the series (30) into Eqs. (23) and (24) leads to a three-term matrix-valued recurrence relation, Here, α n , β n , γ n are all 2 × 2 matrices and can be expressed, respectively, as where c 0 , c 1 , c 2 and c 3 are given by The quasibound frequencies are roots of equation with U n+1 = R n U n and Then, the nontrivial solutions U 0 exist if For physical accepted solutions, the series expansion (30) should converge at spatial infinity. Thus, we may fix a large truncation order N and initialize R N as an identity matrix 2 . Then, Eq.(41) can be used to construct the matrix M 0 (See [48] for more details). Finally, the quasibound state frequencies are the roots of Eq. (42), which correspond to the local minimum points of det |M 0 | in a plane spanned by ω R and ω I . Fig.1 shows det |M 0 | as a 2 Equivalently, we require U N+1 = U N . To obtain the quasibound state frequencies we use a 2D root-finding algorithm. Using this approach, one may easily find that the local minimum in the Fig.1 gives M ω = 0.945159 − 0.398158i, which is the frequency of the fundamental mode.
Due to the spin-orbit coupling, Dirac fields around a BH have a rich spectrum, even for the case in which both the field and BH are neutral. Fig.2 shows the real part ω R (expressed by 1 − ω R /µ in the left panel) and imaginary part ω I (the right panel) of the first few modes, as functions of M µ, for Dirac fields in the Schwarzschild spacetime (Q = 0). In Table I we present some reference frequencies for initial estimations of the root-finding algorithm.
From the left panel of Fig.2, we see that for small values of M µ, the spectrum of modes with the same value of n = n + j + 1 2 are indistinguishable, and they are in good agreement with the analytic formula Eq.(19) (denoted by black dotted lines in the plot). As M µ increases, the spinorbit coupling begins to affect the quasibound spectrum, resulting in a removal of the degeneracy of the same value ofñ. The dependence of ω I (µ) is simpler. The right panel of Fig.2 shows that the absolute value of ω I decreases as M µ decreases. In the limit of M µ → 0, the value of |ω I | also tends to zero, which means that ultralight Dirac field could be arbitrarily long-lived around a Schwarzschild BH.
We now turn to the case in which both the BH and the Dirac field are electrically charged. Without loss of generality, we assume that the charge of BH is always positive Q > 0. We shall show how the electromagnetic interaction changes the spectrum and how long-lived modes are achieved. Fig.3 shows the effect of field charge q on the quasibound state spectrum. Clearly, more negative field charge corresponds to smaller values of ω R but larger values of |ω I |, which suggests that for positive BH charge, Dirac field with negative charge decays faster than the one with positive charge. More interestingly, for positively charged Dirac field, the value of M µ of the quasibound states is bounded below by M µ min = qQ, which is denoted by the vertical lines in the right panel of Fig.3. By decreasing M µ close to its minimum value, i.e., M µ qQ, one gets very long-lived modes since the decay rate |ω I | tends to zero rapidly.
IV. TIME DOMAIN ANALYSIS
Massive test fields around a BH can form both quasinormal modes (QNMs) and quasibound states. Generally speaking, QNMs would appear in early times and damp quickly, whereas quasibound states decay on larger time scales. It follows that given generic initial data, quasibound states would dominate the waveform of massive fields around BHs. This is well understood for scalar and Proca fields [3,14,22,[49][50][51][52][53][54][55][56]. Unfortunately, studies of the time evolution for massive Dirac fields are relatively few [5,8,57].
Here, we consider the time evolution of massive charged Dirac fields around a RN BH. From Eqs.(6) and (7), the Dirac wave equations in the time domain can be written in the form where the tortoise coordinate x = dr r 2 /∆. We use the method of lines with a second-order finite difference stencil for the spatial derivatives, and thirdorder RungeKutta integrator for the time integration. To minimize the boundary reflections, we place the inner boundary very close to the event horizon, typically x = −1000M , and put the outer boundary far away from the BH; furthermore, we suppose that solutions are continuous enough and extrapolate values of R 1 and R 2 at boundary points from the inner points at each time step. In addition, we include a Kreiss-Oliger dissipation to ensure the numerical stability. We choose Gaussian wave packet as the initial data. The time derivatives of R 1 and R 2 are set to zero initially. For convenience, we set r + = 1 and measure all quantities in terms of r + in the time evolution of the Dirac field. The typical results for the time evolution are presented in Fig.4, where we plot the waveforms of R 1 and R 2 at a fixed point x o = 80 for different values of σ in the left panels, and plot the corresponding Fourier spectra in the right panels. The left panels show the beating effect of the Dirac field, which has been found for bosonic fields in Kerr spacetime [49]. Although the evolutions of R 1 and R 2 are different at early times and depend strongly on the initial data we choose (See the insets in the left panels of Fig.4), the beating patterns of them are quite similar at late times of the evolution. As pointed out in [49], such beating effect results from the interference between several modes with different frequencies. Indeed, from the right panels of Fig.4, we see that there are several visible peaks in the Fourier spectra which correspond to the dominating modes at late time stages of the evolutions. These peaks coincide with the quasibound state frequencies. Thus we conclude that quasibound states of Dirac fields could be excited by a generic Gaussian initial data and would dominate the waveforms of long-timescale evolutions. However, one may easily observe that the n = 0 state is missing in the Fourier spectrum. This is reasonable because the absolute value of ω I of the n = 0 state is about two orders of magnitude larger than those of higher overtones, which makes the n = 0 mode decay rapidly and disappear in the Fourier spectrum.
We remark that parameters of the results in Fig.4 obey M µ > qQ. In the M µ < qQ regime, no bound states could be excited in time evolutions. In this case, the Dirac field decays at asymptotically late time as [57] Re(R 1,2 ) ∼ t −5/6 sin µt, t → ∞.
This result is confirmed in Fig.5, where we present the time evolution of the Dirac field with qQ = 1.2M µ.
V. DISCUSSION AND CONCLUSION
We have solved the Dirac equation on the RN background numerically both in frequency and time domain.
Our results indicate that quasibound states of massive Dirac fields can only exist in the M µ > qQ regime, and the long-lived modes exist when M µ qQ, although there is no stationary configuration of Dirac field in this background [37]. Here, we shall show that this is actually the necessary condition for the existence of quasibound states. From Eq.(16), at large distance, the effective potential goes as Notice that this asymptotic behavior is the same as that of the potential of a massive scalar field in the Kerr-Newman spacetime to the first order [26]. To support quasibound states, the effective potential must have a trapping well outside the BH, and its asymptotic derivative must be positive, i.e., dV dr → 0 + as r → ∞ [58]. From Eq.(47), the necessary condition for a potential well is given by Note also that quasibound states decay exponentially at spatial infinity, which are characterized by ω 2 < µ 2 . Thus the necessary condition for the quasibound states is which suggests that f (µ, q) should be smaller than µ. That is, Table II is a collection of these results. The thick vertical lines give ω = µ, which denotes the upper bound of the frequency of the quasibound states.
Simplifying the above inequality leads to the relation M µ > qQ, which is necessary for the existence of quasibound states. Indeed, all the quasibound states we have found obey this relation. The co-rotating modes of massive Dirac fields around a Kerr BH with the frequency obeying the 'superradiance' condition ω < mΩ H could be very long-lived [8]. However, we shall show that such long-lived modes do not exist for Dirac fields around RN BHs, since the 'superradiance' condition ω < ω c ≡ qQ r + is incompatible with the quasibound state condition. First, suppose we have a quasibound state in the 'super-radiance' regime Eq.(51) for Dirac fields on the RN background. Then, from the bound state condition Eq. (49), we must have f (µ, q) < ω c .
Combining this equation and the definition of f (µ, q) and ω c leads to 3 which is incompatible with the quasibound state condition Eq. (49). Thus, it is not possible to find quasibound states in the 'superradiance' regime Eq.(51) for massive Dirac fields around RN BHs, let alone long-lived modes. It would be interesting to study whether the co-rotating modes, with the frequency obeying a 'superradiance' condition, exist for Dirac fields on the Kerr-Newman background.
It should be pointed out that in this paper we only treat the Dirac field as a test field in the RN background. It would be more interesting to study the back reaction and the gravitational radiation (and possibly electromagnetic radiation) triggered by the Dirac field. Finally, it is plausible that the long-lived configurations we have found here could be generalized to Fermions with higher spin. All of these possibilities deserve further studies in the future.
|
2017-08-16T03:38:57.000Z
|
2017-08-16T00:00:00.000
|
{
"year": 2017,
"sha1": "1c26dbec190052b26790a9cbe9233c37ccdc46b4",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/1708.04761",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "1c26dbec190052b26790a9cbe9233c37ccdc46b4",
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"Physics"
],
"extfieldsofstudy": [
"Physics"
]
}
|
258290245
|
pes2o/s2orc
|
v3-fos-license
|
Immune Responses After Vaccination With Primary 2-Dose ChAdOx1 Plus a Booster of BNT162b2 or Vaccination With Primary 2-Dose BNT162b2 Plus a Booster of BNT162b2 and the Occurrence of Omicron Breakthrough Infection
Background Before the omicron era, health care workers were usually vaccinated with either the primary 2-dose ChAdOx1 nCoV-19 (Oxford-AstraZeneca) series plus a booster dose of BNT162b2 (Pfizer-BioNTech) (CCB group) or the primary 2-dose BNT162b2 series plus a booster dose of BNT162b2 (BBB group) in Korea. Methods The two groups were compared using quantification of the surrogate virus neutralization test for wild type severe acute respiratory syndrome coronavirus 2 (SVNT-WT), the omicron variant (SVNT-O), spike-specific IgG, and interferon-gamma (IFN-γ), as well as the omicron breakthrough infection cases. Results There were 113 participants enrolled in the CCB group and 51 enrolled in the BBB group. Before and after booster vaccination, the median SVNT-WT and SVNT-O values were lower in the CCB (SVNT-WT [before-after]: 72.02–97.61%, SVNT-O: 15.18–42.29%) group than in the BBB group (SVNT-WT: 89.19–98.11%, SVNT-O: 23.58–68.56%; all P < 0.001). Although the median IgG concentrations were different between the CCB and BBB groups after the primary series (2.677 vs. 4.700 AU/mL, respectively, P < 0.001), they were not different between the two groups after the booster vaccination (7.246 vs. 7.979 AU/mL, respectively, P = 0.108). In addition, the median IFN-γ concentration was higher in the BBB group than in the CCB group (550.5 and 387.5 mIU/mL, respectively, P = 0.014). There was also a difference in the cumulative incidence curves over time (CCB group 50.0% vs. BBB group 41.8%; P = 0.045), indicating that breakthrough infection occurred faster in the CCB group. Conclusion The cellular and humoral immune responses were low in the CCB group so that the breakthrough infection occurred faster in the CCB group than in the BBB group.
INTRODUCTION
A vaccination program against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which started in Wuhan, China in December 2019, was initiated in South Korea in February 2021. 1 Healthcare workers (HCWs) were one of the first groups to receive the COVID-19 vaccine due to the risk of occupational exposure to SARS-CoV-2 2 ; specifically, they underwent two types of primary vaccination series: a two-dose BNT162b2 (Pfizer-BioNTech) series and a two-dose ChAdOx1 nCoV-19 (Oxford-AstraZeneca; referred to as AZD1222) series. 3 From October 2021, the booster vaccination was administered to the majority of HCWs after having previously received one of the mRNA vaccines, namely BNT162b2. In Korea, there were two major vaccination groups in HCWs-the ChAdOx1/ChAdOx1/BNT162b2 (CCB) and the BNT162b2/BNT162b2/BNT162b2 (BBB) group.
Neutralizing antibody responses against the omicron variant and the occurrence of omicron breakthrough infection have rarely been investigated in the CCB and BBB groups in Korea. Our institution has conducted a cohort study to investigate the vaccine-induced immunities in HCWs since the rollout of COVID-19 vaccines while also tracking COVID-19 breakthrough infections. Therefore, we investigated the correlation between neutralizing antibody responses against the omicron variant and the occurrence of omicron breakthrough infections, as well as the correlation between vaccination groups and breakthrough infection using the observational cohort data.
Study design and data collection
This study was conducted at Chung-Ang University Hospital in Seoul, Korea. At this medical center, COVID-19 vaccination for HCWs began in early March 2021. The first vaccine dose of BNT162b2 was administered from March 11 to 13, 2021; after 3 weeks, the second dose was given from April 1 to 3, 2021. The first dose of ChAdOx1 was administered from March 4 to 17; after 11-12 weeks, the second dose was given from May 20 to June 10, 2021. The booster dose was administered from October 20 to December 28, 2021. Blood sampling for serologic tests was performed on HCWs only if they provided written informed consent to this study. Only HCWs who received the primary series with booster vaccination were included in this study. The first blood sampling was done 184 median days (interquartile range [IQR]: 183-187) after 1st immunization in CCB groups and 185 median days (IQR: 183-186) after 1st immunization in BBB group. The 2nd blood sampling was done 63 median days (IQR: 61-90) after 3rd booster vaccination in CCB group and 61 median days (IQR: 56-63) after 3rd booster vaccination in BBB group. Analysis of anti-spike IgG, neutralizing antibodies against wild type and omicron variants and interferon-gamma (IFN-γ) release assay (IGRA) for wild type virus was performed on each of the collected samples, but the samples collected from the BBB group after the second dose did not undergo IGRA analysis. Investigation of breakthrough infection was performed until June 11, 2022. We collected blood samples twice from the participants for evaluation 6 months after the 1st vaccination dose and 2 months after the booster vaccination. The overall flow of this study is presented in Fig. 1.
All HCWs participating in this study had no prior history of COVID-19 before 1st and 2nd blood sampling. From the 2nd blood sampling until June 11, 2022, point surveillance was
Effectiveness of Hetero/Homologous COVID-19 Vaccination
performed for all participants through questionnaires to determine whether they had a breakthrough infection. The first case of breakthrough infection among participants occurred on January 28, 2022. Since the omicron variant became dominant in the 3rd week of January 2022 in Korea, 4 the breakthrough infections in the HCWs of this study occurred during the omicron-predominant period.
Assessment of neutralizing antibody responses against SARS-CoV-2
Neutralizing antibody responses against wild type SARS-CoV-2 were detected using the GenScript SARS-CoV-2 cPass surrogate virus neutralization test (SVNT-WT) kit (GenScript Biotech Corporation, Piscataway, NJ, USA). The test was modified to detect SARS-CoV-2 neutralizing antibodies against the omicron receptor binding domain (RBD) by replacing the horseradish peroxidase-conjugated recombinant RBD fragment according to the manufacturer's specifications. The neutralization antibody responses against the omicron variant were detected using this modified commercial SVNT kit (SVNT-O). The percentage of neutralization was calculated as follows: (1 − Optical Density [OD] of Sample/OD of Negative Control) × 100.
Assessment of SARS-CoV-2-specific T cell responses
SARS-CoV-2-specific T cell responses were measured by quantification of the IFN-γ release against wild type after stimulation with the SARS-CoV-2 spike protein using the Euroimmun SARS-CoV-2 Interferon Gamma Release Assay (Euroimmun). A set of three tubes was used for each sample: 1) SARS-CoV-2 IGRA BLANK without the IFN activating substance as the individual background stimulation; 2) SARS-CoV-2 IGRA TUBE containing the S1 domain of the SARS-CoV-2 spike protein; and 3) SARS-CoV-2 IGRA STIM containing a mitogen for unspecific IFN stimulation to evaluate the viability and stimulation capacity of T cells and the number of T cells in the participant's blood sample. The response was defined as the IFN-γ concentration in the TUBE sample minus that in the BLANK sample, in international units per milliliter (IU/mL). IFN-γ responses above 200 mIU/mL were defined as positive according to the manufacturer's instructions. 5
Statistical analysis
All statistical analyses were performed using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA) and IBM SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA). Categorical variables were analyzed using the χ 2 test or Fisher's exact test. Continuous variables were described as median values and compared using the Mann-Whitney U test. Statistical significance was set at P < 0.05. Since the time interval between the booster vaccination and blood sampling was different between the two groups ( Supplementary Fig. 1), the time difference was controlled in the statistical models. To adjust the effect of the time difference in comparing the serologic test results between the two groups, we performed multiple linear regression models with confounding variables.
To compare the cumulative incidence of breakthrough infection of the two groups after the booster vaccination, the Gehan-Breslow-Wilcoxon method was used because the data of this study did not satisfy the proportional hazards assumption.
Ethics statement
The Institutional Review Board (IRB) of Chung-Ang University Hospital approved this study (IRB No. 2051-001-415). Informed consent was submitted by all subjects when they were enrolled.
RESULTS
The 177 enrolled HCWs received three doses of COVID-19 vaccination and blood tests were performed at least once after the primary series. Of the 177 HCWs enrolled in the study, 122 HCWs were classified as the CCB group and 55 HCWs as the BBB group. The median age was 38 years (IQR: 30-48) in both the CCB and BBB groups (P = 0.934), and two-thirds of both groups were women (66.4% in the CCB group vs. 67.3% in the BBB group, P = 0.909). After receiving the primary series, 164 HCWs participated in the blood sampling (113 in the CCB group and 51 in the BBB group). After receiving the booster vaccination, 88 HCWs participated in the blood sampling (55 in the CCB group and 33 in the BBB group).
Trends of humoral immunities
The Fig. 2A), but this difference was not significant in a multiple linear regression model, which included the time from the booster vaccination to blood collection (P = 0.704, Fig. 2A).
Although the median value of SVNT-O was higher in the BBB group than in the CCB group after the primary series ( Fig. 2B). In a multiple linear regression model including the time from the booster vaccination to blood collection, there was still a significant difference between the two groups (P = 0.001, Fig. 2B).
The median values of the anti-spike IgG significantly increased after the booster vaccination in both groups ( (Fig. 3A).
Trend of cellular immunities
In the CCB group, the median IGRA values significantly increased after the booster dose from 93.2 mIU/mL to 429.3 mIU/mL over a cutoff of 200 mIU/mL (P < 0.001). The median IGRA value after the booster dose was higher in the BBB group than in the CCB group (550.5 vs. 387.5 mIU/mL, respectively, P = 0.014; Fig. 3B). However, this result was not statistically significant in a multiple linear regression model, which included the time until blood collection after the booster dose as a variable (P = 0.104, Fig. 3B).
5/10
Effectiveness To adjust the effect of the time-difference in comparing the serologic test results between the two groups, the comparison in (A) was not different (P = 0.704), but that in (B) was different (P = 0.001) in multiple regression models * .
Omicron breakthrough infections
During the study period, total breakthrough infections occurred in 84 HCWs (47.5% of 177), and more cases occurred in the CCB group than in the BBB group (50.0% of 122 vs. 41.8% of 55), but there was no significant difference (P = 0.313). Fig. 4 shows the cumulative incidence of total breakthrough infection between the two groups over time, which was significantly different (P = 0.045), indicating that breakthrough infection increased one month earlier in the CCB group than in the BBB group. There was no significant difference between SVNT-WT, SVNT-O, anti-spike IgG, and IGRA values between HCWs with and without breakthrough infections (Supplementary Fig. 2). However, when the same comparison was made between 6 HCWs with and 82 HCWs without breakthrough infections within 30 days of the 2nd blood sampling after booster vaccination, the median values of the anti-spike IgG and SVNT-O were lower in HCWs with breakthrough infections (5.178 vs. 7.950, P = 0.013; 31.0% vs. 58.0%, P = 0.031, respectively) (Fig. 5).
DISCUSSION
The CCB group showed lower levels of neutralizing responses against the omicron variant than the BBB group after the primary series and booster vaccination. Moreover, in the CCB group, omicron breakthrough infection occurred earlier than in the BBB group.
The median neutralizing antibody responses to wild type SARS-CoV-2 in the BBB groups was not higher than those in the CCB group ( Fig. 2A). This is consistent with one of the results of the COV-BOOST study. In that study, when BNT162b2 was administered as a booster for
6/10
Effectiveness Interferon-γ response against wild type, mIU/mL In a multiple regression model, which included time from the booster dose to blood sampling, the comparison in (B) was not statistically significant (P = 0.104) * .
both ChAdOx1/ChAdOx1 and BNT162b2/BNT162b2 groups, the increase in the neutralizing antibody response appeared far higher in the latter group than in the former group. 6 Thus, as with our study, the difference between the two groups disappeared after the booster vaccination. Lower neutralizing antibody responses to wild type SARS-CoV-2 were found after the primary series with ChAdOx1 ( Fig. 2A). Likewise, lower antibody responses were also found to the omicron variant after the primary series with ChAdOx1 than those after the primary series with BNT162b2 (Fig. 2B) is strongly associated with neutralization against the ancestral virus. Furthermore, this phenomenon persisted even after the booster vaccination (Fig. 2B). In the COV-BOOST study, the neutralizing antibody response data for the delta variant showed that the boosting effect of BNT162b2 was less than that of the wild type virus. 6 Considering that the omicron variant has a more superior immune evasion ability than the delta variant, 8 it is plausible that the neutralizing antibody response level of the CCB group to the omicron variant could not reach that of the BBB group despite a booster vaccination with BNT162b2. In addition, vaccineinduced immunity wanes over time. 9 Although Khoury et al. 10 suggested that neutralizing antibody responses are predictive of immune protection from SARS-CoV-2 infection, it has been unclear whether the difference in the neutralizing responses against the omicron variant between the CCB and BBB groups was associated with the difference in the occurrence of omicron breakthrough infection between them. In our study, while there was no difference in the frequency of breakthrough infection between the two groups within 6-8 months after booster vaccination, breakthrough infection accumulated more rapidly in the CCB group than in the BBB group. There might be several reasons for this difference. However, there was no demographic difference between the two groups of HCWs and no particularly notable difference in their behaviors. Thus, the difference in their serostatus should be considered first. Furthermore, considering that the neutralization response to the omicron variant was lower in HCWs with breakthrough infections than in those without breakthrough infections within 30 days of the 2nd serologic testing post-booster vaccination, the lower neutralization response in the CCB group is the most plausible cause of the earlier occurrence of breakthrough infection in this group. The association between the level of neutralizing response and breakthrough infection was also found in previous studies comparing the ChAdOx1/ChAdOx1 and BNT162b2/BNT162b2 groups. In a few studies, including this study, the neutralizing response was lower in the ChAdOx1/ChAdOx1 group than in the BNT162b2/BNT162b2 group. 3,11 Additionally, three large cohort studies conducted in the United Kingdom (UK) directly compared the protective efficacy against COVID-19 between the ChAdOx1/ChAdOx1 and BNT162b2/BNT162b2 groups. 12-14 Although there was no difference in COVID-19 incidence or hospitalization between the two groups in a previous study, 11 the ChAdOx1/ChAdOx1 group had a greater COVID-19 incidence and hospitalization rate than that of the BNT162b2/BNT162b2 group in other studies. 13, 14 Collectively, it is suggested that the difference in the vaccine-induced neutralizing response is associated with the difference in breakthrough infection during the omicron variant era.
This study has several limitations. First, considering the small number of subjects used in this study, further research, such as one of the above-mentioned large-scale studies in the UK, is required. Second, some study participants may have had undetected COVID-19 during symptom-based testing, which may have affected antibody test results among HCWs enrolled in this study. Third, as a neutralizing test using live virus is not clinically applicable, it would be rational to utilize the commercial SVNT kits. Lastly, we had difficulty interpreting the results because we lacked the IGRA values for the BBB group after the primary series. Despite these limitations, the present study has the strength to identify omicron breakthrough infection using neutralizing antibodies against the omicron variant.
In conclusion, HCWs vaccinated with CCB showed a lower neutralization response against the omicron variant than those with the BBB vaccination series. This difference in neutralization response may be responsible for the omicron breakthrough infection
8/10
Effectiveness of Hetero/Homologous COVID-19 Vaccination occurring earlier in the former than in the latter group. To respond to the continuing outbreak of SARS-CoV-2 and the future respiratory virus pandemic, further research is needed to obtain maximum efficacy by appropriately utilizing various vaccine types.
|
2023-04-23T15:09:52.778Z
|
2023-04-20T00:00:00.000
|
{
"year": 2023,
"sha1": "e07802b3796aa46a9e427599bd2391c8ba170981",
"oa_license": "CCBYNC",
"oa_url": "https://doi.org/10.3346/jkms.2023.38.e155",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "0036c1e0dec997d88648afcfb6599f5de4eb6ac5",
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"Medicine"
],
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"Medicine"
]
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|
24108749
|
pes2o/s2orc
|
v3-fos-license
|
Alteration of muscle fiber characteristics and the AMPK-SIRT1-PGC-1α axis in skeletal muscle of growing pigs fed low-protein diets with varying branched-chain amino acid ratios
There mainly exists four major myosin heavy chains (MyHC) (i.e., I, IIa, IIx, and IIb) in growing pigs. The current study aimed to explore the effects of low-protein diets supplemented with varying branched-chain amino acids (BCAAs) on muscle fiber characteristics and the AMPK-SIRT1-PGC-1α axis in skeletal muscles. Forty growing pigs (9.85 ± 0.35 kg) were allotted to 5 groups and fed with diets supplemented with varying leucine: isoleucine: valine ratios: 1:0.51:0.63 (20% crude protein, CP), 1:1:1 (17% CP), 1:0.75:0.75 (17% CP), 1:0.51:0.63 (17% CP), and 1:0.25:0.25 (17% CP), respectively. The skeletal muscles of different muscle fiber composition, that is, longissimus dorsi muscle (LM, a fast-twitch glycolytic muscle), biceps femoris muscle (BM, a mixed slow- and fast-twitch oxido-glycolytic muscle), and psoas major muscle (PM, a slow-twitch oxidative muscle) were collected and analyzed. Results showed that relative to the control group (1:0.51:0.63, 20% CP), the low-protein diets with the leucine: isoleucine: valine ratio ranging from 1:0.75:0.75 to 1:0.25:0.25 especially augmented the mRNA and protein abundance of MyHC I fibers in BM and lowered the mRNA abundance of MyHC IIb particularly in LM (P < 0.05), with a concurrent increase in the activation of AMPK and the mRNA abundance of SIRT and PGC-1α in BM (P < 0.05). The results reveal that low-protein diets supplemented with optimal BCAA ratio, i.e. 1:0.75:0.75-1:0.25:0.25, induce muscle more oxidative especially in oxido-glycolytic skeletal muscle of growing pigs. These effects are likely associated with the activation of the AMPK-SIRT1-PGC-1α axis.
INTRODUCTION
Muscle fibers account for 75~90% of the muscle and are main factors that influence the characteristics of the muscle [1].The major muscle fibers in mammals can be roughly divided into slow and fast-twitch fibers, and further classified into type I, type IIa, type IIx, and type IIb in limb and trunk muscles.Notably, other muscles such as ocular and jaw muscles contain specialized muscle fibers other than these four fibers [2].It is difficult to clearly distinguish the muscle fibers (I, IIa, IIx, and IIb).However, immunohistochemical studies have shown that the four muscle fibers (type I, IIa, IIx, and IIb) mainly contain myosin heavy chain (MyHC) I, IIa, IIx, and IIb, respectively.MyHC1, IIa, IIx, and IIb are encoded by genes Myh7, Myh2, Myh1, and Myh4, respectively [3].Type I fibers (slow-twitch, oxidative) contain greater mitochondria and predominately oxidative enzymes, and metabolize lipids as a source of energy.Type IIb fibers (fast-twitch, glycolytic) are predominantly glycolytic and use glycogen and glucose as fuel, and type IIa (fast-twitch, oxidative) and type IIx (fast-twitch, oxido-glycolytic) are intermediate to type I and IIb fibers [4,5].In animals, muscle fiber type profile is one of major factors affecting lots of the peri-and post-mortal biochemical processes and hence meat quality [1].In humans, muscle fiber type is strongly associated with muscle health and overall well-being [6].For reasons stated above, the regulation of muscle fiber type is of uppermost interest not only in the conversion of animal muscle to meat, but also in molecular medicine for potential therapeutic perspectives.
Adult skeletal muscle shows plasticity and can undergo conversion between different fiber types in response to a myriad of external stimuli [4,7,8].Nutrition has sparked substantial interests as an external stimuli [9][10][11][12].There is a large body of literature supporting nutritional control of the muscle fiber type.For example, a high-protein diet (30%) preserved fiber type distribution, preventing switch from slow-to-fast twitch fibers in rat soleus muscles [9].Consistently, another experiment using rats have also revealed that 4 weeks of a high-protein diet (35%) induced muscle fibers changing from type II to type I in gastrocnemius muscles, accompanied by increased oxidative properties [12].Some important regulators, such as peroxisome proliferator-activated receptor-g coactivator-1α (PGC-1α), constitute a mechanism that may be responsible for the effects of high-protein diets commented above [12].PGC-1α through its interaction with silent information regulator transcript 1 (SIRT1) contributes to regulation of fiber conversion to type I [13].Moreover, chronic activation of AMP-activated protein kinase (AMPK) has also been demonstrated to evoke muscle plasticity and conversion to the slow oxidative myogenic program, potentially associated with upregulated PGC-1α expression [14].These observations indicate that the AMPK-SIRT1-PGC-1α axis may promote the slow, oxidative phenotype.
In recent years, there is growing awareness that in swine production, reducing the dietary crude protein (CP) level of the diet and supplementing it with the first four limiting crystalline amino acid (lysine, methionine, threonine, and tryptophan) are able to reduce N excretion and to improve gastrointestinal and function after weaning.However, impairment of piglet growth will occur in parallel [15][16][17].Thus, it is urgent to identify the next-limiting amino acids that can maintain the growth performance of piglets fed low-protein diets.
Leucine (Leu) is a branched-chain amino acid (BCAA), which also includes isoleucine (Ile) and valine (Val).The chemical structure of Leu is similar to those of Ile and Val, and Leu competes with Ile and Val for the same enzymes that catalyze the first two catabolic steps [18].An excessive supply of Leu in diets might augment the catabolism of all BCAAs and hence boost the nutritional requirements for Ile and Val [19].Therefore, the dietary ratio of individual BCAA needs to be closely managed.We have previously reported that maintaining the dietary BCAA ratio (Leu: Ile: Val) within 1: 0.25: 0.25 -1: 0.75: 0.75 in low-protein diets (17% CP) contributed to improving the growth performance of growing pigs, facilitating the absorption and utilization of free amino acids, thus improving protein metabolism and muscle growth.These effects even caught up to those observed in the positive control group (Leu: Ile: Val = 1: 0.51: 0.63, 20% CP) [20,21].Although the effects of BCAA ratio on muscle protein metabolism have been examined at the molecular level [21], the relationship between the BCAA ratio and muscle fiber types have not been as thoroughly characterized.Based on these observations, we speculated that effects of a low-protein diet (17% CP) supplemented with balanced BCAAs could catch up to those of highprotein diets on muscle fiber types in growing pigs via the AMPK-SIRT1-PGC-1α axis.In an analogy with muscular protein metabolism, it is of great importance to get an overview of relationships between BCAA ratio and signaling molecules for muscle fiber types.
Myofiber type proportions of longissimus dorsi muscle (LM), biceps femoris muscle (BM), and psoas major muscle (PM) are varied due to their anatomical location and thus they have different metabolic properties [22].The percentage of oxidative (type I and IIa) fibers in the three skeletal muscles is as follows: PM > BM > LM.On the contrary, the percentage of glycolytic (type IIb) fibers in muscles is PM < BM < LM [22].Muscle fibers are not static structures and easily adapt to altered environmental factors, such as changes in nutritional input.Thus, the aim of the present study was to explore the effects of varying BCAA ratios in low-protein diets (17% CP) on the mRNA and protein abundance of MyHC in muscles of different muscle fiber composition, and whether these effects were associated with the AMPK-SIRT1-PGC1a axis.
Serum glucose and insulin concentrations
As shown in Table 2, the serum glucose concentrations were not different among the groups (P > 0.05).The serum insulin concentrations in the 1:1:1 (17% CP) group was the same as those in the control (PC group, Leu: Ile: Val = 1:0.51:0.63,20% CP), while the concentrations in other experimental groups significantly increased (P < 0.05), and there was no difference between the three groups.
Gene mRNA abundance of MyHC isoforms
We analyzed the mRNA abundance of MyHC I, IIa, IIx, and IIb in LM, BM, and PM of pigs fed low-protein diets with varying BCAA ratios.As shown in Table 3, the mRNA abundance of MyHC I, IIa, IIx, and IIb was highest in BM relative to that in LM and PM.In particular, the mRNA abundance of MyHC I, IIa, IIx, and IIb in BM was 19.58%, 84.37%, 78.83%, and 23.49% higher than that in LM (P < 0.05), respectively, and was 4.91%, 67.38%, 97.84%, and 17.20% higher than that in PM (P < 0.05), respectively.Moreover, compared with the control, the experimental groups exhibited higher mRNA abundance levels of MyHC I and IIa, with the highest values observed in the 1:0.25:0.25 group (P < 0.05).Although the experimental groups showed a higher mRNA abundance of MyHC IIx than the control, the mRNA abundance of MyHC IIx in experimental groups gradually decreased with the reduction of dietary BCAA ratio, with the lowest value observed in the 1:0.25:0.25 group (P < 0.05).There was a muscle × diet interaction for the mRNA abundance of MyHC IIa, IIx, and IIb in muscles of different fiber types (P < 0.05).
value in PM (P < 0.05).The mRNA abundance of GLUT4 was highest in PM and lowest in LM, with an intermediate value in BM.Furthermore, the mRNA abundance of SIRT1 and UCP3 in 1:0.75:0.75,1:0.51:0.63,and 1:0.25:0.25 was higher than that in the control (P < 0.05), with the greatest upregulation observed in 1:0.51:0.63(17% CP).As to the mRNA abundance of PGC-1α, the 1:0.75:0.75 and 1:0.51:0.63groups exhibited the higher abundance relative to the control (P < 0.05).The mRNA abundance of GLUT4 in the 1:0.75:0.75 group was higher than other groups (P < 0.05), and none of these groups achieved statistical significance.There was a muscle × diet interaction for the mRNA abundance of SIRT1, PGC-1α, UCP3, and GLUT4 in muscles of different fiber types (P < 0.05).
Protein abundance of MyHC I and IIa as well as the AMPK-SIRT1-PGC-1α axis
As shown in Figure 1A, in LM, the protein abundance of MyHC IIa and I was highest in 1:0.75:0.75 and 1:0.51:0.63(17% CP) groups and lowest in the 1:0.25:0.25 group, with an intermediate value in the 1:1:1 and control (P < 0.05).Alteration in the protein abundance of p-SIRT1 and p-AMPKα showed the same trends as that of MyHC IIa and I.The PGC-1α protein abundance in 1:0.25:0.25 group was of the same value as that in the control (P > 0.05), while the 1:1:1, 1:0.75:0.75 and 1:0.51:0.63(17% CP) groups was unable to bring PGC-1α protein abundance to the same level observed in the control group (P < 0.05).In BM (Figure 1B), the protein abundance of MyHC IIa and I in 1:0.25:0.25 group was not different from that in the control, while other experimental groups was unable to bring the abundance to the same level observed in the control (P < 0.05).The protein abundance of PGC-1α was highest in the 1:0.51:0.63(17% CP) group (P < 0.05), and other experimental groups were not significantly different from that in the control.The protein abundance of p-SIRT1 was highest in the 1:0.75:0.75 group (P < 0.05), and other experimental groups were not significantly different from that in the control.The p-AMPKα protein abundance was not affected by the treatments (P > 0.05).In PM (Figure 1C), the experimental groups tended to increase the protein abundance of MyHC IIa and I, with the greatest increase observed in the 1:0.51:0.63(17% CP) group (P < 0.05).Alteration in the protein abundance of p-SIRT1 and p-AMPKα showed the same trends as that of MyHC IIa and I.The PGC-1α protein abundance in 1:1:1 and 1:0.25:0.25 groups was the same as that in the control (P > 0.05), while the 1:0.75:0.75 and 1:0.51:0.63(17% CP) groups was unable to bring PGC-1α protein abundance to the same level observed in the control group (P < 0.05).
DISCUSSION
The skeletal muscle, including LM, BM, and PM, is a highly heterogeneous tissue, mainly comprised of four myofiber types: oxidative (I and IIa), intermediate (IIx), and glycolytic (IIb) [22].There are marked differences in the fiber proportion between these muscles.The porcine LM has a high proportion of type IIb fibers and a small proportion of type I fibers, and is classified as glycolytic skeletal muscle [23].The BM also mainly contain the fiber type of IIb, whereas the oxidative type I in BM is higher than that in LM.Therefore, the BM is classified as oxido-glycolytic skeletal muscle.Compared to the LM and BM, PM contains a relatively higher percentage of type I fiber and a lower proportion of type IIb fibers, and is classified as oxidative skeletal muscle [24].Our studies indicated that the mRNA abundance of both oxidative and glycolytic isoforms especially increased in the BM of pigs in response to the treatments of the lowprotein diets supplemented with varying BCAA ratios (1:0.75:0.75~0.25:0.25),However, compared to LM and PM, the extent of increases (103.95% and 72.29%, respectively) in oxidative isoforms (I and IIa) in BM was higher than that of increases (78.83% and 17.20%, respectively) in glycolytic isoforms (IIb).Therefore, a transformation from glycolytic-to-oxidative fibers may occur in BM in response to dietary treatments.Muscle fiber type phenotype is strongly associated with meat quality and muscle health.Higher amounts of type I and IIa fibers are associated with improved meat tenderness [25] and afford protection against insulin resistance and metabolic dysregulation [7,26].In contrast, increasing percentages of type IIb fibers may increase drip loss and exhibit tougher meat [27].Based on these, our findings suggest that feeding the low-protein diets (17% CP) with varying BCAA ratios (1:0.75:0.75~0.25:0.25)could induce muscle more oxidative which contributes to the improvement of meat quality and muscle health.Fiber type shift from glycolytic to oxidative in BM of pigs is consistent with the function of AMPK in skeletal muscle.The enzyme AMPK is a major energy sensor of myocytes [28].Muscles rich in type IIb fibers may have a greater AMPK activity than muscles rich in type I and IIa fibers to more effectively or rapidly modulate energetic process, thereby conserving energy for ATPase activity and tension cost.Therefore, AMPK activity can be modulated according to the energetic requirements of different muscle fiber types.This indicates an intimate relationship between AMPK activity and muscle function [29].Chronic exercise training in mice augments mitochondrial biogenesis and induces a glycolytic-to-oxidative fiber type transition in skeletal muscle; these transitions are blocked in AMPKα-inactive mice [30,31].Likewise, chronic AMPK activation by injecting AICAR promotes a fast-to-slow fiber type transition in skeletal muscle of rabbits and rodents [14,32].Reports of the effect of AMPKα on muscle fiber transformation have mainly focused on rodents, and the relationship between AMPKα and porcine muscle fiber transformation is not well understood.However, it has been recently reported that maternal dietary linoleic acid treatment enhances the AMPKα mRNA abundance in piglets that have more oxidative muscle fibers [33].These data reveal that AMPKα may be associated with porcine muscle fiber composition.Consistent with the previous studies, we found that lowprotein diets supplemented with varying BCAA ratios (1:0.75:0.75~1:0.25:0.25)specifically stimulated the activation of AMPK in BM, which had more oxidative muscle fibers than LM and PM.In contrast, other studies showed a different patterns of responses.For instance, previous studies showed downregulation of AMPK in rats fed increased Leu [34], or the phosphorylation of AMPK was increased in response to high concentration of BCAAs, but accompanied by inflammation [35].The discrepancies between these findings might be related to several factors including animal model and experimental conditions (i.e., BCAA ratio).Although we show a potential connection between BCAA ratio and AMPK activation, the proximal mechanisms by which AMPK responds to BCAA ratios remain elusive.Overall, muscle fiber type-specific mRNA and protein abundance of AMPKα may promote a shift from glycolytic to oxidative fibers in BM.
It has been reported that PGC-1α, a downstream of AMPK, has been proposed as partially responsible for the effects exerted by AMPK on muscle fiber transformation [3].Experiments in mice have shown that PGC-1α overabundance results in an enhancement of type I fibers [13], while the proportions of type I fibers tend to reduce in PGC-1α muscle-specific knockout mice [36].AMPK may modulate PGC-1α directly or through SIRT1 [37].Consistent with the protein abundance of AMPK, the mRNA abundances of SIRT1 and PGC-1α were higher in BM of pigs fed low-protein diets supplemented with varying BCAA ratios (1:0.75:0.75~1:0.25:0.25).The present results suggest that low-protein diets supplemented with optimal BCAA ratios specifically elevated AMPK abundance in BM of growing pigs and that AMPK may affect muscle fiber transformation by modulating the mRNA abundance of PGC-1α.Previous studies reveal that the AMPK-SIRT1 axis sensing the cellular energy status can modulate muscle fiber type by influencing mitochondria [38][39][40].Mitochondrial function has been regarded as a new mediator of skeletal muscle fiber type, and PGC-1α exerts a particularly robust role in mitochondrial biogenesis and function [41].In addition, mitochondrial biogenesis is involved in the role of Leu in energy metabolism [42].There is compelling evidence that Leu (0.5 mM) can enhance mitochondrial biogenesis in C2C12 myocytes and regulate skeletal muscle energy metabolism by modulating the abundance of SIRT1 and PGC-1α [43].Therefore, it is hypothesized, though not yet tested by the present study, that the AMPK-SIRT1-PGC1a axis contributes to the skeletal muscle fiber type alterations in response to dietary treatments by affecting mitochondrial biogenesis and function.Further studies are required to confirm this hypothesis.In agreement with a typical fast-to-slow fiber type shift, enhancements in GLUT4 abundance generally parallels the activation of AMPK and PGC-1α, thus leading to the enhancement of muscle glucose metabolism and energy utilization [44,45].Moreover, GLUT4 protein content and muscle glucose uptake were increased in response to AMPK activation especially in fast-twitch glycolytic muscles rather than in slow-twitch oxidative muscles [46].In contrast to these previous studies, we did not observe a concurrent increase in GLUT4 and AMPKα abundance in BM upon dietary treatments.Instead, PM exhibited the highest abundance of GLUT4.Our data provide evidence that the capacity of muscle glucose uptake may be improved especially in slowtwitch oxidative muscles.In rodents, GLUT4 content is inherently greater in oxidative type I muscles vs. glycolytic type IIb muscles [47].Consistent with this, our results with pigs showed that oxidative PM contains more GLUT4 than oxidative-glycolytic BM and glycolytic LM, although the activities of AMPK and PGC-1α were significantly upregulated in BM.In contrast, other studies exhibited a different patterns of responses in muscles from other species.Studies using models of rat and humans show that GLUT4 level is not consistently higher in type I than in type II [48].A greater GLUT4 level in type IIb fibers than type I and IIa fibers of equine muscle has been reported [49].Moreover, some studies show that there is little relationship between GLUT4 content and fiber type although exercise training increases GLUT4 level [50].Therefore, GLUT4 may be more in connection with muscle activity than with MyHC isoform, and the precise mechanism by which AMPK modulates muscle fiber typespecific GLUT4 abundance remains elusive.It has been reported that UCP3 abundance is closely related to glucose metabolism in skeletal muscle.Overexpressing UCP3 in L6 myotubes promotes glucose uptake through an increased recruitment of the glucose transporter GLUT4 to the cell surface [51].UCP3 and GLUT4 mRNA abundance have been shown to increase in parallel after endurance exercise [52].One proposed mechanism by which UCP3 could influence glucose uptake via GLUT4 translocation is via AMPK [53].Experiments using humans also show that UCP3 is expressed more abundantly in glycolytic type IIb muscle fibers than in oxidative type I muscle fibers [54].In contrast to the previous findings, we found that in our in vivo experimental conditions the mRNA abundance of UCP3 was highest in oxidative-glycolytic skeletal muscle (BM) and lowest in glycolytic skeletal muscle (LM) of growing pigs.The pattern of UCP3 mRNA abundance was similar to that of AMPKα protein abundance in skeletal muscles.However, GLUT4 mRNA abundance did not increased in parallel with UCP3.Experiments using mice also show that overexpressing UCP3 reduces plasma glucose and insulin levels [55].However, in the present study, no difference in serum glucose concentrations among all groups was noted, and serum insulin concentrations were increased in pigs fed diets with varying BCAA ratios (1:0.75:0.75~1:0.25:0.25).A peculiar characteristic of skeletal muscle is that it is composed of different types of muscle fibers, with different capacities to respond to external stimuli [56].Therefore, although the reason for this discrepancy is not clear, it is possible that difference in the experimental approaches such as animal breeds, exercise or not, and diets could have contributed to the observed difference.Overall, our data provide evidence that AMPKα activation increased the PGC-1α mRNA and protein abundance levels, and it consequently increased UCP3 abundance especially in oxido-glycolytic skeletal muscles (BM).
In summary, we herein demonstrated that lowprotein diets supplemented with optimal BCAA ratio, i.e. 1:0.75:0.75-1:0.25:0.25,induced a muscle fiber transformation from glycolytic to oxidative fibers especially in oxidative-glycolytic skeletal muscle of growing pigs.These effects were likely attributed to the activation of the AMPK-SIRT1-PGC-1α axis.In addition, such treatment also increased the UCP3 mRNA abundance in the oxidoglycolytic skeletal muscle.It is speculated that this adaptation may be due to an increased AMPKα activity.On the other hand, the pattern of GLUT4 mRNA abundance in selected muscles may be independent of the AMPK pathway.
MATERIALS AND METHODS
All procedures followed in the present experiment were approved by the committee on animal care of the Institute of Subtropical Agriculture, the Chinese Academy of Sciences.
Animals and diets
A total of forty pigs (Large White × Landrace) with a mean initial weight (9.85 ± 0.35 kg) were chosen and randomly allotted into five dietary treatments.Each treatment had eight replicates (n=8).Animals were housed individually in cages.Diets were corn and soybean mealbased and formulated to differ in CP and AA quantities (Supplementary Table 1).All diets were fortified with lysine, methionine, threonine and tryptophan to provide recommended levels according to the National Research Council (NRC, [57]).The diets of positive control (PC) group contained 20% CP with a Leu: Ile: Val ratio of 1:0.51:0.63 according to the recommendation of the 2012 NRC [57].In the four experimental groups, the dietary CP level was reduced to 17%, and the Leu: Ile: Val ratios were 1:1:1, 1:0.75:0.75,1:0.51:0.63,and 1:0.25:0.25,respectively.The total BCAA amount was equal in all treatments.All the experimental diets were formulated to be isoenergetic and to meet the nutritional requirements for growing pigs (Supplemental Table 1).Pigs were fed with the experimental diets ad libitum, and had unlimited access to clean drinking-water.The experiment lasted for 45 d.
Tissue sample collection
Before slaughter, blood samples were collected into 10 ml tubes from the jugular vein puncture for the determination of serum biochemical indices.Serum was separated by centrifugation at 3,000 g for 10 min at 4°C and then stored at -80°C until analysis.At the end of the feeding test, all the pigs were fasted overnight and slaughtered by electrically stunning (250V, 0.5 A, for 5~6s) and exsanguinating as described in our previous study [58].Immediately, skeletal muscle samples including LM, BM, and PM were rapidly excised from the left side of the carcass.The samples were then placed in 10% neutral buffered formalin or placed in liquid nitrogen and then stored at -80°C, respectively, until further analysis.
Measurement of serum glucose and insulin concentrations
The concentrations of serum insulin were measured using commercial ELISA kits (Cusabio Life Science Inc., Wuhan, China).Circulating glucose concentrations were determined using commercial kit from CIBA Corning (OH, USA).www.impactjournals.com/oncotarget
Reverse transcription and real-time quantitative PCR
The reverse transcription and real-time quantitative PCR were conducted as previously described [22,58].Briefly, total RNA was extracted from skeletal muscles using Trizol reagent (Invitrogen, Carlsbad, CA, USA).Primers for the selected genes were designed using the Oligo 6.0 software (Table 1).RT was performed using the AMV Reverse Transcriptase Kit (Promega).The relative expression levels of the target genes were determined using quantitative real-time PCR, performed with an ABI 7900 PCR system (ABI Biotechnology).The final volume of the reaction mixtures (20 μL) contained diluted complementary DNA and SYBR Green I (Molecular Probes) as a PCR core reagent.The housing-keeping gene β-actin was used as internal control to normalize the expression of target genes.The relative quantification of gene amplification by RT-PCR was performed using the value of the threshold cycle (Ct).Relative expressions of target genes were determined by the 2 -∆∆Ct method [58,59].
Statistical analyses
Data of serum parameters and the fiber size obtained from this study was analyzed by the One-way analysis of variance (ANOVA) using SAS 8.2 software (Cary, NC, USA) followed by a Duncan's multiple comparison test.Other data in the present study were performed by ANOVA using the general linear model procedures of SAS appropriate for a 2 × 2 factorial design (SAS Inc., Cary, NC).The statistical model included the effects of muscle (LM, BM, or PM), diet (1:0.51:0.63(20% CP), 1:1:1 (17% CP), 1:0.75:0.75(17% CP), 1:0.51:0.63(17% CP), and 1:0.25:0.25 (17% CP)), and their interactions.The differences among treatments were evaluated using Tukey's test.Results are presented as means with standard errors.Differences between significant means were considered as statistically different at P < 0.05 and a trend toward significant at P < 0.10.
Figure 1 :
Figure 1: Protein abundance levels of MyCH I, MyCH IIa, and p/t-SIRT1, p/t-AMPKα, PGC-1α in the longissimus dorsi muscle, biceps femoris muscle, and psoas major muscle of growing pigs fed low-protein diets supplemented with varying BCAA ratios.Data were normalized to the value of corresponding total protein or the inner control β-actin and expressed as means ± SE (n = 8).Values within a row with different superscripts differ significantly (P < 0.05).
Table 4 .
The mRNA abundance of SIRT1, PGC-1α and UCP3 was highest in BM and lowest in LM, with an intermediate
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2018-04-03T05:58:21.355Z
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2017-10-31T00:00:00.000
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1 VITORIA BAY POLLUTION STUDY IN THE FRAME OF TAGUBAR RESEARCH PROJECT – GEOCHEMISTRY OF THE SEDIMENTS OF ESPIRITO SANTO BAY
The Bay of Espirito Santo is located in the Espírito Santo State, in the eastern part of Brazil. It is surrounded by the city of Vitoria on one side and by the Atlantic Ocean on the other. Superficial sediments of Espirito Santo Bay were analyzed at 12 (western shallow silt sediments) + 8 (eastern sandy sediments and relatively deep sampling stations) = 20 uniformly distributed sampling points where geochemical analysis was performed. Nineteen elements were analyzed: Mo, Cu, Pb, Zn, Ag, Ni, Mn, Fe, As, U, Th, Sr, Cd, Sb, Bi, V, Cr, Ba, and Al. This selection was made based on the most representative heavy metals present in this area and according to the results obtained from the geochemical analysis. Their concentrations were compared with metal contamination benchmarks like Screening Quick Reference Tables (SQuiRTs), Effects Range-Low (ERLs) and Effects Range-Median (ERMs), TELs (Threshold Effects Levels), PELs (Probable Effects Levels), ERM (Effects range median), and AETs (Apparent Effects Thresholds). Results indicate that there is no particular pollution condition able to alter the condition of any part of this water body. The Authors conclude that the Espirito Santo Bay is only moderately polluted and some elements are virtually absent.
Introduction
TAGUBAR Project derives its origins from a Brazilian Government decision to tackle the planning and management challenges related to the restoration of some degraded aquatic ecosystems like the Guanabara (State of Rio de Janeiro) and the Vitoria-Espirito Santo (State of Spirito Santo) bays.This was performed by using the successful outcomes of a previous MFA/DGCS (Ministry of Foreign Affairs/Direttore Generale alla Cooperazione allo Sviluppo, i.e., Directorate General for the Cooperation and Development) cooperation program involving the Project NIKE in Vitoria (Espirito Santo Bay).Such relevant multi-year collaboration between Italian and Brazilian scientific institutions allowed the exchange of the acquired methodologies and of the corresponding data-bases.The general objective of the programme was to contribute to the economic and social development of population living around Guanabara, Vitoria and Espirito Santo bays while promoting the conservation of their natural resources.This objective was supposed to be achieved by consolidating local Authorities' ability to plan and implement a re-conditioning program within a systemic management framework in severely polluted ecosystems.The proposed project was named TAGUBAR (TAngential GUanabara Bay Aeration and Recovery, following the pattern of the VENICE's Logical Framework) developed by IDEAS (institution belonging to the University of Venice, Italy) and applied to Guanabara Bay.In the frame of TAGUBAR Project, the MFA/DGCS and the Government of Brazil decided to perform a specific parallel research to check the methodology applied on Rio de Janeiro Bay and to compare the results with two other different tropical environments.The Vitoria Bay and Espirito Santo Bay were selected to be used as comparison.
The study area: the main physical and geographical aspects of the Vitoria Bay
Vitoria City was founded in 1535 and originally was called Vila Nova ("New Town") do Espírito Santo.Today it is a sort of Rio de Janeiro in miniature and capital of the Espírito Santo State, located in the eastern part of Brazil (Figure 1).The city of Vitória lies on an island in front of the coast (Figure 2).areas near the beach, but the central business district is limited to a cove of 5 km in the inland.It is connected to the mainland by a bridge.
Vitoria constitutes an important commercial center of the State, with exports of sugar, coffee, lumber, rice, and manioc.Nowadays (2018), it has a population of about 360 thousand inhabitants and its bay is one of the most industrialized region in the State of Espírito Santo.As a result of the urban growth, large quantities of raw industrial and urban sewage containing heavy metals enter the estuarine system [1,2,3].
The Espírito Santo Bay presents a high degree of exposure to tides, winds, and waves.On the north, it is surrounded by the Tubarão Mountain while on the south by the Moreno do Moreno Mountain and the Camburi Beach, the most renowned beach of the municipality of Vitoria.Anthropic influence contributed heavily over the decades to decrease of the mangrove area around the island of Vitoria [4], as well as in the proximity of the communities of Sao Pedro, Caieiras, Santo Antonio, and Maria Ortiz.Carmo [5] and Jesus [6,7] suggested that the main causes of such decrease could be landfills, industries, and port activities.
Currently, mangroves occupy an area of only 18 km 2 , which represents 20% of the total mangroves present in the Espirito Santo State [4].The northwestern part of the estuary system includes the mouth of the Santa Maria River (4 km 2 of mangrove), Bubù River (3 km 2 ), and Lameirao Island (Municipal Biological Reserve, 4.9 km 2 ) [6].From the naturalistic point of view, it is the most preserved area and it displays a high mangrove density [4].
The Vitoria Bay receives the inputs from the rivers Aribiri, Bubu, and Santa Maria.The farms located in the estuaries of these rivers provide to the market considerable amounts of fish and shellfish used for human consumption.Therefore, the evaluation of the contamination of the marine environment by heavy metals becomes urgent [2,3,7,8].The Bay of Vitória is protected from the ocean waves while the tidal wave crosses the entire bay, rising up above the Santa Maria Delta and entering the Canal da Passagem (Pass Channel).It presents extensive shallow waters where mangroves flourish [4].Its SW-NE orientation favors the prevailing winds in the region.
The Canal of Porto (Port Channel) is an integral part of the Bay of Vitória, being the main channel of connection between the Bay of Espirito Santo and the Bay of Vitoria.It is a region greatly altered by landfills and dredging activities while, as it is already suggested by its name, it is the access channel to the Port of Vitoria, justifying therefore to be treated separately with respect to the Bay of Vitoria.
The surroundings of Espirito Santo and Vitoria Bays are characterized by a high rate of urbanization and industrial activities, especially regarding tourism infrastructures (big buildings and hotels) and shipping activities, which can be considered indicative of a fast city growth.Presence of large ships and boats can be observed specially at the end of the Canal do Porto as a consequence of the trading with American and European Countries.
The area is characterized by the presence of hilly islands (granite composition) densely vegetated.Some of them are getting also densely populated due to the facilities offered by transportation and good communication with the mainland.On the other hand, the surroundings of the Canal do Porto, at least large part of it, are populated by dense mangrove flora which is considered a good water and sediment depurator [4,9].Mangroves also represent a marine environment rich of life and biodiversity [4].The natural reserve "Lameirão" is located in this area.Presence of small houses can be also noticed in some parts along the Canal do Porto, which most probably belong to the fishermen living in this area, who benefit of fishes and mollusks characteristic of this kind of vegetation [8].Along some of these places it is also possible to see some mangrove "deforestation" caused by house construction [4].
The Canal da Passagem is located in the estuary system of the Santa Maria da Vitória River, in the municipality of Vitória-Espìrito Santo (20° 19' S and 40° 20' W).It could be classified as a coastal plain estuary and, regarding stratification, it can be considered "well mixed".At the beginning of Camburi Beach, it connects the Espírito Santo Bay to the northern portion of Vitoria Bay, receiving the influence of the tide at both ends, tide influence that involves much of the existing mangroves [4].Its bathymetry is variable, showing both shallow channels that dry at the time of the ebb tide and locations as deep as 7 m.Its average width is about 80 m, with the smallest width, 35 m, under the Passage Bridge [10,11].It is a shallow, windy channel with extensive mangrove areas [4].It characterized by a curious hydrodynamic behavior caused by the barotropic convergence of the tidal wave, resulting from the meeting of the tidal fronts that propagate through the different channels.It receives a large supply of domestic sewage, mostly untreated [1].The rest of domestic sewage comes from several neighborhood treatment plants of Vitória, such as CESAN (Companhia Espirito Santo Santana de Saneamento) André Carlone, Camburi, and Nova Palestina [1].Then, it reaches the Bay of Espírito Santo during the ebb tide [3].Because the sewage treatment is largely insufficient, this makes the partially treated sewage the main contributor to the degradation of the water quality [1,2,3,10].The sediments of this region presented the highest levels of organic matter as compared with the rest of the estuary.At the innermost points of the Canal, the sedimentation rate is high because the small flow velocity due to the presence of the mangroves [4,12].The inversion of flow direction, as consequence of the tide, favors the deposition of loamy sediments and organic matter [12,13].
Sampling points
Sampling points were uniformly distributed along the Espirito Santo Bay and part of the Vitoria Bay, with special emphasis on the Canal do Porto.The type and location of each station was previously determined according to the characteristics of each bay.Depending on the location and characteristics (depth) of the sampling sites, two boats were used for this campaign.The first boat used for the sampling at the Espirito Santo Bay was fully equipped with a professional navigation system including an accurate GPS and echo sounder.The other boat used for sampling at the canal (shallow waters), contained a portable GPS in order to know the correct coordinates of the sampling points.Also in this case, the water depth was measured by the professional echo sounder.For each sampling point, regardless the type of station, the following data were always recorded: geographic co-ordinates (longitude, latitude), water depth, time, and type of sediments.The sediments were collected, mixed carefully to obtain homogeneous samples, frozen, and then immediately transported to the laboratory for analysis.The samples were dried under nitrogen atmosphere to avoid oxidation and stored in sealed glass containers.This procedure was previously tested in our laboratory [14,15,16].
Sediment sampling stations
Sampling consisted on the collection of sediment and water samples for geological, chemical (water and sediments) and biological analysis [17].Each sampling took about 40 minutes to be completed since there were several different activities related to the sampling techniques used in each case.Briefly, sampling activities comprised the collection of bottom sediments using a Van Veen grab having a capacity of approximately 5 liters.Only one launch of the grab was adequate to collect enough amount of sample.All sediment samples were then deposited on a plastic tray and well mixed till homogenization (Figure 3).The sediment samples collected in this type of stations were analyzed geologically (grain size, mineralogy and geochemistry analyses) [18] and chemically (on sediments).The sediment samples deposited on the plastic tray were then stored in plastic containers for geological analysis, and glass bottles for chemical analysis.A bigger container was used for storing samples for mineralogical and grain size analysis [18] while for geochemical and chemical analysis smaller bags and jars were used.
Different types of sediments were found on the bay sampling points, like for example: sand, mud, and mixed mud + sand (Figure 4 left and right).In more detail, in Figure 4 (right) the sediments looks heterogeneous because it is possible to see the black mud (bottom) and the more clear sand (top).Vice versa, on the left of Figure 4 the sediment appears homogeneously black.Different sediments could be an indication either of the different activities taking place on the surroundings (like urban vs. industrial discharges) or of the different hydrodynamic situations typical of that specific site influenced by marine currents or bottom morphology.As it can be seen in Figure 5, two different areas can be clearly indentified: RING A made of 12 stations (i.e., stations 1a, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, and 16) close to the coast and therefore rich of organic matter and RING B made of 8 stations (i.e., stations 1b, 13, 3b, 11, 12, 17, 4b, and 14) where the sandy component prevails.
The ERL and ERM are measures of toxicity in marine sediment.The ERL indicates the concentration below which toxic effects are scarcely observed or predicted, while the ERM indicates the concentration above which effects are generally or always observed.AET is defined as the sediment concentration of a given chemical above which statistically significant effects (e.g., sediment toxicity) are always observed.AET may be more sensitive than PELs or ERM since these last ones incorporate several endpoints in their determination.Despite we reported five benchmarks, special emphasis has been given to TEL and AET, being the first one the most protective and the second one the more statistically significant value.Unfortunately, for some elements TEL, ERL, PEL, and ERM are not defined.Particularly, they are not defined for aluminum, barium, iron, manganese, nickel, antimony, and vanadium.
Geochemical analyses. Instrumentation
For geochemical analysis, Inductively Coupled Plasma Emission Spectroscopy (ICP-ES) methodology was used.Nineteen elements were analyzed: Mo, Cu, Pb, Zn, Ag, Ni, Mn, Fe, As, U, Th, Sr, Cd, Sb, Bi, V, Cr, Ba, and Al.For all of them, concentrations are here expressed in μg g -1 (micrograms per gram of dry sediment), except for Fe, and Al, which are reported in the % (by dry weight) form.Twelve elements were selected as the most important and more appropriate to be either representative or trace indicators of the level of chemical pollution and therefore deeply analyzed and discussed in the 2D diagrams which here follow.These elements are: Ag, Al, As, Ba, Cr, Fe, Mn, Ni, Pb, Sb, V, and Zn.This selection was made on the basis of the results obtained from the geochemical analysis.In addition, 17 elements (Ag, Al, Bi, As, Cd, Cu, Fe, Pb, Mn, Mo, Ni, Sb, Sr, Th, U, V, and Zn) were selected to be represented by 3D diagrams in order to detect the possible pollution sources.
Results and discussion
The following two figures illustrate the metal distributions by two different set of diagrams: 2D and 3D diagrams.The first group of diagrams (Figure 6) shows in two dimensions the different metal concentrations (μg g -1 or %) present in the sediment of each station (stations are named "VIT-", after "Vitoria").Each of these diagrams is subdivided in two parts, left and right, respectively.The concentrations measured in each station (on the right) are compared with the five quality aforementioned benchmarks (on the left).Also, sampling stations are grouped according to their geographical distribution as it is depicted in Figure 5, i.e., those belonging to RING A (stations VIT-1a, VIT-2, VIT-3, VIT-4, VIT-5, VIT-6, VIT-7, VIT-8, VIT-9, VIT-10, VIT-15, and VIT-16) are close to the coast and are rich of organic matter while for those inside RING B (stations VIT-1b, VIT-13, VIT-3b, VIT-11, VIT-12, VIT-17, VIT-4b, and VIT-14) the sandy component prevails.
The second group of diagrams (Figure 7) reports in three dimensions on the z-axis the concentration (μg g -1 or %) of the 17 considered elements (Ag, Al, Bi, As, Cd, Cu, Fe, Pb, Mn, Mo, Ni, Sb, Sr, Th, U, V, and Zn) against the geographical coordinates (longitude on the left and latitude on the right), so that the prevailing contaminated spots become evident.From the two dimensional diagrams presented in Figure 6 it is possible to see that only 6 out of the total 12 reported elements show a regular behavior that would allow a rational interpretation.In more detail, the following 6 elements: arsenic, barium, manganese, copper, antimony, and vanadium suggest that the potential pollution sources could be located between station 6 and station 8, being probably station 7 the closest to the actual contamination source.As far as manganese and iron, it seems that there could be a substantial contribution from sources located very close to the industrial area, where specific metal industrial processing is normally carried out.
On the other hand, metal industrial processing does not seem to contribute significantly to the arsenic concentration which is probably more dependent from the Canal de Passagem and the Canal de Camburì inputs.However, this looks bizarre because it requires that such element gets to the Baia de Espirito Santo through the Canal de Passagem, i.e., an area densely populated by mangroves which normally should be a priori chemically uncontaminated [9].Therefore, such results should be more deeply analyzed in the future taking into account also the hydrodynamics of the involved ecosystem.
A deeper analysis of the bidimensional diagrams reported in Figure 6 could suggest which might be the origin of the inputs of the polluting metals discharged in the bay.Particularly, it is possible to identify at least four main potential sources of pollutants, which, briefly will be here named α, β, γ, and δ, corresponding to the Espirito Santo Bay regions of South-East (see for example station VIT-4b, but also , East/North-East (see for example stations VIT-8, VIT-9, VIT-10, VIT-11, and VIT-12, but also VIT-15 close to Ilha do Frade bridge), North (see for example stations VIT-5, VIT-6, and VIT-7), and West/South-West (see for example stations VIT-1a, VIT-1b, and VIT-2), respectively.The source α may represent, on one hand, the pollution coming out from (and collected by) the Vitoria Bay and, on the other hand, that coming from the urban discharges of the Vitoria City suburbs like Bento Ferreira, Jesus de Nazareth, Praia do Suà, Santa Helena, and Santa Lucia but also from Centro de Vila Velha.As far as β, this source should describe the Canal de Passagem outputs but also those coming from Vitoria City quarters like, again, Santa Lucia and also Praia do Canto, Jardim de Penha as well as Ilha do Frade.The pollution originated by the activities located along the Camburì beach and the Jarim de Camburì urban site, is monitored by γ stations.Last, the Tubarão harbor area pollution situation is illustrated by δ.In such contest, Ba shows a large and high peak along β (stations VIT-8, 265 μg g -1 ; VIT-9, 311 μg g -1 ; VIT-10, 273 μg g -1 and VIT-15, 159 μg g -1 ), a secondary peak along γ (stations VIT-5, 276 μg g -1 ; VIT-6, 252 μg g -1 and VIT-7, 239 μg g -1 ) and sharper peaks along δ (station VIT-3, 253 μg g -1 ) and α (station VIT-16, 227 μg g -1 ).On the other hand, Cr displays practically only one interesting peak, namely that corresponding to station VIT-10 (β direction, 84 μg g -1 ).The picture is completed by two peaks corresponding to stations 2 (73 μg g -1 ) and 4 (73 μg g -1 ).As far as the other elements, they will be discussed later, when 3D diagrams will be addressed.
In terms of environmental impact, specifically in terms of AET and TEL benchmarks, experimental data put in evidence that the safety value AET, with the exception of very few stations (like in the case of As and Mn) constantly surpasses the measured concentrations for all stations located within RING A for aluminum, arsenic, barium, manganese, and vanadium, while inside RING B only in very few cases this happens for barium, arsenic, manganese, and vanadium.However, it is interesting to note that TEL (for those elements where TEL is defined) is surpassed within RING A by elements like silver, arsenic, and chromium, while it is only sporadically reached by lead and zinc.On the contrary, in RING B only arsenic presents values that sometime exceed that threshold.Tridimensional diagrams (Figure 7) show that the most relevant immissions concern 10 out of 17 elements: silver, aluminum, bismuth, copper, lead, molybdenum, nickel, vanadium, uranium, and zinc.They seem to come from South-West, i.e., from the final portion of the Vitoria Bay and from the Canal de Passagem.
These are followed by the Camburì beach (North) immissions which concern arsenic, cadmium, manganese, antimony, strontium, and vanadium.Finally, in the Tubarão harbor area (North-East), where several industries are located, it is possible to detect clear indications concerning the immission of cadmium, iron, nickel, antimony, vanadium, and zinc.Apparently, in several cases there are multiple streams with no clear predominance of one of them over the others.This can be observed in the cases of silver, aluminum, bismuth, lead, nickel, antimony, and zinc.In some cases it seem possible to spot a single immission point, like for example for cadmium and thorium (N-W) or for molybdenum (S-W).
Obviously, the aforementioned discussion is necessarily limited by the approximation of the graphic elaboration and by the relatively limited number of sampling stations chosen to adequately depict the different typology of sediments.However, an analysis of the sediment typology would nicely explain why the center of each 3D diagram looks almost always (with the exception of Bi an U) flat and homogeneous (i.e., RING B plateau).Most probably this could be due to the prevailing sandy component of the sediment: sand would bind metal ions much less efficiently than the organic component of the sediment [23], which characterizes RING A.
Conclusions
On the basis of the aforementioned data reported in the diagrams, it seem possible to conclude that the bay of Espirito Santo presents only a modest contamination level due to heavy metals with some critical values for arsenic, manganese, and vanadium.Particularly, such criticity can be detected in those areas close to the coast.This may be due to the fact that sediment composition near the coast is such that it allows the pollutants to be bound more tightly to the sediment because the presence of organic substances like humic acids or because the presence of clays that fix the ions to the sediment [23].On the other hand, some elements like arsenic (see Figures 6 and 7) and barium (reported in Figure 6) show surprising high concentrations which are difficult to be explained, especially when taking into account their possible and probable origin, as it was here discussed.
According to our understanding, a correct evalutation of the presence of such elements (with the purpose of indentify and then eliminate the possible pollution sources) appears necessary to complete the research.This would include an appropiate analysis of the ecosystem dinamics, particularly with respect to the bay marine currents which could transport the pollutants in a different way with respect to those evaluated by the simple model of linear diffusion from the potential sources.
Figure 2 .
Figure 2. Detailed maps of the study area: on the left, the physical map shows urban and industrial areas while details of the different water bodies are reported on the right.
Figure 3 .
Figure 3. Storage of sediment samples depends on type of analysis to be performed later on.
Figure 4 .
Figure 4. Sediments sampled from different stations: mud, which constitutes the bottom part of the sample, looks black while the more clear sand stays the top (right).
Figure 5 .
Figure 5. Location of the sediment sampling stations in the Baia of Espirito Santo (Municipio de Vitoria -ES).Arrows are oriented in the North direction.Sampling stations are located at the top of each arrow.
Figure 7 .
Figure 7. 3D diagrams of Ag, Al, Bi, As, Cd, Cu, Fe, Pb, Mn, Mo, Ni, Sb, Sr, Th, U, V, and Zn distribution in Espirito Santo Bay diagrams oriented after the bay map (see upper left).
|
2018-10-27T09:45:24.565Z
|
2018-10-24T00:00:00.000
|
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53842468
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pes2o/s2orc
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v3-fos-license
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318. Treatment Outcomes of Prosthetic Joint Infections: An Internal Assessment of Adherence to Best Practice Guidelines
Abstract Background The impact of prosthetic joint infections (PJI) on patient outcomes and health systems is extensive. Patients with PJI may receive nonpreferred antibiotic therapy due to ease of administration, cost, and drug interaction profile. Our objective was to compare treatment of PJI to internal guideline-recommended therapy and assess treatment outcomes. Methods To reduce heterogeneity of PJI treatment within a large, integrated health system, our antimicrobial stewardship program and orthopedic surgeons created an internal best-practice guideline for treatment of PJI based on published literature. The guideline is organism and surgery specific (Figure 1). Patients who had total knee arthroplasty (TKA) or total hip arthroplasty (THA) and subsequently developed PJI from July 2016 to June 2017 were identified retrospectively. Recurrent infections were defined as recurrence of primary infections or new infections with other organisms. Rates between patients treated with guideline-concordant and guideline-discordant regimens were compared. Results Among 36 TKAs complicated by PJI, fewer patients who received guideline-concordant therapy experienced recurrent infection than patients who received guideline-discordant therapy (1 of 16 patients [6.25%] vs. nine of 20 patients [45%], P = 0.0219). Among 25 THAs complicated by PJI, there was a trend toward fewer recurrent infections when patients received guideline-concordant therapy (2 of 12 patients [16.7%] vs. 5 of 11 patients [45.5%], P = 0.1775). Common deviations from the guidelines included daptomycin use for methicillin-susceptible Staphylococcus spp. with implant retention due to ease of administration in outpatient settings and avoidance of rifampin due to tolerability or drug interactions. Conclusion Deviation from treatment guidelines for PJI following TKA and THA may increase the risk of recurrent infection. Barriers to utilizing guideline-recommended antibiotics in the outpatient setting should be addressed. Institutions should develop internal consensus on PJI treatment with prospective surveillance.Figure 1. Treatment recommendations for Staphylococcus spp. After Debridement and Implant Retention (DAIR)—one element of the comprehensive internal guideline Disclosures All authors: No reported disclosures.
Background. Fungal PJIs are rare and often associated with poor outcome. Risk factors are not well described and thus, we sought to determine such risks among patients cared for at two large academic hospitals.
Methods. This was a retrospective case-control study among patients with PJI from 2006 to 2016. Each fungal PJI case was matched 1:1 with a bacterial PJI control for joint location (hip, knee, and shoulder) and year of diagnosis. We compared demographics (age, sex, and race), co-morbid conditions (BMI, diabetes, immunosuppression, renal disease, and antibiotic use), and clinical characteristics (joint age, wound factors, laboratory data, previous joint surgeries, and previous PJI) between fungal and bacterial PJI groups using chi square/Fisher's exact or Wilcoxon rank-sum test. Risk factors statistically (P < 0.05) or clinically significant were included in a multivariable logistic regression (MVR) model in stepwise fashion (SAS 9.4,Cary,North Carolina).
Results. Forty-one fungal PJI occurred over the study period and 61% were due to Candida albicans. Median age was 64.7 years, 51% were females, and 87% were White.
Conclusion. In our study, Candida albicans was the most common species in fungal PJIs. The presence of wound drainage for more than 5 days and receipt of antibiotics within the past 3 months were independent risk factors for fungal PJI among a cohort of PJI patients.
Disclosures. All authors: No reported disclosures. Background. The impact of prosthetic joint infections (PJI) on patient outcomes and health systems is extensive. Patients with PJI may receive nonpreferred antibiotic therapy due to ease of administration, cost, and drug interaction profile. Our objective was to compare treatment of PJI to internal guideline-recommended therapy and assess treatment outcomes.
Treatment Outcomes of Prosthetic
Methods. To reduce heterogeneity of PJI treatment within a large, integrated health system, our antimicrobial stewardship program and orthopedic surgeons created an internal best-practice guideline for treatment of PJI based on published literature. The guideline is organism and surgery specific (Figure 1). Patients who had total knee arthroplasty (TKA) or total hip arthroplasty (THA) and subsequently developed PJI from July 2016 to June 2017 were identified retrospectively. Recurrent infections were defined as recurrence of primary infections or new infections with other organisms. Rates between patients treated with guideline-concordant and guideline-discordant regimens were compared.
Results. Among 36 TKAs complicated by PJI, fewer patients who received guideline-concordant therapy experienced recurrent infection than patients who received guideline-discordant therapy (1 of 16 patients [6.25%] vs. nine of 20 patients [45%], P = 0.0219). Among 25 THAs complicated by PJI, there was a trend toward fewer recurrent infections when patients received guideline-concordant therapy (2 of 12 patients [16.7%] vs. 5 of 11 patients [45.5%], P = 0.1775). Common deviations from the guidelines included daptomycin use for methicillin-susceptible Staphylococcus spp. with implant retention due to ease of administration in outpatient settings and avoidance of rifampin due to tolerability or drug interactions.
Conclusion. Deviation from treatment guidelines for PJI following TKA and THA may increase the risk of recurrent infection. Barriers to utilizing guideline-recommended antibiotics in the outpatient setting should be addressed. Institutions should develop internal consensus on PJI treatment with prospective surveillance. Background. In 1998 Modic described changes in vertebral body marrow with magnetic resonance imaging, and related those changes to pathological findings in the
|
2018-12-12T08:49:50.389Z
|
2018-11-01T00:00:00.000
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240534993
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pes2o/s2orc
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v3-fos-license
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PIECC: Point Inversion algorithm for Elliptic Curve Cryptology to Secure IoT Data Communication
In the Internet of Things (IoT), the internet-connected objects send the Collected data and act on the received data. Encryption controls a large number of structured and unstructured data protection during transmission. Inadequate memory and processing capacity of IoT devices demand Elliptic Curve Cryptography (ECC) for simple, secure functionalities. Scalar Multiplication frequently uses Modular Inversions that impact significantly on ECC-based applications with low resource usage with the enhancement of reliable IoT System availability. The Point Inversion algorithm for Elliptic Curve Cryptology (PIECC) enhances security and reduces the Computation time of Modular Point Inversion of Elliptic Curve using High-Speed Split Multiplication and Squaring. The use of limited intermediate registers for Cryptographic functions optimizes the Storage. The proposed algorithm reduces the Computation Time of the Cryptographic operations in terms of Clock cycles using chain Fermat-based Inversion compared with High-Speed multiplication and Product Scanning algorithms with lower Space Complexity.
INTRODUCTION
The varying worldwide development in Modern Technology shows the converging of computation and communication. The distributed Smart devices with a remote connection substitute the Personal Computers with a wired network in almost all the sectors. It necessitates information protection and security measures. Data security and confidentiality are currently the requisite for Banking applications (mobile, SMS, UPI, etc.,) on Phones, wearable Healthcare devices, work-from-home, etc. Internet of Things (IoT) drives the concept of bringing the world together via universal connectivity. The fundamental requirement of IoT is to secure information and data communication providing robust availability with optimized resource usage. The embedded systems are highly domain-specific; The domains expand for such systems. The purpose of the washing machine is to inlet and outlet water at a controlled time using a programmed microcontroller. The fully automatic washing machines provide the option of fuzzy, pre-set hot water washing and other fabric-dependent programming features. The cell phone acts as a router and the Television screen as a Smart screen using Hotspot and WifiDirect facility. Every device viz., laptop, printer, refrigerator provides functionality and maintenance status. The internet of Things provides connectivity of the underlying devices with least or without human intervention. The connected embedded devices transfer information and need network security in a resource-constrained environment. The small key size with a high level of protection causes Elliptic Curve Cryptography [?] effective in an IoT scenario with reduced Storage, and Time overhead minimizes the power consumption. It increases the life span of IoT devices. The primary component of IoT is Device-to-Device communication. Wireless sensor networks are application-specific. The sensor nodes or motes design in compliance with the application. IoT and Device-to-Device communication use existing motes. The functionality of sensors integrates into the chipset of the specific device leads to a device-specific implementation. The Sensor nodes of the IoT and WSN nodes vary in IP connectivity. Cooja simulator implements an IP stack into sensor nodes e.g., TELOSB, Tmote, Micaz, etc., Sensor nodes in IoT are IP-enabled with a 6lowPAN IP address allowing them to interact with remote Edge-to-Edge motes. The characteristics of the sensor node or mote are non-replaced battery, low data transfer capacity, restricted computational and operational efficiency. IoT environment requires the highest performance of the nodes consuming the least energy. Motivation: Finite Field Arithmetic is extensively used in numerous fields viz., Combinatorics, Coding, String Theory, and Cryptology, Logic Gate Theory, etc., A wide range of Public-Key Crypto Applications implemented are over a high-order Finite Fields [?]; accordingly, multiplying and dividing operations influence the processing time of encrypting and decrypting functionality. Thus, designing and developing a faster approach to carry out such operations is essential.
BACKGROUND WORK
Researchers have designed Scalar Multiplication with Inversion algorithms for specific Elliptic Curves providing mathematical models and validations for various motes in an IoT scenario. Liu et al., [?] implemented MoTE Curves to secure IoT device communication. The Curve models resist the Power Analysis attack. Table 1 gives the comparison of related background works on the Elliptic Curve Cryptographic operations.
Elliptic Curve Cryptography
The Elliptic Curve Point representation of the data provides tamper-resistance data transfer. (1).
where A, B curve coefficients belong to a Finite Field Prime F p with (A 2 − 4).B resulting in a non-zero number. Twisted Edwards Curve E t /F p is given in equation (2).
where A, D ∈ F p and A.D.(A − D) a non-zero integer. The Additive Policy of Elliptic Curve [?] states that sum of any two Points P i , P j on the E t curve results in a Curve Point subject to √ A is a positive Integer with A ∈ F p and √ D is a real number i.e.,
Fermat-Euler theorem
The Fermat-Euler theorem is given in the equation (3), (4) is used to Inverse the Prime number exponentiation representation of an Elliptic Curve.
where ρ is Prime, N + is the set of Positive Integer Numbers and a, b, ρ ∈ N + ; i.e.,
Problem Statement
The Encryption/Decryption technique provides secure information exchange in a stable framework.
Assumptions
The heterogeneous IoT devices or the Sensor nodes: i) Handle unexpected failure.
ii) Report on session failure.
iii) Portable within a network range of varied topologies.
PROPOSED POINT INVERSION ALGORITHM FOR ELLIPTIC CURVE CRYPTOLOGY (PIECC) FOR SECURE DATA COMMUNICATION IN IOT
The Cryptographic Elliptic Curve Point Encrypt and Decrypt functions perform Point Addition, Doubling, Inversion, etc.,. The Prime numbers chosen depend on the Curve properties to satisfy. There is no division arithmetic function for the prime group, a division is performed by finding Inversion of the denominator and then multiplying with the numerator. Table 2 provides the definition of the notations used in the Section.
and Twisted Edwards curve given in the equation (6).
Modular Squaring
The Modular Squaring function performs multiplication of two same operands based on HSSM.
end for 6.
end for 12.
The Function 1 plays a vital role in the Modular Inversion algorithm as the frequency of multiplying in others is more. So the efficiency of this Function increases the efficiency of incorporated algorithms in many folds. The symmetrical factor of the large Integer Multiplicand and Multiplier in the Square Function, P 1 provides better performs compared to asymmetric Integer Multiplication function. In a regular Multiplication based Squaring, all interjacent outcomes of the form P 1κ × P 1ν with ν! = κ computed twice. The Function 1 computes these interjacent outcomes merely one time and later shifts words to the right to obtain the Doubled result by shrinking excess processing cost. The two nested loops in the Function compute the Double of interjacent outcomes P 1κ × P 1ν equivalent loops in multiplication algFuncorithm used in this HSSM. The initial and the final interjacent outcomes are attached to the interleaved blocks, and reduction in the frequency of inner blocks repetition differs the Squaring Function from the regular Doubling. The modified exit-control statements reduce the overall Multiplications carried out by the dual repetitive blocks from η 2 − 2 to (η 2 − η)/2.
Modular Reduction
The Multiplication and Squaring of two numbers of bit length η results in 2η length of the result. Since the value of η is large, subtracting the prime number from the result consumes much time. The Reduction is a process of minimizing the result of 2η bit length to η bit length less than the prime number chosen, which is the same as reducing the result belong to the selected Prime Field.
The Modular Reduction Function can be implemented in two methods efficiently. In Function 2, the first method, the value of Z H is left-shifted n times, where n is the bit length of the integer d. The intermediate results and the value Z L are added and reduced further using subtraction. In Function 3, the second method, the usual multiplication algorithm is used instead of the left shift, and further reduction is obtained by subtraction. 1: a 2 ← a 2 2: a 9 ← (a 2 ) 4 .a 3: x 1 ← 2; t ← (a 2 .a 2 ) x 1 .a 9 4: x 2 ← x 3 ← 2 5 ; t ← (t) x 2 .t 5: for i from 1 to 4 do 6: The total number of squaring and multiplications required to calculate Inversion for the prime number p = 2 223 − 235 is 173 squaring and 12 multiplications. As the squaring of a number is considerably faster than multiplying the number twice, Squaring Function is called wherever feasible to make it more efficient. The algorithm 1 described, calculates the Inversion of any number belonging to the Finite Field chosen significantly faster than most previously implemented versions. The inversion algorithms for the prime numbers p = 2 191 − 19, p = 2 159 − 91 and p = 2 255 − 19 follows the same methodology with few required changes required accordingly.
CONCLUSIONS
In an IoT environment, devices store and transmit data over communication media must be protected from illegitimate usage. Encryption controls for Data security at rest and in transit are essential. Elliptic Curve Cryptography enhances IoT security providing secure data communication in the Internet of Things Application. The proposed Point Inversion algorithm for Elliptic Curve Cryptology uses High-Speed Split Multiplier and HSSM based Squaring in Scalar Multiplication to secure IoT Data Communication. The PIECC optimizes the data and code Storage, improving the performance of IoT devices using limited intermediate registers to carry out Cryptographic functions. The proposed algorithm is almost two times faster than LHS and three times than RPSM. The PIECC uses 6% less memory than the LHS and 41% than RPSM by the reduction in intermediate memory
|
2021-10-20T15:17:23.436Z
|
2021-09-18T00:00:00.000
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237935035
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pes2o/s2orc
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v3-fos-license
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Medical Genetics, Genomics and Bioinformatics Aid in Understanding Molecular Mechanisms of Human Diseases
Molecular mechanisms of human disease progression often have complex genetic underpinnings, and sophisticated sequencing approaches coupled with advanced analytics [...].
Molecular mechanisms of human disease progression often have complex genetic underpinnings, and sophisticated sequencing approaches coupled with advanced analytics. Modern computational approaches for the search and analysis of potential drug targets critically depend on the ability to reconstruct gene networks and to model the protein structure. New biomarkers derived from transcriptomics analysis or the standalone mining of associative networks aim to aid physicians to consider individual cases. This Special Issue continues the collection of papers on "Medical Genetics, Genomics and Bioinformatics" published in this journal in the wake of a series of medical research conferences held in Russia in 2020. The presented analytic techniques were discussed at the medical forum coordinated by I.M. Sechenov First Moscow State Medical University, and at the "Systems biology, bioinformatics and biomedicine" symposia held during BGRS-2020 biannual computational biology meeting in Novosibirsk, highlighting recent advances at the biomedical frontier. This collection of papers showcases insights in the field of human genomics, transcriptomics and proteomics, as well as some work conducted in model organisms. The current Special Issue contains eight research manuscripts and two reviews, each concerning some model or a pipeline applied to extract information useful for understanding molecular underpinning for a human disease and suggestive of a mechanism-specific treatment. We continue the previously published set of paper collections with an overarching theme of bioinformatics and medical genetics, which began in 2019 (https://www.mdpi.com/ journal/ijms/special_issues/Medical_Genetics_Bioinformatics, accessed on 25 August 2021) [1]. This Special Issue is focused on the deciphering of molecular mechanisms underpinning common chronic diseases, including mental disorders, and emphasizing searches for drug targets.
Computational models for systemic human disorders are now in high demand. Regulation of the so-called "normal" state of the cell is extremely complex. Gene expression may be controlled at transcriptional, post-transcriptional, and translational levels, and in the gene networks and pathway levels as well. This list is as endless as the research on this field will be. The disease models derived from this research are, however, very practical. New biomarkers derived from the transcriptomics analysis or standalone mining of associative networks aim to aid physicians to consider individual cases. The current series of post-conference journal Special Issues started with a coverage of Bioinformatics of Genome Regulation and Structure (BGRS) conferences and related Schools on Systems Biology and Bioinformatics (SBB) held in Novosibirsk, Russia [2][3][4][5][6]. This particular Special Issue contains the manuscripts that have followed on from oral and poster presentations discussed at the "Systems biology, bioinformatics and biomedicine" (SbioMed-2020) symposium in Novosibirsk, as well as the conferences at I.M. Sechenov First Moscow State Medical University in Moscow [6]. The majority of these papers present some insights into the molecular mechanisms of various human diseases, and their progression.
We open this collection of papers with gene network modeling studies. The paper by Olga Saik and Vadim Klimontov describes a gene network related to glucose level variability in diabetes [7]. A growing body of evidence indicates that excessive glucose fluctuations serve as a risk factor for microvascular and macrovascular diabetic complications [8]. Accordingly, glucose variability is increasingly recognized as a therapeutic target [9]. Recent data indicate that deteriorative effects of glucose variability in the target organs are realized through the up-and down-regulation of a large set of genes [10]. In such cases, the analysis of gene networks can provide valuable information for a comprehensive understanding of disease pathogenesis. To reconstruct relevant networks in the automatic mode, authors employed the ANDSystem (Associative Network Discovery System), which is based on text mining, an automatic knowledge extraction from the texts of scientific publications [11][12][13]. The reconstructed gene network of glucose variability consists of 37 genes associated with both hyperglycemia and hypoglycemia. The identified genes are involved in insulin secretion, glucose homeostasis, as well as some signaling pathways which regulate cellular metabolism, cell cycle, and cell-cell interactions. Interestingly, the genes associated with glucose variability turned out to be hubs in gene networks describing diabetic vascular complications. A number of new candidate genes, promising for experimental verification of their role in glucose variability, have been identified as well.
Anna V. Glyakina and colleagues presented a spatial model of filamentous actin (F-actin) organization in eukaryotic cells [14]. First, they employed electron microscopy, limited proteolysis, mass spectrometry, X-ray diffraction, and structural modeling to show that the double helical molecules of filamentous actin are inconsistent with the observed ladder-like stacking of G-actin into F-actin, which is evident from the EM images. Therefore, a novel model of stacking actin monomers in filamentous actin is proposed, where actin monomers form one filament to make the F-actin core as inaccessible to the solvent as possible.
The topic of protein structure modeling is continued by Dmitry Karasev and coauthors [15]. They developed an approach for the fuzzy classification of protein sequences based on the ligand structural features to analyze ligand-protein interactions for new therapies. The current study extended the topic of the protein-ligand models published recently in the Special Issue "Medical Genetics, Genomics and Bioinformatics" [16]. The protein kinase family case demonstrated the effectiveness of the proposed technique.
Larisa Litvinova et al. [17] studied the secretory activity of mesenchymal stem cells upon an in vitro contact with calcium phosphate coatings, with an important biotechnological consequence. The manufacturing of specific biomaterial surfaces may directly induce osteogenic differentiation in cells. Cellular and molecular reactions of mesenchymal stem cells with the plastic with a double-sided calcium phosphate coating show that there are correlations between the mRNA expression levels for the selected genes and the secretion of cytokines and chemokines that may potentiate the differentiation of these cells into osteoblasts [18].
Evgeny A. Ermakov and co-authors [15] studied the biochemical underpinnings of schizophrenia and have linked this condition to immunity and inflammation via immunoglobulins hydrolyzing histones. Schizophrenia is known for its association with chronic low-grade inflammation. Extracellular histones and nucleosomes may trigger systemic inflammatory and toxic reactions. The authors presented the first evidence that polyclonal IgGs of patients with schizophrenia effectively hydrolyze five common types of histones.
Immunohistochemistry research was continued by the work of Anastasiya V. Snezhkina and colleagues [19]. The authors studied succinate dehydrogenase (SDHx) genes in carotid paragangliomas, a type of rare neuroendocrine tumor. SDHB gene immunohisto-chemistry could be useful for the initial identification of patients potentially carrying SDHx mutations necessitating genetic testing. This work extends the previous study of paraganglioma performed with bioinformatics methods [20] and presented at the post-conference Special Issue on bioinformatics [21].
Olga Redina et al. [22] dissected the molecular mechanisms of behavior in laboratory animal models. The authors used RNA sequencing to identify gene expression changes in the ventral tegmental area of mouse brains in animals undergoing agonistic interactions (fighting), a known model for the study of excitation of brain neurons and the formation of social behavior patterns. The author revealed a set of differentially expressed genes playing a role in the maturation of dopaminergic neurons under the influence of social stress.
Molecular mechanisms of behavior disorders were analyzed in the study by Marco Ragusa and colleagues [23]. This group analyzed associations among the alteration of salivary miRNAs, saliva microbiome structure, and autistic spectrum disorder (ASD). When the relationship between brain functionality and saliva bacterial populations is perturbed by pathological conditions, ASD may result. The authors present a statistical association of both miRNAs and microbes with neuropsychological scores related to social interaction anomalies. This work continues the studies of autism predisposition genes previously described in an IJMS Special Issue [24].
The topic of possible mechanisms of childhood-onset neurodegenerative disorders was continued by Elena Shematorova and George Shpakovski [25], who reviewed molecular mechanisms promoting the juvenile form of neuronal ceroid lipofuscinoses (Batten disease). This malady is caused by mutations in the CLN3 gene, which is highly conserved in the evolution of all mammalian species [26]. Detailed analysis of recent genomic and transcriptomic data indicated the presence of human-specific features of its expression.
The review by Simone Donati et al. concludes the Special Issue [27]. The authors discuss the potential role of microRNAs in multiple endocrine neoplasia type 1 syndrome, a rare inherited tumor disease, characterized by the development of multiple neuroendocrine tumors. Deregulation of certain miRNAs species has been associated with this syndrome. The potential roles of miRNAs as future non-invasive diagnostic and prognostic biomarkers is discussed.
Thus, the current Special Issue on medical genomics shows that bioinformatics tools for the systems analysis of human diseases are in high demand, as could be seen from recently published post-conference papers [28,29]. The outputs of disease model analysis come in the form of sets of genes and protein markers which represent a particular interest to medical practitioners [30]. The guest editors are happy to announce that the next Special Issue topic at MDPI IJMS will be on medical genomics (https://www.mdpi.com/journal/ ijms/special_issues/Medical_Genetics_2021, accessed on 25 August 2021).
Based on the readers' interest in the topic, we are continuing to focus on the materials in this scientific field based on novel computational approaches, digital medicine technologies, networks and metabolic pathways analysis.
Funding: The publication has been prepared with the support of the RUDN University Strategic Academic Leadership Program (recipient Y.O.). The article was prepared within the framework of the state assignment of the RICEL-branch of IC&G SB RAS.
Acknowledgments:
The authors are grateful to all the reviewers who helped review and validate this thematic Special Issue. The authors thank the Sechenov University in Moscow, the BGRS\SB-2020 Organizing Committee, Novosibirsk State University, and the Institute of Cytology and Genetics SB RAS in Novosibirsk for providing platforms for these conferences.
Conflicts of Interest:
The authors declare no conflict of interest.
|
2021-09-28T05:18:56.692Z
|
2021-09-01T00:00:00.000
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236814930
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pes2o/s2orc
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v3-fos-license
|
A framework for decision making of migraine treatment with anti- CGRP monoclonal antibodies: a guide for real-world practice and public policies
Anti-CGRP monoclonal antibodies have been developed for migraine preventive treatment. There is evidence of good efficacy and safety of these medications; however, cost is a factor that interferes with the choice of treatment. This paper proposes a framework in order to better assist the decision-making processes on the use of these drugs in developing countries without coverage of health care costs for migraine. The framework was built after reviewing phase II and III studies on episodic and chronic migraine treatment with erenumab, galcanezumab and fremanezumab. Mario Fernando Prieto Peres mariop3r3s@gmail.com Edited by Marcelo Moraes Valença Received: August19, 2020 Accepted: September 14, 2020
Introduction
M igraine is a common and debilitating disorder, which affects a significant proportion of the population worldwide. 1,2 Abortive and preventive treatment strategies are often combined using pharmacological and nonpharmacological methods. The most commonly used medication categories are: antidepressants, beta blockers and anticonvulsants. 3,4 The poor quality of life of these patients interferes in their functional status. There are losses in work performance, school, family, relationships, in addition to affecting daily concentration, generating fatigue and mood alterations. These medications lead on average to a 50% frequency reduction in 50% of patients, and their use are limited due to side effects or contraindications. 5 Four monoclonal antibodies (mAbs) have been developed: one targeting the calcitonin gene-related peptide receptor (erenumab) and three targeting the calcitonin gene-related peptide (eptinezumab, fremanezumab, and galcanezumab). 6 Erenumab, galcanezumab, fremanezumab and eptinezumab have yielded positive results concerning episodic and chronic migraine prevention. 7,8 The new drugs have already been approved in the United States and other countries worldwide. Erenumab was approved in May, 2018 in the US, in August, 2018 in Europe, while galcanezumab and fremanezumab were approved in September, 2018 in the US, and in November, 2018 in Europe. Eptinezumab was approved in February, 2020?
Headache care lacks universal coverage even in developed countries. 9 From the estimated 1 billion migraine sufferers across the globe, at least half do not have full coverage even of essential health services.10 About 100 million people worldwide have been pushed into extreme poverty because they have to pay for health care. 11 Migraine treatment and health care policies should be planed according to patients' access.
Although studies on phase II and III clinical trials have revealed a protocol or a way of prescribing migraine by administering subcutaneously injections every month for 3 to 6 months, there is scarce information regarding when to stop medication, dosing strategies, management involving refractory patients or non-responsive patients, and other issues yet to be examined. 12 Administration and use of new monoclonals are expected to vary according to the environment, physicians experience, patient's responses, and financial aspects. Headache related health care policies are not available worldwide, and patient access is still a matter of intense debate. Medical systems across the globe have country specific regulations for patient access, reimbursement and price policies. 13 Therefore, CGRP monoclonal antibodies protocols should consider not only the pre-fixed protocols studied in clinical trials, but should be customized for the real-world practice, individualized according to the medical system.
In order to account for socio-economic factors, a decision tree algorithm for migraine preventive treatment with mAbs should be generated. In this paper we suggest a framework for improving the decision making process in real life and for public policies.
Methods
This is an opinion article in which the authors propose a framework for improving the decision making in choosing steps for the management of preventive treatment of migraine with monoclonal antibodies. This could be a ground for gathering opinions and collecting data towards cost-benefit analysis, aiding decisions in the context of limited financial resources. The framework was built after reviewing phase II and III studies on episodic and chronic migraine treatment with erenumab, galcanezumab and fremanezumab.
Information regarding dosage, timing, half-life and administration protocol were reviewed. Eptinezumab data were excluded due to its approval in limited countries.
The authors created an algorithm that summarizes strategies for the treatment of migraine with anti-CGRP monoclonal antibodies.
Evaluating the response pattern according to the percentage of reduction in pain days, three categories were considered: Group 1: Poor response, less than 25% reduction; Group 2: Partial response, reduction between 25% and 75%; Group 3: Good response, more than 75% reduction.
The framework was set in order to facilitate the opinion on the following questions: 1. When should treatment be offered? 2. How long to maintain treatment in cases of good response? 4. How long to maintain treatment until no response is established? 5. In cases of partial response, what steps can be taken in order to increase efficacy? ASAA Gama RN, Lima TAC, Dangoni Filho I, Peres MFP A framework for decision making of migraine treatment with anti-CGRP monoclonal antibodies: a guide for real-world practice and public policies After analyzing data, we modeled an algorithm so a framework could be the basis for clinical migraine prevention decision-making in different health care scenarios.
Results
We propose an algorithm to clarify the phases in migraine management through anti-CGRP monoclonal antibodies. The decision tree is intended to support the clinical practice and was developed according patient's response. We tried to generate an approach that is based on efficacy data but also could be taken different economic scenarios into account. Erenumab, galcanezumab and fremanezumab half-life is around 25-30 days, regarding subcutaneous injections perfomed in the abdomen, thigh, or upper arm. The recommended dose are: erenumab, 70 mg or 140 mg monthly; fremanezumab 225 mg monthly or 675 mg every three months; and galcanezumab 120 mg monthly. Framanezumab can be also administered quarterly. Galcanezumab can be started with a loading dose of 240 mg (Table 1).
Treatment with monoclonal antibodies should be offered to what kind of patients? Should only to those who have failed two classes of preventive treatment: antidepressant, anticonvulsant or beta-blocker? Once the treatment with monoclonal antibodies is decided, following the protocol studied is the obvious indication, therefore galcanemuzab would be administered with a loading dose (240 mg). But what if the medication cost is dose dependent? Should starting with 120 mg be cost effective?
The next step is to measure the clinical response and classify the patient in the three groups. For group 3, in which the patient presents substantial improvement in the first month, we suggest repeating the dose as soon as reducing the effectiveness for less than 50%.
For group 2, in partial response, should the same dose be repeated within a month? For group 1, with no response, twice the dose of erenumab and the same dose of galcanezumab and fremanezumab should be prescribed? In the following month, patients must be reclassified, according to the response rate. Patients who remain in group 1 would be advised not to continue treatment, within a context in which financial resources are limited? How much more time should we insist in the trial? In this group, patients should be reassessed in an attempt to confirm the diagnosis and identify other factors that contribute to pain refractoriness, such as mood disorders, sleep, postural errors, physical inactivity or exposure to triggering factors.
For groups 2 and 3, the optimal or partial response is an opportunity to optimize non-pharmacological treatment measures and the pharmacological treatment kept for how long? At least six months for chronic migraine? Three to six in episodic migraine (Figure 1).
Discussion
Data from available clinical trials indicate that erenumab, fremanezumab and galcanezumab are safe and effective for the prevention of episodic chronic migraine.
Monoclonal antibodies present a good tolerability profile, low incidence of side effects and easy application, which may lead to patients who prefer such prophylaxis. The high cost, however, does not allow their use as first choice option in most cases. 14 Regarding the question about when should treatment be offered, most studies of prophylactic use of monoclonal antibodies have as exclusion criteria patients that have failed with two or more prophylactic medication. However, an analysis in the subgroup of chronic migraine with erenumab showed effectiveness of the medication, even in patients that failed the previous prophylactic treatments, showing that such medications can be considered in case of refractory migraine. 15 In our opinion, to achieve a cost effective treatment for migraine, we should first try a preventive treatment with low cost medication.
The loading dose is propose to galcanezumab, but in the context of limited resources, after analyzing clinical outcomes, one may have to evaluate the cost benefit of 120 mg and 240 mg doses of galcanezumab. 16 [16][17][18] These patients responded in a modest way in the first dose application and have reached better responses in the following months. 19 In another open clinical trial 20 that verified the satisfaction of participants with the use of galcanezumab, it was shown an enhancement in the positive response in the visits of first, sixth and twelfth month.
Although the studies concluded the main outcome for longer periods, improvement can start to be observed as early as the first week; therefore, we consider that by the end of the first month it is possible to classify patients in group 1, 2 or 3.
Future directions
Ideally, this framework should be field tested, and clinical trials be done. The opinion of general practitioners, family physicians, policy makers, neurologists, headache specialists in several countries should besought and studied.
Divergence or controversy is expected as for what steps of the algorithm should be followed. This is, however, only the first steps in clarifying this issue.
Conclusion
The framework is intended to provide an easier approach for a better decision making in real life and regulatory affairs.
Author's contributions: the authors contributed equally in data collection, writing and editing.
|
2021-08-04T00:04:33.731Z
|
2020-09-30T00:00:00.000
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12589872
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pes2o/s2orc
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v3-fos-license
|
Evidence for dual superconductivity of QCD ground state
A discussion is made of the strategy to check dual superconductivity of the vacuum as a mechanism of colour confinement. Recent evidence from Lattice is reviewed.
Introduction
No reliable analytic approach exists to QCD at large distances. The usual perturbative quantization leads to an S matrix which is not Borel summable. For reasons which are not understood the perturbative expansion works anyhow at small distances, where a few terms correctly describe experiments. It fails at large distances, where the coupling is large, in particular in describing confinement of colour.
Attempts have been made to describe the degrees of freedom relevant to confinement by effective models. Particularly attractive from the theoretical point of view, is the possibility that vacuum behaves as a dual superconductor 1,2 . Dual Meissner effect would accordingly produce confinement by constraining the chromoelectric field into Abrikosov flux tubes, with energy proportional to their length.
The mechanism is appealing because it relies on a symmetry property. Superconductivity is a Higgs mechanism, by which a charged field acquires a non zero v.e.v., the order parameter in the Landau Ginzburg free energy. The ground state has no definite charge, the U (1) related to charge conservation being spontaneously broken.
For QCD magnetic charges should condense in the confined phase, and break some magnetic U (1) symmetry. A dual order parameter, a disorder parameter in the language of statistical mechanics, would then describe this change of symmetry.
Only a non perturbative quantization, like lattice, can help in cheking if the above mechanism is at work. The simplest strategy to do that consists of two steps 1. Identify the relevant magnetic U (1).
Check by a disorder parameter if it breaks spontaneously.
2 Identifying monopoles: the abelian projection.
Monopoles in non abelian gauge theories were first discovered 3,4 as solitons in the Higgs phase in a gauge theory with gauge group SO(3) coupled to a scalar field in the adjoint representation 5 It was shown in ref's 3,4 that monopoles exist as static solutions (solitons) in the Higgs phase of the model, i.e. for µ 2 > 0, Φ 0 = Φ = 0.
In the hedgehog gauge the monopole has the form with f (r), h(r) ∼ 1 as r ≫ 1/µ. A gauge transformation to the unitary gauge, U ( r), is defined up to a residual U (1) gauge group of rotations around the z axis. U ( r) is singular at the zero of Φ( r), r = 0. U ( r) is usually called an abelian projection. For the monopole solution the abelian field of the residual U (1) in the abelian projected gauge is the field of a Dirac monopole. F µν can be written in a gauge invariant form as Calling Eq.(6) identifies an U (1) magnetic symmetry. The corresponding charge Q is a colour singlet and is equal to two magnetic units for the monopole solution. Also F µν and F * µν are colour singlets. More generally, an abelian projection U ( r) can be performed which is defined by eq.(3) on a generic configuration. U ( r) is singular at the zeros of Φ( r). Around these points the field has the topology of an abelian monopole 6 .
We could think of a slightly more general model in which an additional Higgs field Φ ′ is present, e.g. with the same potential as Φ We can define an abelian projection which brings Φ to the unitary gauge, as in the simple model above, and define the gauge invariant field F * µν , and the corresponding magnetic U (1). We can play the same game with Φ ′ , and this will in general bring to a different abelian projection and to a different U (1). Both magnetic charges are gauge invariant. On a given field configuration the zeros of Φ and Φ ′ will not coincide in general so that the two abelian projections define different monopoles. However the theory is totally symmetric under the exchange Φ ↔ Φ ′ and hence the two monopole species defined by the two abelian projections must be physically equivalent.
There is in the literature a misuse of terminology: the abelian projections are named from the abelian projected gauge, so that the two monopole species defined in the above example are called monopole in the gauge Φ and monopoles in the gauge Φ ′ : a possible difference of physics, e.g. if the two fields have a different potential is called gauge dependence. This terminology is misleading: usually, e.g. in QED, as gauge dependent is meant a quantity which does not depend only on the physical fields, F µν , but could depend on the choice of the gauge. Monopole charges defined by any abelian projection are instead physically well defined and gauge invariant quantities.
In QCD there is no Higgs field. However there exist infinitely many fields transforming in the adjoint representation, and each of them can define an abelian projection and with it a monopole species.
On the lattice any parallel transport along an arbitrary path C coming back to the starting point defines an abelian projection and a monopole species. The corresponding monopoles are different in number and located in different sites, configuration by configuration. A possible guess is that they are all physically equivalent 6 , in the same way as the two monopole species of the model eq.(7). For each of them it is anyhow possible to investigate condensation in the vacuum and dual superconductivity. Some results will be presented in the next section.
An alternative attitude is that some abelian projection is better than others. This attitude is popular among the practitioners of the so called maximal abelian gauge. This is an abelian projection for which the operator Φ is im-plicitly defined by maximizing numerically the quantity with respect to the gauge transformation Ω(x). The numerical output is that in the new gauge all the links U µ (x) are practically aligned along σ 3 , within 10−20%. A remarkable observation 7 which is a consequence of this fact is the so called "abelian dominance". Quantities like e.g. the string tension, when computed in the U (1) residual gauge, agree within 10 − 20% with the exact result. In addition the abelian monopole part, corresponding to integer number of 2π in the abelian plaquettes, saturates the abelian approximation to within 90% again.
Dominance is interpreted as special relevance of the specific monopoles in the long range physics.
From theoretical point of view we find more significant and anyhow necessary to investigate the symmetry of the vacuum, i.e. the condensation of different monopole species in connection with confinement.
In the language of statistical mechanics the main issue of the problem is duality: the gauge field of monopoles presents non trivial connection or topology. A creation operator for monopoles has the form of a translation of the field in the Schrödinger representation by a monopole configuration. In U (1) gauge theory 8 µ carries non zero magnetic charge. Eq.(9) is the analog of the elementary translation E is the conjugate momentum to the field. Some technical modifications will be needed to keep the compactness of the theory into account 8 on the lattice, and some extra care to perform the shift in the abelian projected U (1) for non abelian gauge theory 9 .
For different monopole species we have measured µ or better ρ = d dβ ln µ , as a function of temperature, on asymmetric lattices N S ≫ N T . ρ contains the same information as µ and has less numerical problems in its determination.
Since µ β=0 = 1, The typical behaviour of ρ vs β = 2N c /g 2 is shown in fig.1 for SU (2) There is no practical difference between different abelian projections, in agreement with the guess of t'Hooft's 6 .
The strong negative peak occurs at the deconfining transition, and, by eq.(11), indicates a rapid drop to zero of µ .
At large β's ρ is computed by perturbation theory giving at the leading order ρ = −c 1 L S + c 2 . As the spatial size L S → ∞, ρ → −∞ and µ = 0, as expected for any disorder parameter in the thermodynamical limit. For β ∼ β c a finite size scaling analysis with respect to L S can be performed.
Since the transition is second order for SU (2) and weak first order for SU (3), the correlation length ξ goes large at β c , with some effective critical index ν By dimensional arguments This implies by eq.(12) The scaling law is obeyed for the appropriate values of ν and β c . Fig.4 shows how scaling works. The output for SU (2) is ν = .62 ± .02 to be compared with the expectation, the critical index of 3d Ising model ν = .631(1).
For SU (3) we find a similar value, contrary to the expectation which should be 1/3. However our volumes are not sufficiently large, and further investigations are on the way.
Conclusions
A disorder parameter can be defined to investigate condensation of monopoles in the vacuum of QCD. QCD vacuum is a dual superconductor. Different monopole species look equivalent, and condense in connection with confinement, in agreement with the conjecture of t'Hooft's 6 . This is an important information on the symmetry of vacuum, which must be explained by any model of confinement.
|
2014-10-01T00:00:00.000Z
|
1998-09-08T00:00:00.000
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252466137
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pes2o/s2orc
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v3-fos-license
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Using a discrete choice experiment to develop a decision aid tool to inform the management of persistent pain in pharmacy: a protocol for a randomised feasibility study
Introduction In an era of personalised healthcare, it has become increasingly important to elicit individual-level preferences. While discrete choice experiments (DCEs) are widely used to measure patient preferences in the delivery of healthcare, the focus has been sample-level analysis. Using the DCE methodology, this project has designed a digital decision aid tool (DAT) with the potential to estimate individual preferences in real time to inform clinical consultation decisions in persistent pain management. Methods Using a feasibility randomised control trial, this study aims to assess the feasibility of using this Understanding Persistent Pain (UPP) DAT in a pharmacy-based clinical setting and to test processes for a future definite randomised trial. Community and practice-based pharmacists (up to 10) will be recruited in The National Health Service (NHS) Grampian and trained in the use of the digital UPP DAT. Pharmacists will recruit up to 60 patients who are living with persistent pain. Patients will be randomised to one of two groups: using the UPP DAT or usual care. Pharmacists will follow-up patients as needed according to clinical need and following standard practice. DCE response data collected by the UPP DAT will be analysed using the penalised logit model, allowing estimation of individual preferences in real time. We will follow-up pharmacists and patients who use the UPP DAT to gather feedback on their experiences. Ethics and dissemination This study received ethical approval from the North of Scotland Research Ethics Committee (21/NS/0059) and received Research & Development Management Permission to proceed from NHS Grampian (2021UA003E). The study has been registered in the ClinicalTrials.gov database. Findings will be disseminated in peer-reviewed publications, presentations and newsletters and made available in the University of Aberdeen and Pharmacy Research UK websites. Participants gave informed consent to participate in the study before taking part. Trial registration number NCT05102578; clinicaltrials.gov.
INTRODUCTION
Decision aid tools (DAT) can facilitate shared decision-making and help deliver patient-centred care. 1 DATs are resources designed to help people make informed choices about healthcare that consider their personal values and preferences. 2 Studies have found that DATs can improve patients' knowledge and make them feel better informed about their preferences and values. 3 4 Furthermore, DATs can improve health literacy concerning the underlying condition, resulting in more efficient interventions. 5 Many DATs have been developed, varying in format (eg, leaflet, video or online website), type of information presented (eg, clinical problem, outcome probabilities), methods used to clarify patients' values (eg, ranging from simple information to exercises to help them clarify what matters most to them) and degree of participation in decision-making. A fundamental drawback of most existing DATs is that they fail to explicitly ask patients to consider trade-offs between the treatment characteristics, deviating from how patients normally and intuitively make decisions in real life. 6 7
STRENGTHS AND LIMITATIONS OF THIS STUDY
⇒ We use the discrete choice experiment methodology to develop a decision aid tool (DAT) capable of estimating individual preferences in real time. ⇒ Our Understanding Persistent Pain (UPP) DAT generates a personalised report to help inform treatment choices. ⇒ Development of UPP DAT was informed using extensive patient and relevant stakeholders' input. ⇒ It is not feasible to incorporate all features that may affect patient's pain management preferences in the DAT. ⇒ The study will be undertaken in The National Health Service (NHS) Grampian in Scotland and may not be generalisable to other regions or primary care areas.
Open access
Discrete choice experiments (DCEs) are a widely used method to elicit preferences in healthcare delivery. [8][9][10] DCEs are rooted in economic theory, thus providing an analytical framework that can incorporate multiple and competing criteria in a way that mimics real-life decisionmaking processes. 11 DCEs assume that services (or goods) can be described by a set of characteristics or features, which vary systematically to form alternative packages. Individuals are asked to compare and choose between competing alternatives, thus implicitly trading off the features of each, in several choice tasks. Through the individuals' repeated choices, it is possible to estimate the relative importance of each feature and obtain quantifiable measures of preferences. 12 In other words, it is possible to work out what features are liked and disliked, and by how much relative to each other.
While DCEs offer a salient mechanism to intuitively estimate the trade-offs and relative importance of different treatments' features (eg, benefits, risks), to our knowledge, there are only two studies using this approach within a DAT framework. Dowsey et al 13 used a DCE as part of a decision aid for patients undergoing total knee arthroplasty. They conducted a randomised control trial (RCT) to determine whether completing a DCE prior to surgery influenced patient expectations, health outcomes and satisfaction. They focus on the value of the completion process of the DCE to inform the patient. Hazlewood et al 14 evaluated a proof-of-concept DAT for patients with early rheumatoid arthritis, which included a DCE to assist respondents in making a choice of initial treatment. DAT responses were combined with data from a previous DCE study to infer the patient's preferred treatment.
In this study, we use the DCE methodology to develop a DAT that directly estimates preferences at the individual level in real time without relying on a previous data set. Our application is in persistent pain, estimated to affect 28 million adults living in the UK and which has been highlighted as a national priority. 15 The digital Understanding Persistent Pain (UPP) DAT uses the patient DCE responses to create a personalised report that interprets the trade-offs and relative importance of different features of persistent pain management strategies. Pain is a subjective experience and highly preference sensitive. 16 Research has shown that patients with persistent pain value personalisation of care. 17 Furthermore, there is substantial preference heterogeneity in patients' choices for support for persistent pain management . 18 This is unsurprising since individuals will have different experiences, health needs, expectations and treatment preferences. In clinical and research practice, pain intervention approaches (pharmaco-driven and non-pharmaco-driven) tend to focus on average pain intensity. 19 However, this may not be the most important outcome to patients. As such, the management of persistent pain should take a patient or person-centred approach and involve shared decision-making. [20][21][22] At the same time, there is growing evidence that patients would benefit from pain management strategies that actively involve pharmacists. [23][24][25][26] This paper describes a research protocol to investigate the feasibility of using the digital UPP DAT as part of a pharmacy-led pain consultation. There is, however, a lot of variation in existing pharmacist-led pain consultations and scarce guidance on how to ensure that these can lead to shared decisionmaking and patient-centred care. [27][28][29][30] This feasibility RCT will assess the feasibility of using the UPP DAT in a pharmacy-based clinical setting and to test processes for a future definite RCT.
METHODS
The primary aim of the study is to examine the feasibility of using the digital UPP DAT in clinical consultations between pharmacists and adults with persistent pain. We will also inform future parameters for a future RCT and assess the feasibility of the collection of secondary outcomes. The Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines, adapted for feasibility studies, were used to guide the preparation of this protocol. 31 32 Table 1 summarises SPIRIT applied to our protocol; in what follows we provide more detail. The trial methods are also summarised in the WHO Trial Registration Data Set (online supplemental material table A1). The study procedures for prescribing and non-prescribing pharmacists are outlined in online supplemental material figure A1,A2.
Study design
The design is an RCT, unblinded, with two parallel groups and a simple randomisation until target recruitment or the study end date (whichever happens first). A 2:1 (intervention:control) allocation ratio will be applied to test the UPP DAT with more patients.
The intervention group will be asked to take part in a pain consultation using the digital UPP DAT and the control group in a pain consultation following usual care (ie, without a digital DAT.) The study is set in the NHS Grampian region (Aberdeen and Aberdeenshire, Scotland). The consultation can either take place face-to-face or remotely (eg, using the NHS Near Me platform). 33 34 The remote option is included considering the ongoing COVID-19 pandemic and potential restrictions to face-toface interactions and to account for any postpandemic rise in remote consultations in health services. 35 36 For face-toface consultations, the UPP DAT will be completed using any internet-enabled device in the consultation room. For Near Me consultations, the UPP DAT will be completed using an on-site computer, with the pharmacist sharing the screen with the patient.
Pharmacist recruitment
Registered pharmacists, based in a community pharmacy or General Practitioner (GP)-practice, with or without an independent pharmacist prescribing qualification, with an interest and/or experience of managing persistent pain in NHS Grampian, are eligible to take Open access part. Expressions of interest will be sought following an email to all general practices in NHS Grampian, sent by the NHS Research Scotland Primary Care (NRS Primary Care) network, social media alerts from the Royal Pharmaceutical Society in Scotland and the NHS Grampian Pharmaceutical Care Services. All participating pharmacists will receive training, including a Good Clinical/ Research Practice course, enrolment in a Continuing Professional Development eligible course in Musculoskeletal and Chronic Pain, 37 a webinar session with a pain consultant (highlighting biopsychosocial approaches to pain management, covering self-management and pharmacological management of pain in depth) and a session on the study procedures and UPP DAT use provided by the research team.
Patient recruitment and consent
Pharmacist will identify patients to take part in the study from personal knowledge and opportunistically as they present. Inclusion and exclusion criteria are defined below: Patient inclusion criteria ► Above 18 years old. ► Suffering from non-malignant persistent pain (defined as pain lasting more than 3 months). ► Being managed entirely within a primary care setting.
Patient exclusion criteria: ► Not fluent in English. ► Have concomitant severe mental health problems or terminal illness.
► Suffer from pain caused by cancer or other malignancy ► Are not able to give informed consent (eg, because of mental state). ► Taking part in another research study.
Pharmacists will take informed consent from patients. For face-to-face consultations, the patient will complete and sign a consent form (see online supplemental material). For remote consultations, the pharmacists will complete and record a verbal consent form.
Sample size
We will recruit 10 pharmacists and 60 patients. Sample sizes are based on available funding, resources (eg, pharmacist time to deliver the UPP DAT) and study duration and pilot study norms.
Randomisation
Pharmacists will randomise to intervention or control at patient level using a study-specific online randomiser, designed by the research team and hosted by the software company Qualtrics, after informed consent has been given and before the pain consultation.
The intervention-DAT
The intervention is the use of the digital UPP DAT. This tool follows the principles of the National Institute for Health and Care Excellence decision aids process guide. 38 The DAT's wrapper application is coded and hosted by the company Clinvivo. Intervention group: using decision aid tool. Assessments
Open access
The UPP DAT has three main sections. The first section asks people about their current pain levels, current management plans and impacts on their life and aims to establish a structured clinical pain history spanning biological, psychological and social domains.
The second section includes the DCE component with a series of questions that ask users to choose between different pain management plans. The plans are described by features and corresponding levels, which include broad categories of guideline-based pain management strategies routinely available in clinical practice. 39 The feature descriptors and their format were informed by a systematic literature review 40 and qualitative research study that involved semistructured interviews with 9 GPs, 10 pharmacists and 24 patients living with persistent pain from across Grampian. 41 Following this, the eight features described in table 2 were identified as important and relevant to patients living with persistent pain. These relate to both the actions they need to take and the expected outcomes.
We used experimental design methods to identify a manageable set of choices to present to individuals. 42 43 This design combines the attributes and levels into management plans that differ systematically across the choice tasks, aiming to present realistic combinations that maximise the precision of the parameter estimate for the main effect of each feature when analysed using a penalised logit regression model that can be analysed in real time (see below). 44 The experimental design resulted in 12 choices, each offering a choice between two hypothetical pain management plans. Figure 1 shows an example choice set.
We will ask patients to take a fresh look at their pain management and choose between the different management plans, described by the actions they need to take and expected outcomes on their life. When patients make these types of choices, they implicitly trade off the different attributes described in table 2, which allows the estimation of quantifiable measures of preference for each feature using econometric models. 45 The third section is a personalised report, which includes the patient's answers to the first section's questions and the results of the analysis of the DCE responses showing the features of a management plan they like and/ or how important they are to them. As shown in figure 2, the report will display the order of importance of the different features and visually illustrate the magnitudes of each in terms of the others (eg, how much a feature is liked or disliked with respect to the others). The UPP DAT will then prompt the patient to discuss the preference report with the pharmacist in a shared decision-making context, so that it can inform that discussion and ultimately the management plan. Pharmacists and patients will be able to review the raw data (eg, responses to the questions). In case the statistical model fails to converge, or the participant withdraws in the middle of the consultation, the report will instead display a summary of the responses to the previous questions. The UPP DAT will not make a medical recommendation.
Agreed management plans will be based on strategies routinely available in local clinical practice and will depend on the prescribing qualifications of the Open access pharmacist. Where a prescription medicine is deemed beneficial, pharmacists who are qualified independent prescribers will authorise this directly. Pharmacists without this qualification will, with the patient's agreement, book an appointment with the GP and send a letter to the GP with their recommendation for a prescribed medicine. Adherence to the consultation outcome and management plan will be up to the patient. Pharmacists can arrange a follow-up consultation according to clinical need and if deemed beneficial to the patient. The UPP DAT has been pretested to check ease of comprehension of its content and presentation. This involved feedback sessions with local patient groups and members of the study's Patient Advisory Group (PAG), consultations with healthcare professionals (pharmacists, GPs and specialised pain consultants) and opportunistic think-aloud sessions carried out by the research team with colleagues. This stage identified issues concerning the DCE choice tasks and difficulty understanding the results. We made changes to the design, going from threealternative to two-alternative tasks, to make the choice questions more intuitive and easier to complete. We also edited the personalised report format, which previously used positive-negative bar and pie charts to present results, to one which lists the relative ranking and illustrates using a normalised bar chart the relative magnitude of the parameter estimates, grouped by actions and outcomes. Furthermore, after a suggestion from healthcare professionals, we added extra clinical screening questions that could help guide the consultation. We also made changes to wording and rearranged content to make instructions clearer and easier to understand. The UPP DAT was also stress tested to verify its stability and reliability across different devices (eg, ensuring the tool can serve responses to the expected user count).
Control
The control group will undertake the consultation without the use of the digital UPP DAT.
Study outcome measures
Primary outcome measures are related to: ► Technical performance of the UPP DAT-does the UPP DAT generate individual-level preference estimates, does the model converge, is an individual report produced, face validity of parameter estimates. ► Pharmacist views about potential usefulness in the clinical setting. All pharmacists will be invited to a telephone debrief interview to explore their experience of taking part in the study, using the UPP DAT in their clinical setting and the pain consultations in general. The interview will be audio recorded and fully transcribed. ► Patient participants allocated to the intervention group will be invited to a debrief telephone interview with a member of research team to explore their experience of using the UPP DAT. The interview will be audio recorded and fully transcribed.
To inform a future RCT, we will also assess: (1) feasibility of recruitment processes, (2) response and retention rates and (3) timeline and resources required to collect and analyse data. Data on recruitment and consultation details will be recorded by the pharmacists in a patient activity log.
Secondary outcome measures are related to the feasibility of collecting the following outcome measures collated into a paper or electronic survey form to be completed immediately following the pain consultation and after 4 to 6 weeks. ► Personal Well-Being Scale: developed by the Office for National Statistics, it contains questions measuring four domains of well-being, namely, life satisfaction, worthiness, happiness and anxiety. 46 ► EuroQol five dimensions questionnaire (EQ-5D): an instrument that measures health outcomes on five dimensions, namely, mobility, self-care, usual activities, pain/ discomfort and anxiety/depression. 47 ► Chronic Pain Grade (CPG): a 7-item scale, which assesses pain severity on two domains (disability and intensity). The CPG scale classifies pain according to level of intensity and disability ranging from I (low disability-low intensity) to IV (high disability-severely limiting). 48 ► Decisional Conflict Scale: a validated instrument to evaluate patients' decision-making processes and satisfaction with their choice, aiming to assess the interactions using the UPP DAT and the overall shared decisionmaking process 49 (only included in postconsultation survey). Pharmacists will ask patients to complete the survey at the end of their pain consultation. Patients will be followed up by the research team by post and asked to complete the survey again, online or by post (see table 1).
Patient and public involvement
The UPP study has a patient representative as part of the research team who has contributed to the study aim, design, methods and dissemination plan. The study also has a PAG who meet regularly to provide feedback on findings from the study's preliminary stages and help inform this study design. The UPP DAT's DCE component was informed from a qualitative stage that actively involved patients in NHS Grampian. Patients involved in this stage were provided a summary of the results and given the opportunity to provide feedback to ensure the findings matched their experience. The UPP DAT was presented to local patient groups to obtain feedback on its usability and content as part of its design stage. PAG members used the UPP DAT in a mock consultation with a GP with expertise in pain management (member of the research team) and provided feedback that helped design and edit its content and format. Findings and dissemination plans will be discussed and agreed with the PAG.
Open access
Data analysis DCE responses will be modelled under the Random Utility Maximisation framework 50 , using variants of the multinomial logit (MNL) model. This framework assumes respondents make choices described by: where participant (n) at choice task (t) selects the alternative (j) yielding the highest level of utility (U ntj ). The utility is divided into an observed deterministic component (V ntj ), which is described by the preferences for the features' levels and an unobserved random component (ε ntj ). The deterministic component is an addition function of the features and their respective parameter estimates, such that: where β are the parameter estimates for marginal changes in the levels (X) for the features (k) described in table 2. The errors assumed to be independently and identically distributed (iid) as type 1 extreme values, leading to the MNL model. A limitation of this model to describe individual-level preferences is that it often does not work in small samples, such that it may require more observations (eg, choice tasks) than it is feasible to present to the respondent.
We overcome this small sample limitation by using a penalised MNL (pMNL) model. First proposed by Firth 51 and introduced to DCEs by Kessels et al 52 , this model uses a bias term in the standard likelihood function to obtain a penalised likelihood function, such that: is the likelihood and I is the Fisher Information matrix. This penalisation aims to decrease estimation bias and help stabilise the model when small sample bias is likely to occur. Crucially, it allows to estimate parameters from a manageable number of choice tasks without relying in prior samples. Thus, using a pMNL allows us to estimate preference parameters for each feature at the individual level in real time, which can be used to inform a personalised report. As well as assessing if the model converges to estimate parameters, consideration will be given to the face validity of estimated parameters.
Transcription of debriefing interviews with pharmacists and patients will be overseen by the research team. Analysis will begin as soon as data collection begins and involve an iterative process beginning with independent reading and immersion in the data followed by identification of recurring themes, coding and categorising of the data. The themes and subthemes identified from the interview data will be organised and analysed using the Framework approach. 53 To ensure the validity of the analysis, two or more research team members will code and extract data from a random subset of interview transcripts independently. Emerging coding frameworks will be compared, and any disagreements resolved through team discussion.
Data on the length and type of consultation (eg, faceto-face vs remote), recruitment and retention rates and response rates to outcome questionnaires will be recorded at the pharmacist level using a patient and activity log. The effectiveness of the digital UPP DAT on secondary outcomes will not be assessed.
Data management
Data management and monitoring will follow the Sponsor's (University of Aberdeen) Standard Operation Procedures, Health Research Agency Research Governance Guidance and the NHS Code of Practice on Protecting Patient Confidentiality.
ETHICS AND DISSEMINATION
This study has undergone internal and external peer review as part of the funding process. This study received ethical approval from the North of Scotland Research Ethics Committee (21/NS/0059) on 1 June 2021 and received Research & Development Management Permission to proceed from NHS Grampian (2021UA003E) on 3 June 2021. The study was registered in the Clinical-Trials. gov database on 1 November 2022. The study findings will be used for publication in academic journals and presentation at scientific meetings. Participating pharmacists will be sent a summary of the study key findings. A lay summary of findings will be made available at the Health Economics Research Unit (http://www.abdn.ac. uk/heru) and other University of Aberdeen websites. A technical report will be prepared for Pharmacy Research UK, which will be available in their website. The PAG will be consulted on dissemination decisions.
Study status
Recruitment of pharmacists began in January 2022 and of patients in May 2022 and is expected to continue until September 2022.
Twitter Luis Enrique Loría-Rebolledo @luisloria, Mandy Ryan @MRyan_HERU, Christine Bond @christinebond20, Peter Murchie @CAPCAberdeen and Rosalind Adam @rosadamaberdeen Open access collection, management, analysis or interpretation of the data and will have no input to the writing of the report or decision to submit for publication.
Competing interests None declared.
Patient and public involvement Patients and/or the public were involved in the design, or conduct, or reporting, or dissemination plans of this research. Refer to the Methods section for further details.
Patient consent for publication Consent obtained directly from patient(s)
Provenance and peer review Not commissioned; peer reviewed for ethical and funding approval prior to submission.
Data availability statement No data available.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.
|
2022-09-24T06:18:19.267Z
|
2022-09-01T00:00:00.000
|
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14942802
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pes2o/s2orc
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v3-fos-license
|
Lepton Number Violating Radiative $W$ Decay in Models with R-parity Violation
Models with explicit R-parity violation can induce new rare radiative decay modes of the $W$ boson into single supersymmetric particles which also violate lepton number. We examine the rate and signature for one such decay, $W\rightarrow \tilde l\gamma$, and find that such a mode will be very difficult to observe, due its small branching fraction, even if the lepton number violating coupling in the superpotential is comparable in strength to electromagnetism. This parallels a similar result obtained earlier by Hewett in the case of radiative $Z$ decays.
In the simple Minimal version of the Supersymmetric Standard Model(MSSM), both baryon(B) and lepton(L) numbers are simultaneously conserved quantities due to the imposition of a discrete symmetry called R-parity. One can then assign to each Standard Model(SM) particle and its SUSY partner a multiplicatively conserved quantum number, given by R = (−1) 2S+3B+L , where S is just the particle's spin. In addition to the gauge symmetries and the assumption of minimal particle content, R-parity conservation severly limits the possible interactions among the usual SM fermions and their superpartners as well as the properties of the SUSY partners themselves. The two most important phenomenological consequences [1] are well-known: (i) The SUSY partners of the conventional SM particles which carry negative R-parity can only be produced in pairs and (ii) the lightest supersymmetric particle(LSP), is an electrically neutral, color-singlet and is stable. This second property is the one which is conventionally used experimentally to search for SUSY particles, i.e., once they are pair-produced their decays involve final states which include the LSP that only appears as a missing energy signature in a collider detector. If R-parity were broken both these conclusions would be invalidated leading to an entirely new phenomenology.
Of course this minimal approach may not be that realized by nature. In particular, it is possible to construct phenemenologically viable models wherein R-parity is violated either spontaneously [2], through the acquisition of a vacuum expectation value(vev) by a sneutrino, or explicitly via the existence of additional terms in the superpotential [3] constructed out of the conventional superfields. These additional terms are possible as the gauge symmetries alone do not forbid their existence. If such new interactions are present they can lead not only to a destablization of the LSP but also new production modes for SUSY partners not present in the MSSM and thus forces us to a re-evaluate of the traditional search techniques for these particles. Clearly, the breaking of R-parity implies that these new interactions will violate L and B. In such a R-parity breaking scenario this more general form of the superpotential, W , can be written as where ijk are generation indices, and the h's and λ's are a priori unknown Yukawa couplings. [4] that if one assumes only the particle content of the MSSM and the lack of rapid proton decay, then two unique discrete symmetries are possible: R and B, the so-called baryonparity. Thus the most reasonable scenario to consider for our purposes is one in which B is conserved while both R and L are violated; this corresponds to setting all of the λ ′′ terms to zero in W . In principle, this class of models should be considered as to be just as likely a scenario for the realization of SUSY as is the more conventional MSSM with the same particle content. This is the scheme we consider below.
The interaction Lagrangians that result from W can be written as and Once B-parity has been imposed, many of the remaining couplings can be constrained via various phenomenological considerations using these Lagrangians as has been done by several groups of authors [5]. Combining all of their results, one finds that the only potentially large couplings, i.e., ones that are capable of being of the same strength as electromagnetism, are [6]: λ 131 , λ ′ 3jk , λ ′ 121 , λ ′ 222 , λ ′ 223 , λ ′ 232 , and λ ′ 233 . Note particularly that interactions involving third generation particles are least likely to be constrained by existing data.
Although one can look for the direct influence of R-parity violating interactions, it may be possible to search for their indirect effects through, e.g., loop diagrams. One such possiblility, recently examined by Hewett [7], is the loop-induced decay Z →νγ, with a rate that might be only an order of magnitude smaller than that for Z → Hγ in the SM.
In this paper we wish to examine the corresponding process in W ± decay, i.e., W →lγ.
Unfortunately, as we will see, the branching fraction for this process is found to be much smaller than in the Z case due to helicity supression and the form of the superpotential, W .
The diagrams responsible for the W →lγ decay are shown in Fig. 1 and involve one R-parity violating vertex. For simplicity, we have assumed that only one of the two ∆L couplings is non-zero, i.e., we ignore the possible contributions of the λ terms in the superpotential and concentrate on the λ ′ terms since more of them can be large. We note, however, that since λ ′ 131 can be sizeable, a loop involving first generation leptons might yield a significant contribution. In fact, one finds that such contributions are suppressed due to the small masses of these particles. This being the case, it is the couplings λ ′ 233 and λ ′ 333 which are relevant here. In the former case, thel is a smuon whereas in the latter it is a stau. We, of course, do not know the size of this active λ ′ coupling so we simply scale it to the electomagnetic strength as is customary, i.e., we write the effective tbl interaction as where F is an unknown parameter which may be as large as unity. With this normalization, the amplitude for the W →lγ decay process can be written as with q(k) being the momentum of the photon(W ). In terms of the form factors F 1,2 , the decay width is given by with ml being the slepton mass. Definining the mass difference, δ = m 2 l − M 2 W , we find that F 1,2 can be written as where N c =3 is the usual color factor, m b is the b-quark mass, g is the conventional weak coupling constant, Q u,d are the electric charges of the up-and down-quarks, and I i can be expressed as sums of parameter integrals: where the G n are given by It is important to note that both F 1,2 are proportional to m b and not m t . This comes about due to the fact that the W charged current interactions are purely left-handed, as is the coupling in Eq. (4), and that unless a mass term from a propagator of an internal quark line is picked up, the result will vanish since a trace over an odd number of γ-matrices will then be taken. In the actual calculation only the b-quark mass term is picked up so that instead of increasing in magnitude as m t increases, the rate for W →lγ will decrease for large m t due to supression from the top-quark propagator.
Using m b =5 GeV, α −1 =127.9, and M W =80.15 GeV as numerical input, we can calculate the partial decay width as a function of ml for different values of m t and the parameter F . The result of this calculation, expressed as the branching fraction(B) for the W →lγ process, is shown in Fig.2. As advertised, even for F =1, we see that B is less than about 10 −8 for all values of m t and ml and, as anticipated, falls with increasing m t . This rate is sufficiently tiny that this reaction will be impossible to observe. If the helicity structure of the R-parity violating interaction had been opposite, the rate could have been larger by a factor of m 2 t /m 2 b ≃ 10 3 and potentially observable since the overall nuerical factor in the amplitude would then have been proportional to m t instead of m b .
Unfortunately, our result implies that signals for R-parity violating interactions must be sought elsewhere than in radiative W decays.
|
2014-10-01T00:00:00.000Z
|
1992-11-10T00:00:00.000
|
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225459506
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pes2o/s2orc
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v3-fos-license
|
English for IELTS test preparation: the needs analysis
Received 03 May 2020 Revised 04 July 2020 Accepted 07 July 2020 Available online 31 July 2020 The present study tried to modify IELTS Needs analysis based on the participant’ learning competence. The participant was a student with IELTS Score 4.5 and he was required to have minimum score 5.5 in order to enroll an overseas university. A skill-based analysis was used in the paper to identify the student. Questionnaire along with the placement test was conducted to get the skills that the participant needed to improve. From the analysis, it was seen that the participant needed to improve his productive skills. Slow learning competence from the student made the instructor had to change the situation of learning process. The final result of the paper is an IELTS course preparation analysis adapted from the participant’s need. Materials, practices, assessment and Evaluation were also added in this analysis. The writer expected that such needs analysis can be employed to IELTS preparation course institution so that better learning outcomes can be achieved.
Introduction
As the growing nation, Indonesia is one of the countries that often send its students and workers to study or work overseas. Indeed, Indonesian people consider studying and working abroad is important to extend their knowledge and experience as well as sharpen their foreign language. However, there are several requirements for those who want to study or work in the English speaking countries, one of them is passing the given score of IELTS test.
The International English Language Testing System (IELTS) assesses the English language ability of the student or worker candidates based on the four language skills. The test is designed to reflect how the students or workers will use English at school or university, at work, and at home, in their new life abroad. IELTS is a standardized test that is considered valid and reliable by the researchers (Spolsky, 1995;More & Morton, 2005;O'Loughlin, 2008;Weir et al., 2008). Therefore "IELTS is the most widely accepted English language test that uses a one-on-one speaking test to assess your English communication skills" (British Council, 2010). Many English speaking countries such as USA, the UK, Canada, and New Zealand require IELTS test for the foreign learners and workers if they want to live there.
Since each English speaking country has different requirement score of IELTS test, it is sometimes not easy for the candidates to pass the given score of IELTS test if the countries they want to go need a high score for the candidates. Furthermore, IELTS is designed to assess the language ability of the candidate in all four skills; the candidates must at least possess the fluency of the four skills at the rate of daily communication. This also becomes one of the difficulties for the candidates to pass the test particularly for Indonesian candidates who do not use English as daily communication.
In order to help the candidates to get the given score of IELTS test, many English courses nowadays provide IELTS preparation courses for those who need high score of IELTS test. The courses commonly provide both intensive and extensive classes, private and regular classes for the learners based on how fast they have to get the IELTS results. The courses also provide up to a hundred hours for the learners who really need English from the very basic level, usually for the lower level learners. During the course, the learners will study English in all four language skills and in the rate of daily communication and academic field. It occurs because the learners are expected to be familiar not only in English daily nature but also in English academic nature as they need to be familiar in both fields when they are in the university or in the work field.
However, because IELTS preparation learners have different level of English proficiency, it is not easy for IELTS preparation instructors to provide the materials and the activities that are really appropriate for the learners, particularly for the instructors with a large number of students. It is even more difficult for the teacher because, with only tens to a hundred hours they get, they have to make the learners master all four language skills of English. It is especially difficult to teach the productive skills, writing and speaking, as the students do not usually produce words in English. Therefore, having an appropriate course design for the IELTS preparation class is necessary in order to get what the learners really need in mastering all four languages needed for IELTS test.
In this paper, the IELTS preparation course analysis is made for the private class which has students who need to pass a given score of IELTS test in order to enroll a university in Norway. The student needs to pass at least 5.5 in each band (each language skill score) and he only wants to have a 50 hours class. For that reason, the need analysis is necessary to be conducted so that the learner can get what he really needs and how he can learn it in order to pass the IELTS test.
Literature Review
It is known that to make IELTS Test learners achieves a good score, the teachers need to analyze learners' English competence, ability, and drawbacks before starting the class. One of the way to do it is by doing the diagnostic test and placement test. The placement test is held in order to place the right level of the class and to seek the learners' competence. However, it is sometime not proficient to only have the placement and diagnostic without having any learning background of the student. It occurs because many of the learners still have difficulty to cope the IELTS materials. One of the reasons is they are not familiar with IELTS specifically, and English generally. For that reason, learner's needs analysis is important to be conducted in order to make learners feel comfortable to learn IELTS.
In defining the needs analysis, Graves (2000) stated that needs analysis is a systematic and continuous process of combining information about what students need and identifying the information in order to have an effective course to meet the needs. A systematic and continuous process here means when it needs time and effort to analyze the learner needs. A thorough analysis is necessary to do in order to find a comprehensive result of student needs. Therefore, a placement test and diagnostic test are not quite proficient if we want to analyze the IELTS learners' needs.
A previous study about students' needs analysis was conducted by Cahyono & Widiati (2015). They analyzed students' vocabulary, grammar, punctuation and correct structure of English before starting an English class. They conducted the study arguing that it was very rare to find those kinds of analysis used as the ground of conducted the class. It came out that the analysis could enhance the students' motivation to learn based on the competence they need to improve. Eshtehardi (2017) also conducted a learner's need analysis study focusing on vocabulary in reading skills of IELTS learner. The results revealed that unknown vocabulary and lack of employing reading skills are their main weaknesses in Reading. Lastly, Daud, Daud, & Kassim (2016) conducted an analysis study of learners' writing skill before starting the IELTS class. They focused on the writing skill because IELTS writing test gave large scale high stake in evaluating the test performance of the test takers. Two different kinds of tasks make IELTS writing test become a great enemy of the test takers. Many of them have failed the test because of the writing task. The results came out that Students' writing performance is in accordance to anxiety especially when students do not know what to write and what to do. Therefore, they spent a lot of time only doing the brainstorming in writing process.
From those previous studies, researcher wanted to seek the chance to analyze her learners' needs before taking the IELTS class. The present case study was conducted in order to find an alternative solution to enhance the student's IELTS score as it is needed for the student to get a good score.
Needs Analysis
There were two different kinds of method to analyze the learners need conducted in this study, first, the analysis of the placement test which has done by the learner and second, the questionnaire given for the learner after taking the placement test. As Dudley-Evans and Saint John (1998) stated in their book that there are three different kinds of needs analysis in ESP; target situation analysis, present situation analysis, and learning situation analysis (p. 123-125). It is expected that those three kinds of needs can be attained through these two methods.
Placement Test done by the learner
The placement test is chosen as one of the needs analysis method because the test can be a tool to analyze the learner's present situation (PSA) in learning English as Dudley-Evans and Saint John (1998) claimed that "Testing may form part of a pre-course PSA" (p. 137). The form of the placement test is similar to the real ILETS test which is divided into four parts based on the language skills. Because there are four language skills assessed in IELTS test, it is important to have the needs analysis based on the four skills. Each skill is analyzed through each part of the test.
Listening Skill
According to the result of the listening part, the learner seems to have a good command in listening skill as he has a good score in this part. He only made 10 mistakes from 40 questions given to him. However, from this part, it is found that he has a difficulty in answering the summary task. The summary task is in the form of gap filling which there no option is given for the test taker. In the summary task, the learner had to listen to a complete monologue as well as checking the summary of the monologue in the question sheet. Because what the speaker talked was not the same with what in the summary, he probably could not get the real message of the monologue and it affected to confusion in answering the summary. Beside the summary completion, it is complicated for him to answer the completion task with a complete phrase. There are some questions that require the learners to answer with more than one word to have a clear idea of the answer. Yet he seemed could not give the clear answer. Next, it seems not easy for him to listen to the singularity and the plurality of the nouns. There are some answers need to have plural nouns whereas he made in singular. Indeed, it is important to make him know that some questions require plural nouns for the answer. Lastly, similar with the summary completion, he had a hard times listening to the long monologue while checking the answer. He could not get the key words of each question and could not grasp the important message of every utterance in the monologue. Therefore, he was not able to answer almost all questions in monologue parts.
Reading Skill
Unlike the listening skill, the learner did not have a good score in reading skills. From 40 questions given, he only succeeded 20 questions of them in which he only made 50% of questions correct. Almost all of types of the questions had got wrong by him; completion, matching, and multiple choices. Multiple choices with multiple answers type is one of the question types where he made it all wrong. He also had the difficulty in answering the matching of headings where he had to decide which heading options characterize certain paragraphs. Identifying the writer's views part seems also complex for him where he had to decide whether the statements given in question represent the writer's views or not. Next, not only in the listening part, the learner also could not answer the summary completion in reading section even though he can take a look to the text for several times. Indeed, summary completion is very important to be learnt and reviewed by the learner during the course. True, false, and not given is also the part that he could not answer correctly. It seems hard for him to get the information from the statement that is being paraphrased with the text. Moreover, another kind of completion, sentence completion frightened the learner the most. All questions in sentence completion are wrong as the questions are being paraphrased. Therefore, paraphrasing should be put in the course design as he had the difficulty in understanding it.
Writing Skill
There are two parts in IELT writing skill, the first part is about describing the graphs, process, or map and the second part is giving argumentation and reason of a topic given to the learner. From those parts, the learner is generally unfamiliar with the structuring the paragraph in IELTS writing parts. He also tended to repeat the same vocabularies and sentence structures in which it shows that he still has a limited number of vocabularies. He also could not be aware of presenting the tenses structure in both writings. Regarding with the content of writing, he seemed cannot develop the topic well so that he wrote those part less than the required number of words. Lastly, he still had a difficulty in making the correct verb agreement. He mostly wrote an inaccurate auxiliary verb which is not matched with the verb he mentioned.
Speaking Skill
Unlike the writing part, the learner made a better performance in speaking part in which he could speak fluently and not being nervous in front of the panel. However, his lacks are quite similar with the writing task as both skills are about producing the words and sentences. He could not develop the topic in every question given so that he spoke less than the time provided. It makes his score became not satisfying. He also made the same mistakes with the verb agreement which it is seen as he made the mistake several times. Lastly, he seems had the difficulty in pronouncing English word. It is important for him to pronounce the words clearly so that the panels can understand what he said as most of the panels are natives of English.
Questionnaire for the learner
A questionnaire is given to the learner after he knew the overall result of his placement test. The questionnaire is contained the questions related with his learning situation (LSA) and target situation (TSA) analysis. The questionnaire is divided into five parts. Those questions represent the LSA, TSA, and part of PSA of the learner as seen on table 1. From the topic related with IELTS, it is known that the learner need the score 5.5 for minimum score in each skill. He needs that kind of score because it is the required score if he wants to continue his study in Norway. He need the IELTS score in a month as the university enroll time will be started after a month of the study. He never had IELTS test before and he is actually unfamiliar with IELTS test.
In the column of education, he has just graduated from a high school in Makassar and wants to spend his holiday to learn IELTS in Jakarta. He wrote that he likes the physical exercises subject but he is not really keen to any foreign language subject. Because he wants to enroll petroleum department, he is interested to study anything about oil industry. Therefore, giving him the reading text and the writing topic related with oil industry may attract him to learn better.
In the section of language experience, the learner does not speak other language beside Indonesian. Because he has just moved from Makassar to Jakarta, he currently does not have any teacher to teach him English. He likes to listen to English songs and he often watches English movies even though he uses English only in English class. Lastly, during the school he used English textbook from Longman Publisher.
The learner generally has little time to study English; he stated that he only learns English in the classroom during English subject. The English subject in his school is three times a week in one and a half hour per meeting. He started to study English since the elementary school and it has been twelve years he studied English. He does not have self-study of English and he is not a person who is really interested in learning English.
Besides in school, his own time to learn English may be from listening to English songs and watching English movies. He does not have any foreign friend although he has an aunt who lives in Norway and eventually asks him to study there to sharpen his English and he actually plans to go there and live with his aunt.
From the result of analysis above, it can be inferred that the target situation (TSA) of the learner requires the instructor to make a one scale higher score than his pretest (overall 4.5). Moreover, the learner is not familiar with the IELTS test since he never took the test before. Thus, the course should introduce all elements of IELTS test to the learner. Meanwhile, LSA of the learner does not really support him to learn English as much as possible and as quick as possible. Therefore, the instructor needs to design the instruction that can stimulate and motivate the learner to learn IELTS in a practical way and in effective time duration.
Type of the course analysis
The type of the syllabus which is used in the course is skill-based syllabus as the materials and activities executed in this course will be based on the four language skills. Railey (1988) found that "the content of the skill-based language teaching is a collection of specific abilities that may play a part in using language" (p. 1). It is easier to divide the syllabus based on the language skills since IELTS test is also separated into those language skills. Moreover, Rane (2010) also claimed that skill-based instruction is basically for Language for Special Purpose (LSP). "The example of skill based instruction application is in life skills and immigrants and refugees or language programs preparing students for academic work" (p. 5).
There will be several combinations of the skills (integrated skills) when there are some materials of learning have similar strategy of learning for different skills. Paraphrasing, for instance, is needed in almost language skills; reading, listening, and writing. The student needs to understand how to get the message from the paraphrase statement in listening and reading part. Meanwhile, the learner has to be able to paraphrase his sentences in order to avoid the repetition. Therefore, there will be several meetings which join some skills in one material.
Listening Skill
The materials in listening skills are split up according to the question types in IELTS listening part. There are seven different types of listening question, they are, form/notes/table/flow-chart/summary completion, multiple choice, Short answer question, sentence completion, labeling the diagram, map, or plan, Classification, and Matching. Each of the questions commonly has its own strategy to answer it. However, due to the limited time and the consideration that the learner has a good proficiency in this skill, there are several types of question which are taught in the same meeting and only some types of it will be taught in separated meeting. Summary completion is one of the materials that is taught in one meeting as it will be combined with summary completion in reading task. It occurs because the learner had difficulty to answer the summary completion in both reading and listening skill.
Reading Skill
Reading skill instruction will also be taught based on the question types, however, it has more types than in listening skills. Therefore, the number of hours will be more than listening skill. There are eleven types of question in reading skill; some of them are similar with listening part. They are multiple choices, identifying information, Identifying writer's views or claims, matching heading, matching information, matching features, matching sentence ending, sentence completion, form/table/notes/flow-chart completion, labeling the diagram, and short answer question. According to the PSA analysis, the learner needs to spend more meetings in reading skill since he had a hard time to answer the reading question.
Writing Skill
There are two writing parts in IELTS test. The first part is reporting the graphs, process, and maps and the second part is making an essay of the topic given. The course will have both part separated since they are different in genre. Writing task one will be divided into six different kinds of figure; line graph, bar graph, pie chart, table chart, process, and map. While writing task two is based on the way of giving argumentation and reasoning; agree/disagree, advantage/disadvantage, discuss two views, cause and effect, two part questions, and giving opinion. Since the learner had a difficulty in structuring the paragraph, there will a time in the beginning of the writing session to learn about structuring the paragraphs in both writings. He will also get a session to learn the vocabularies of describing trends and numbers which is related to writing task one. As for his grammar, he will be given some exercises related to the verb agreement before he starts to write.
Speaking Skill
Speaking test consists of three parts which each of parts contain different topics and different time provided. Speaking part one is related to personal life of the learner and the general topic related with daily nature, speaking part two is named the topic card where the learner will be given a topic in a card contained some questions related with the topic, while speaking part three is about the issues-oriented question related with the topic in part two. The learner needs to develop his answer with reason and example so that many practices and feedback is chosen as activities which are mostly used in this session.
The Future Course Details
The development of course details is presented on table 2.
Student's Analysis
Because IELTS test is considered as public examination (Dudley-Evans & Saint John, 1998, p. 214) and it has standardized assessing system, it is important for the course to have similar kinds of assessment in order to have the same conclusion of proficiency measurement. IELTS test uses band descriptor for each skill and the results are given on a scale number, from 1 to 9. All skills in IELTS test have their own system of assessment, the receptive skills assessment are based on the total numbers of correct questions, while the productive skills use four major linguistic criteria of producing text. The four criteria in writing test include task achievement, coherence and cohesion, lexical resource, and grammar range and accuracy. While the speaking test criteria consist of fluency and coherence, lexical resource, grammatical range and accuracy, and pronunciation. As the same with the receptive skills, each of the criteria is measured using the scale from 1 to 9. The score from all criteria will be collected and divided by four to make a final score of the skills. The course will execute the same assessing system with the real IELTS test using two kinds of final test, mock test and final test. The mock test is Course duration : The form of the course is intensive course with 2 hours per meeting every Monday to Saturday within a month. The class is held in the morning from 10 am to 12 pm. After the end of the course there are several IELTS test practices conducted until the day of the real IELTS test comes.
Course Length : The course has 50 hours long. After the course finished, the student will spend three hours for practices every Monday to Saturday to have the practices of IELTS test.
Student
: It is a private class where there is only one student as the participant of the class. His name is Erzal Prayuda. He was already graduated from high school and plan to get his Bachelor Degree in Norway.
Objectives
: After completing the course, the student is expected to have the following competences: 1. Understanding and using a range of around 5,000 general and academic vocabulary 2. Understanding and using a range of sentence structures and grammar features. 3. Comprehending English in a conversation or monologue and answering questions related to them (listening skill). 4. Comprehending Reading passages and answering questions about the main ideas or details from the passages (reading skill). 5. Describing and/or comparing graphs/tables/diagrams (writing skill task 1) 6. Writing essays of presenting and justifying opinions (writing skill task 2) 7. Talking about personal details, familiar topics, and offering arguments about abstract issues (speaking skill).
Resources
: The course will use several references related to IELTS test such as: done after the instructor gives the review in all parts of IELTS test, while the final test is purely done without the interference of the instructor. The instructor even cannot be the panel of the speaking test in order to avoid the subjective judgment of the student's assessment. The course will only use the final test (summative test) without having any formative test since it is an intensive class which only has a month as the class duration. Therefore, instead of having the summative test, the course will have the practices carried out after the student finishes his study of each skill. There will be no formal assessment during the practice time; yet, the instructor will have some final feedback given to the student before starting to learn the new skill.
One of the TSA of the students related with the requirement score is that the student needs to have 5.5 for minimum each band (skill) while his PSA regarding with his placement test score is 4.5 with the lowest score is in the writing part with 4. It is one of the teacher responsibilities to make the student reach the required TSA in order to make him pass the enrolment requirement. For that reason, the mock test and the final test will be used as the monitor whether the student is ready to join the test or not. If he failed to have the minimum score for each band in his mock and final test, the instructor will give another feedback during the practice time available before the day of the real test. In the case that he still cannot pass the minimum score in the final practice, it is better to recommend the student to not take the real IELTS test and to have another session of IELTS preparation to avoid wasting money to pay for the test.
The Skills Assessment
IELTS preparation course is the course where the students mostly expect to pass the IELTS test with the score that they intend to have. Therefore, the IELTS preparation instructors bring a high responsibility to satisfy the students during the course. In this present course, the course evaluation will be based on two methods of evaluation, assessment and the student's questionnaire. Assessment evaluation is based on the formal summative test conducted through the final test, in the case that the student takes the real IELTS test; the score of the IELTS test is being the assessment of the course evaluation. If the test results to the required score, it can be considered that the materials, activities, and the teaching methods executed in the course can satisfy the learner's need. If not, the instructor needs to get some changes in some parts of the course and teaching methods in order to reach the satisfaction of the student's need.
The second course evaluation is taken from the student's questionnaire done after the final test. The questionnaire is collected and modified from imscience.edu.pk and from Moore et al. (2009) which provided the questionnaire of the IELTS course evaluation for the students in their study of IELTS in Cambodia. The questionnaire includes the learning activities, materials range based on the language skills, teacher's performance, and teaching aids.
Discussion
From the result, it was found that the learner was lacking in several skill of language which needs to be improved if he wanted to succeed in IELTS test. Reading skill, for instance, was one of the skills that the student was very lacking. He could not get the matching questions right and he did give a great summary for summary completion. And one of the reasons is the lack of vocabulary knowledge. He seemed confused with what he read and he even did not know what to write in doing the summary. The result corresponded to the previous study from Eshtehardi (2017) that the vocabulary building was being a great weakness for the students of IELTS reading test. Therefore, an additional materials and time consuming for reading skill was needed to improve the learner competence in reading. Next is writing skill. It was also found that the student needed more time to improve writing skill since the learner was very new in doing the writing task of IELTS. He could not structure the paragraph coherently and he was not able to develop his topic of the paragraph well. The analysis was in one way the same with what Daud, Daud, & Kassim (2016) found that the students not only need more time to learn the grammar of writing task, but also the time to learn the process of brainstorming and make it short so that it did not consume so much time only for brainstorming. In this case, the failure of writing test gave a much lower score for the learner. Hence, the teacher needed to add more time to teach writing test to the learner. Though the learner was lacking in reading and writing skill, he was better in doing the listening and speaking task so that the learning time for those skill can be reduced in order to improve the skills that he needed to improve more.
Conclusion and suggestions
To be concluded, based on the TSA of the student, the course duration can take only 50 hours, because the student has only a month to prepare before the IELTS test and before the enrollment time is closed. For the same reason, the intensive course is decided to make the most of his available time. Each meeting holds 2 hours long since the student need to prepare other necessities regarding his enrollment such as making a passport, enrolling the IELTS test, etc. Skill-based syllabus is chosen as the types of the syllabus since the IELTS test examines the student's proficiency based on the four language skills. This decision is also supported by Rane (2010) who stated that skill-based syllabus is suitable for the course which its participants are from foreign language country such as immigrants and refugees. In the skill-based syllabus, the materials and the topics are based on the specific area of language skills such as writing a good introduction, discussion data, etc. (Dudley-Evans & Saint John, 1998, p. 164). In the case of the present course, the materials and the topics are based on the task types and the question types from which commonly exist in the IELTS test. The listening and reading skill instruction are based on the question types while writing and speaking skill are taught from the task types. Each of the types generally has its own strategy to handle the questions or the tasks. Therefore, the strategies used for handling the task and the questions can be practiced and discussed directly through the exercises. This, according to Dudley-Evans & Saint John (1998) can first, be the learning support which makes the pattern of answering the questions and be the stimulation for the learner when they face the certain types of tasks or the questions from the test (Dudley-Evans & Saint John, 1998, p. 171-172).
IELTS test is considered as the in-house test development since from the result of the test, the student will know whether he passes or fails to enroll the intended university (Dudley-Evans & Saint John, 1998, p. 221). The standardized assessing system of IELTS test requires the student to perform as well as possible. Therefore, the course implements the same system of assessment in order to raise the responsibility of the student to perform well when they face the real test. Feedback is always given to the student after he does both practices of the final test.
The present study surely has its benefits and drawbacks for the course. When the benefits are planned carefully, there must be the drawbacks comes at the same time. Firstly and mainly, since the intensive class obliges the student to learn English every day, it is already expected that the declining of the motivation to learn will come (Dudley-Evans & Saint John, 1998, p. 147). There will be sometime when the learner feel bored of learning IELTS. Therefore, the instructor is allowed to exchange the skill instruction or introducing the new skill instead of continuing the same skill in order to recharge the learner's motivation. Secondly, it is seen in the syllabus that the activities are mainly in the form of discussions and practices. It occurs because it is expected that the more practices that the learner does, the more ready the learner to face the real test. It certainly makes the student feel bored of the repeated activities. However, both the learner and the instructor cannot avoid facing it since the course is basically a test-oriented course and the duration of the course is only for a month. Lastly, the course does not use the materials and the topics related with the real life because the real life topics are considered less useful for the test-oriented course. However, some terminologies and vocabulary related with the common core of real content will be taught related with the texts, questions, and the topic card (speaking) in the materials.
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2020-08-13T10:10:04.213Z
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2020-07-31T00:00:00.000
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Overview of the Maturation Machinery of the H-Cluster of [FeFe]-Hydrogenases with a Focus on HydF
Hydrogen production in nature is performed by hydrogenases. Among them, [FeFe]-hydrogenases have a peculiar active site, named H-cluster, that is made of two parts, synthesized in different pathways. The cubane sub-cluster requires the normal iron-sulfur cluster maturation machinery. The [2Fe] sub-cluster instead requires a dedicated set of maturase proteins, HydE, HydF, and HydG that work to assemble the cluster and deliver it to the apo-hydrogenase. In particular, the delivery is performed by HydF. In this review, we will perform an overview of the latest knowledge on the maturation machinery of the H-cluster, focusing in particular on HydF.
[Fe]-hydrogenases, found only in methanogenic Archaea, are the smallest group and are restricted to a single function, they catalyze reversible reaction of methenyltetrahydromethanopterin with H 2 to methylenetetrahydromethanopterin and H + .
[FeFe]-hydrogenases have been found in some unicellular green algae, such as Chlamydomonas reinhardtii, as well as in strict anaerobes, fungi and protists. For the most part, hydrogenases likely function either in recycling reduced electron carriers that accumulate during anaerobic fermentation through proton reduction or in coupling H 2 oxidation to energy yielding processes. Indeed, both [NiFe]-and [FeFe]-hydrogenases catalyze the reversible conversion of protons into hydrogen 2H + +2e − H 2 in conditions of strict anaerobiosis; [FeFe]-hydrogenases are usually involved in the forward reaction, while [NiFe]-hydrogenases in the backward reaction, although there are a number of exceptions [13,14]. Both classes of enzymes are oxygen sensitive, and hydrogen production is strongly inhibited by aerobic conditions, whereas [FeFe]-hydrogenases are irreversibly inactivated during catalysis by trace amounts of O 2 , in most cases [NiFe]-hydrogenases react reversibly with O 2 , giving rise to a mixture of inactivated states [15][16][17][18]. This sensitivity to molecular oxygen is one of the most critical drawbacks that have seriously limited the use of recombinant hydrogenases as stretching modes sensitive to the iron oxidation state have allowed to identify the EPR-silent cluster states.
In this work, we will review the updated knowledge on the maturation machinery of the Hcluster, highlighting the questions that are still open, focusing in particular on the structure and catalytic mechanisms of HydF. A first section will be devoted to the individual enzymes and a second one to the discussion of the overall process. [FeFe] hydrogenase. On the left side, we show the structures and active sites of the enzymes involved in the maturation: purple, HydG from T. italicus (pdb.id 4WCX) with the first active site, the SAM cluster on top, and the second active site on the bottom; pink, HydE from T. maritima (pdb.id 3CIW); blue, HydF) from T. melaniensis (pdb.id 5KH0); green, HydA, from C. reinhardtii (pdb.id 3LX4), without the complete active site. On the right side, a more detailed scheme of the reactions involved in the process. The enzymes are indicated as squares (the color scheme is conserved); the products of the enzymes are shown as ovals of the same color of the enzymes producing them. Some relevant chemical structures are reported, from top to bottom: the synthon; the [2Fe] F cluster; the [2Fe] H cluster; and the active site of the holo-HydA with the full H-cluster. The question mark denotes unknown substrates/products; the asterisk denotes the attachment point of the [2Fe] F sub cluster to HydF. For the acronyms, please refer to the abbreviation section.
The principal methods that have been used to characterize hydrogenases and their active sites are crystallography, fourier-transform infrared spectroscopy (FT-IR), electron paramagnetic resonance spectroscopy (EPR). Crystallography beside providing the molecular structures of the enzymes, also helped in the identification of the molecular mechanisms through co-crystallization experiments with putative substrates. However, most of the information on the mechanisms of action of the hydrogenases and on their maturation process, has been obtained through spectroscopic methods, mainly EPR methodologies and FT-IR, often coupled together. Conventional EPR spectroscopy (i.e., continuous-wave EPR) provides a wealth of information on FeS clusters of the enzymes: different kinds of FeS clusters have different EPR spectra. Additionally, since EPR is only sensitive to paramagnetic states, it can help to define the oxidation state of the metal centers in different conditions. When coupled to isotopic labeling or mutagenesis experiments, pulsed EPR methods allow to identify the residues bound to the metal centers of the active site in the different functional states through the detection of the electron-nuclei hyperfine coupling. Finally, EPR has been coupled to the site-directed spin labeling method (SDSL) to study the changes in structure and dynamics of the enzymes in solution. FT-IR resulted to be unique in its ability to differentiate both the nature of the H-cluster ligands, and their mode of binding. By a careful analysis of the molecular stretching modes, the CO and CN − ligands and their orientation have been defined. In addition, the stretching modes sensitive to the iron oxidation state have allowed to identify the EPR-silent cluster states.
In this work, we will review the updated knowledge on the maturation machinery of the H-cluster, highlighting the questions that are still open, focusing in particular on the structure and catalytic mechanisms of HydF. A first section will be devoted to the individual enzymes and a second one to the discussion of the overall process.
HydE
HydE is the least characterized of the three maturation enzymes, while its structure has been solved and HydE has been assigned to the radical SAM superfamily of enzymes, its substrates and products are still uncertain. While the exact role of HydE in the maturation process is not well-defined, it has been suggested to provide the bridging di (thiomethyl) amine ligand of the H cluster [38].
The structure of HydE has been solved only for the enzyme from Thermotoga maritima (pdb.id 3CIW) [39], see Figure 2. HydE from T. maritima adopts a distorted triose-phosphate isomerase (TIM)-barrel fold, where 8 α-helices and 8 parallel β-strands (shown in dark pink and green, respectively, in Figure 2) alternate along the peptide backbone, these last forming a large internal cavity where a molecule of SAM and additional substrates can be accommodated. Relative to the standard TIM-barrel fold, HydE has additional helices at the N-terminus and an additional strand at the C-terminus (shown in pale pink and dark blue, respectively, in Figure 2). The protein hosts one [4Fe-4S] cluster in the N-terminal part which is highly conserved and has been identified as the active site; this cluster faces the internal cavity of the enzyme, and it is coordinated by a SAM molecule in the crystal structure. A secondary [FeS] cluster binding site is present, about 2 nm away from the SAM cluster, but this secondary site does not seem to be essential for the maturation process, the cysteines of the secondary site are shown as spheres in Figure 2. The stoichiometry of the secondary cluster is not exactly defined in the crystal structures, varying from [2Fe-2S] to [4Fe-4S] [40], and EPR spectroscopy data have shown that full cluster reconstitution leads to two [4Fe-4S] clusters in the enzyme [38].
denotes the attachment point of the [2Fe]F sub cluster to HydF. For the acronyms, please refer to the abbreviation section.
HydE
HydE is the least characterized of the three maturation enzymes, while its structure has been solved and HydE has been assigned to the radical SAM superfamily of enzymes, its substrates and products are still uncertain. While the exact role of HydE in the maturation process is not welldefined, it has been suggested to provide the bridging di (thiomethyl) amine ligand of the H cluster [38].
The structure of HydE has been solved only for the enzyme from Thermotoga maritima (pdb.id 3CIW) [39], see Figure 2. HydE from T. maritima adopts a distorted triose-phosphate isomerase (TIM)barrel fold, where 8 α-helices and 8 parallel β-strands (shown in dark pink and green, respectively, in Figure 2) alternate along the peptide backbone, these last forming a large internal cavity where a molecule of SAM and additional substrates can be accommodated. Relative to the standard TIMbarrel fold, HydE has additional helices at the N-terminus and an additional strand at the C-terminus (shown in pale pink and dark blue, respectively, in Figure 2). The protein hosts one [4Fe-4S] cluster in the N-terminal part which is highly conserved and has been identified as the active site; this cluster faces the internal cavity of the enzyme, and it is coordinated by a SAM molecule in the crystal structure. A secondary [FeS] cluster binding site is present, about 2 nm away from the SAM cluster, but this secondary site does not seem to be essential for the maturation process, the cysteines of the secondary site are shown as spheres in Figure 2. The stoichiometry of the secondary cluster is not exactly defined in the crystal structures, varying from [2Fe-2S] to [4Fe-4S] [40], and EPR spectroscopy data have shown that full cluster reconstitution leads to two [4Fe-4S] clusters in the enzyme [38].
Based on the crystal structure, the access to the internal cavity has been hypothesized to be regulated by a region of the protein directly above the active site, a loop-helix-loop structure acting as a "lid" (shown in light blue in Figure 2) with a strictly conserved aromatic motif, YXXY. The protein surface on this region shows one main access point in the "lid" region and a second one that is open in the absence of the secondary cluster (indicated by the red arrow in Figure 2). Both accesses are narrow, but, combined with dynamic conformational fluctuations of the protein in solution, could allow substrates and products to diffuse to the active site. A third access to the internal cavity, closed in the static protein structure, rests on the other side of the cavity relative to the active site, covered by the C-terminal loop. In magenta (helix) and green (strand) the TIM-barrel fold; in pale pink the N-terminal helices; in dark blue (on the other side of the protein) the C-terminal loop; in light blue the putative "lid" region governing the access to the active site; the [4Fe-4S] cluster forming the active site is in sphere representation; and the cysteine residues not binding the cluster are also in sphere representation. Right, same view as before but with the surface shown: the accesses to the internal cavity are in the center of the light blue zone and close to the two cysteines as shown by the red arrow.
Based on the crystal structure, the access to the internal cavity has been hypothesized to be regulated by a region of the protein directly above the active site, a loop-helix-loop structure acting as a "lid" (shown in light blue in Figure 2) with a strictly conserved aromatic motif, YXXY. The protein surface on this region shows one main access point in the "lid" region and a second one that is open in the absence of the secondary cluster (indicated by the red arrow in Figure 2). Both accesses are narrow, but, combined with dynamic conformational fluctuations of the protein in solution, could allow substrates and products to diffuse to the active site. A third access to the internal cavity, closed in the static protein structure, rests on the other side of the cavity relative to the active site, covered by the C-terminal loop.
While it is certain that the dithiolate bridge comes from HydE, the exact substrates and products of HydE are unknown. HydE belongs, like HydG, to the radical SAM superfamily of enzymes, which perform a homolytic cleavage of the C5 -Sδ bond of SAM at a [4Fe-4S] cluster, to yield a reactive 5 -deoxyadenosyl radical species (5 -dA•), used to usually extract a hydrogen atom from a substrate, thus generating a highly reactive carbon centered radical which is used in further synthetic steps. At variance with the general mechanism, it has been shown that HydE can react directly on a sulfur atom to form a C-S bond without passing through the hydrogen abstraction [41]. However, without knowledge of the actual substrate, it is unclear if this unusual reaction is part of the physiological mechanism involved in the maturation process. Nevertheless, it has been suggested that the substrate of HydE contains a thiol functional group [38]. Additionally, the SAM chemistry is performed on the main cluster, but the role of the secondary, less conserved, cluster in the function of the enzyme is still unknown.
HydG
HydG has been well-characterized, its crystal structure has been solved for different organisms and its function has been recently established in detail. HydG, belonging as HydE to the radical-SAM superfamily, is a bifunctional enzyme performing the synthesis of a Fe-containing complex with CO and CN − ligands that acts as a synthon of the [2Fe] H cluster.
The structure of HydG has been solved for two organisms, Carboxydothermus hydrogenoformans (pdb.id 4RTB) [42] and Thermoanaerobacter italicus (pdb.id 4WCX) [43], the latter shown in Figure 3. HydG, similarly to HydE, has a distorted TIM-barrel fold, 8 α-helices and 8 β-strands (shown in dark pink and green, respectively, in Figure 3), with additional helical domains at the N-terminus and C-terminus (about 80 amino acids long, shown in pale pink and dark blue, respectively, in Figure 3). It has a large internal cavity at the edge of which two distinct active sites, both iron-sulfur clusters, are present. The first is a [4Fe-4S] cluster with the conserved structural motif of a radical-SAM active site, and it is located at the top of the cavity of the TIM-barrel fold. The second site is a peculiar [4Fe-4S] cluster bridged by a µ 2 sulfide ion (or a non-proteic Cys) to a labile dangling Fe atom, and it is located at the bottom of the TIM-barrel cavity about 2.4 nm away from the first cluster, bound to the additional helical domain at the C-terminus. The second cluster is the site at which a Fe(CO) 2 CN synthon is assembled (see the next paragraph). The internal cavity of HydG is connected to the protein surface by a channel through which the products and substrates are thought to diffuse, shaded in light blue in Figure 3. The opening/closing of the channel located at the top of the cavity, close to the SAM active site, is thought to be governed by a loop region with a conserved Arg residue that partakes in Tyr binding, and is analogous to Streptomyces actuosus tryptophan lyase [42,43]. synthon is assembled (see the next paragraph). The internal cavity of HydG is connected to the protein surface by a channel through which the products and substrates are thought to diffuse, shaded in light blue in Figure 3. The opening/closing of the channel located at the top of the cavity, close to the SAM active site, is thought to be governed by a loop region with a conserved Arg residue that partakes in Tyr binding, and is analogous to Streptomyces actuosus tryptophan lyase [42,43]. HydG has long been recognized to provide the CO and CN − ligands of the [2Fe] H cluster. Recently, mostly through a combination of isotopic labeling and pulsed EPR experiments the role of HydG in the maturation pathway has been further expanded [41,[44][45][46][47]: it has been shown to synthesize the synthon of the [2Fe] H cluster, a low-spin iron complex, Fe(CO) 2 (CN)Cys. The assembly of the synthon is a multi-step process involving both active sites of the enzyme. The first active site utilizes the radical-SAM mechanism similarly to HydE (see above) to cleave a tyrosine into p-cresol, one molecule of CO, and one of CN − via a dehydroglycine intermediate. p-cresol migrates out of the protein cavity following the opening of the active site, while the other products migrate to the bottom of the cavity at the second active site. The second active site assembles the synthon: the dangling Fe atom, initially coordinated by a free cysteine, water, and histidine from the backbone, is coordinated by two CO and one CN − produced at the first active site while the second CN − cleaves the formed synthon from the [4Fe-4S] cluster. Note that in the proposed mechanism the stoichiometry of the overall formation of the synthon involves the conversion of two molecules of tyrosine at the first site to produce all the necessary ligands.
HydF
HydF holds a central role in the maturation pathway, but while its homodimeric structure has been crystallized, and its dual role, as a scaffold upon which the [2Fe] H cluster is assembled and carrier of the cluster into the apo-HydA, is universally recognized, a detailed knowledge of its mechanisms of action is still missing.
The structure of a recombinant HydF from Thermotoga neapolitana (pdb.id 3QQ5) has been solved without the [4Fe-4S] cluster [48], which is known to be part of the holo-protein as derived by EPR of the protein in solution-state [37,[49][50][51][52][53][54][55], and more recently complete with the cluster from Thermosipho melaniensis (pdb.id 5KH0, used for Figure 4) and from T. maritima (pdb.id 5LAD) [56]. HydF is a homodimeric protein with three domains per monomer. The N-terminus holds the GTPase domain, the central domain is the dimerization domain, and the C-terminus holds the [4Fe-4S] cluster-binding domain. While HydE and HydG are globular, the homodimeric structure of HydF adopts an open fold with a skewed inverted V shape, with the two GTPase domains located at the outermost part of the dimer and twisting away from the plane formed by the other two domains (as shown in Figure 4). This fold creates a large open "cavity" just below the dimerization interface with the active [4Fe-4S] cluster site of each monomer in its center. The cluster is solvent-exposed as befits an active site that needs to interact with several different proteins, as shown in Figure 4. change from a β-sheet to a structured loop region with specific interactions with K + and Mg 2+ ions mediated by conserved Asn residues [58]. This structural change is highly conserved, and likely adopted by HydF as well. On the other hand, the switch 2 region, which in HydF comprises a loop ending in an α-helix, has a high variability among the proteins belonging to different species and thus the structure adopted by HydF in the presence of GTP is not easily predicted starting from that resolved in the absence of GTP. Based on these considerations, we here present a model of the whole protein in the absence of GTP constructed with the UCSF Chimera package [59]. The model is based on the structure of HydF from T. melaniensis, pdb.id 5KH0, on top of which the two switch regions have been modeled. The switch 2 region was taken from the structure from HydF from T. neapolitana (pdb.id 3QQ5) where it is resolved. The switch 1 region was taken from the GDP-bound soluble Nterminal domain of FeoB from Streptococcus thermophilus (pdb.id 3LX8), a membrane protein that imports Fe 2+ [60], taken as the reference structure of a K + -activated GTPase. The two reconstructed regions are highlighted in Figure 4 (bottom): switch 1 (residues 31-46) in purple and switch 2 (residues 68-86) in dark orange. To highlight the homodimeric assembly of the protein only one monomer has been colored according to secondary structure: in magenta the α-helices, in green the β-strands, the [4Fe-4S] cluster is in sphere representation. Bottom, reconstruction of the whole protein structure in the absence of GTP based on HydF from T. melaniensis (see text) including the missing amino-acid stretches. The left monomer has the reconstructed stretches colored in purple (switch 1 region) and dark orange (switch 2 region). The right monomer has been colored to highlight the three protein domains: GTPase domain in orange, dimerization domain in blue, and cluster-binding domain in green.
GTPase Domain
The GTPase domain at the N-terminus (orange in Figure 4), is the least resolved in the crystal structures, it has high B factors, and is not stabilized in the crystal by direct contact with other molecules [56]. Its fold is similar to that of other GTPases: six β-strands, five parallel and one anti-parallel, assemble into a large sheet, with three α-helices flanking this sheet on one side and two α-helices on the other. Recently, through sequence and structure analysis, it has been shown that the GTPase domain has a high homology to analogous domains of other small K + -dependent GTPases, and as such it should act as a molecular switch triggering a conformational change [57]. Two regions have been identified as switch regions: switch 1 (residues 31-46, located directly above the nucleotide binding site) not solved in any crystal structure, and switch 2 (residues 68-86), solved only for HydF from T. neapolitana. Switch 1 is the most conserved in function and sequence among different K + -dependent GTPases. Upon GTP binding, the switch 1 region of K + -dependent GTPases exhibits a change from a β-sheet to a structured loop region with specific interactions with K + and Mg 2+ ions mediated by conserved Asn residues [58]. This structural change is highly conserved, and likely adopted by HydF as well. On the other hand, the switch 2 region, which in HydF comprises a loop ending in an α-helix, has a high variability among the proteins belonging to different species and thus the structure adopted by HydF in the presence of GTP is not easily predicted starting from that resolved in the absence of GTP. Based on these considerations, we here present a model of the whole protein in the absence of GTP constructed with the UCSF Chimera package [59]. The model is based on the structure of HydF from T. melaniensis, pdb.id 5KH0, on top of which the two switch regions have been modeled. The switch 2 region was taken from the structure from HydF from T. neapolitana (pdb.id 3QQ5) where it is resolved. The switch 1 region was taken from the GDP-bound soluble N-terminal domain of FeoB from Streptococcus thermophilus (pdb.id 3LX8), a membrane protein that imports Fe 2+ [60], taken as the reference structure of a K + -activated GTPase. The two reconstructed regions are highlighted in Figure 4 (bottom): switch 1 (residues 31-46) in purple and switch 2 (residues 68-86) in dark orange.
Dimerization Domain
The second domain in the sequence is responsible for the formation of the HydF dimer (blue in Figure 4). An extended stretch of residues, about thirteen amino-acids long, connects the dimerization to the GTPase domain. The domain is composed of four parallel β-strands and three α-helices. The four-stranded parallel β-sheets of each monomer are coupled in an antiparallel way to form a continuous eight-stranded β-sheet. Additional stabilization comes from the interactions between the neighboring α-helices and the loop regions, to a degree that depends on the species. Note that, so far, it is not clear why HydF needs to be dimeric to partake in cluster maturation, since a single [4Fe-4S] cluster is potentially sufficient to anchor the [2Fe] H cluster. On the other hand, a close look at the HydF structure indicates that the total buried surface due to the dimerization is large enough to support the existence of a stable physiological dimer, which assumes a sort of left-handed helical shape leaving both the putative FeS cluster and GTP-binding sites exposed to the solvent and giving a large protein surface for contacts with possible partners, such as the other two maturases and/or the apo-[FeFe]-hydrogenase. Thus, the dimer could be essential to provide the central open cavity to bind alternatively HydE, HydG, or the domain of HydA containing the H-cluster. This issue will be discussed in greater detail below.
Cluster Binding Domain
The C-terminal domain hosts the putative enzyme active site, where the [4Fe-4S] cluster is predicted to act as an anchor for the [2Fe] H assembly and delivery (green in Figure 4). The domain is composed of four β-strands and five α-helices arranged in a complex way to bring the three highly conserved Cys residues belonging to the highly conserved iron-sulfur cluster-binding motif (CxHx46-53HCxxC) spatially close [48,56]. Site-specific mutagenesis analysis coupled to EPR spectroscopy have shown that while cysteine residues are essential for the cluster assembly of HydF, the conserved histidines are not, and do not belong to the cluster coordination sphere [52]. However, the histidines are essential for the [2Fe] H cluster assembly, possibly partaking in this process via hydrogen bonding to the synthons. The crystal structure of HydF from T. melaniensis revealed that the fourth ligand of the cluster in the absence of the synthon comes from a highly conserved acidic residue (Glu in this organism) coordinating via the carboxylate [56]. HYSCORE spectroscopy studies had already shown that the fourth ligand is an oxygen in a variety of organisms and that this ligand is easily exchangeable, as expected since the fourth position is the one where the [2Fe] H cluster is anchored [52,54,55]; to date, the only known exception coming from the cluster of Clostridium acetobutylicum, where a nitrogen was found, however the possibility that the His-tag at the N-terminus of the recombinant protein, which is located spatially close to the cluster, interferes with the natural coordination must be taken into account. The putative binding pocket of the [2Fe] H cluster is located in a cleft at the interface between the second and third domains and is characterized by a positively charged surface. The loop that holds the fourth ligand likely changes conformation upon de-coordination of the acidic residue for the binding of the synthon, a model has been proposed comparing the two existing crystal structures, with and without the cluster [56].
In addition to the [4Fe-4S] cluster, there is evidence from EPR spectroscopy that as-isolated and chemically reconstituted HydF can also host a [2Fe-2S] cluster [61][62][63][64][65], which is otherwise lacking in all solved HydF crystal structures [48,56]. Given this discrepancy, it is still unclear whether the presence of a [2Fe-2S] cluster represents a physiological state, a state of partial cluster maturation, or simply arises from an incomplete reconstitution due to the in vitro conditions. Since there are no other binding motifs in HydF, and its relaxation properties suggest that the [2Fe-2S] cluster is located at least 2.5 nm away from the main cluster, it is likely bound in the same binding site in a different monomer, with a mixed occupancy of the sites in the two monomers, or a subset of proteins binding only a single type of cluster [62]. Finally, while there is a consensus about the role of the [4Fe-4S] cluster as the anchor for the [2Fe] H cluster, the role of [2Fe-2S] cluster is completely undefined.
The role of HydF as a scaffold and carrier in the maturation process is widely accepted based on both structural and functional studies [24,36,48,66], but several unclear points on its function are still present.
Tetrameric Form
HydF adopts in the crystal structures and in the purification process a tetrameric form (or more precisely forms a dimer of dimers), less abundant than the native dimeric form. The quaternary structure of the tetramer is uncertain since in the three available crystal structures the tetrameric assembly differs greatly and it is likely influenced by the crystal packing [56]. It is possible to separate the two forms in the recombinant protein via chromatographic methods, but the dimer and tetramer are in dynamic equilibrium [64]. A regulatory role has been attributed to the tetrameric form of HydF, acting as a switched-off state of the enzyme, since the tetrameric fractions are much less active in assembling the cluster [62].
[2Fe] H Cluster Precursor
In a recent work [65], the precursor to the [2Fe] H cluster present in HydF (called [2Fe] F cluster) has been suggested to be structurally different from the one present in HydA, based on FT-IR data (see Figure 1 for its structure). [2Fe] F has not been detected by EPR spectroscopy suggesting it is diamagnetic. The [2Fe] F cluster form is coordinatively saturated and bridged to the [4Fe-4S] cluster of HydF by a cyanide ligand. Relative to the [2Fe] H cluster, the [2Fe] F cluster would lack the bridging CO molecule and the relative orientation of the CO and CN-ligands relative to the dithiolate bridge is different, see Figure 1. Another difference that has been highlighted is the different redox state of the sub clusters, which would imply that the [4Fe-4S] cluster of HydF might be redox active and involved in the change of the redox state going from [2Fe] F to [2Fe] H , a step that might play a role in the transfer of the cluster to HydA.
Role of the GTPase Domain
Site-specific mutagenesis analysis revealed that the HydF GTPase consensus motifs are essential for the [FeFe]-hydrogenase maturation and activation [34,66]. NTPases are commonly involved in the assembly of metal cofactors of FeS proteins [67], mediating either the metal delivery to the active site or the transfer of the whole cluster to the target apoprotein. Experimental evidences excluded a role of HydF GTPase activity in the transfer of H-cluster precursor to the [FeFe]-hydrogenase [68], and an involvement of the GTPase domain in the interaction with the two other maturases has been suggested [66,68]. As reported above, the HydF crystal structures showed that this domain includes a flexible loop region which could rearrange upon GTP binding, thus facilitating the interaction with the maturation partners (see next paragraph). The changes in structure and backbone dynamics in the presence of GTP were investigated by EPR spectroscopy coupled to site-directed spin labeling (SDSL) and CD spectroscopy both in the full enzyme [57] and in the isolated domain [69]. EPR spectra in the presence of non-hydrolysable analogues and transition state mimics of GTP showed that GTP binding, and not its hydrolysis, triggers the switch. The binding of GTP causes a change in backbone dynamics diffused throughout the whole protein ( Figure 5): the largest changes are in the GTPase domain (S38, V71, R88), at the interface between the GTPase and cluster-binding domain (D340), or between the GTPase and dimerization domains (I175), while they are nonexistent in the core of dimerization domain proper (V261). All buried residues that were labeled do not change spectra upon GTP addition, suggesting that they do not get exposed irrespective of the domain (A89, T164, L341). DEER (Double Electron-Electron Resonance) experiments, measuring the distances between couples of spin labels, carried on double mutants of the isolated domain [69] and in the full enzyme [57] showed that neither the folding of the GTPase domain nor the dimeric quaternary structure are largely altered by GTP addition. The secondary structure changes in the whole enzyme evidenced by CD spectroscopy were modest, showing that the overall folding does not change. Taken together, all these results suggest that GTP binding induces local conformational changes in the domain and changes in protein backbone dynamics that radiate to the active site. These subtle changes in conformation and backbone dynamics likely play a key role in the regulation of the interaction with the other maturases and/or hydrogenase. Intriguingly, it has been reported that the presence of GTP affects the EPR spectral properties of the HydF [4Fe-4S] cluster [68], suggesting a communication between the GTPase domain and the FeS cluster domain. Thus, another possibility is that the GTP-induced conformational switches could instead modulate the coordination and orientation of the [4Fe-4S] cluster, switching from the four-coordination state with the carboxylate bound to a three-coordination states ready to bind the [2Fe] H precursors. Further work is needed to have more information on the actual mechanism.
between the GTPase domain and the FeS cluster domain. Thus, another possibility is that the GTPinduced conformational switches could instead modulate the coordination and orientation of the [4Fe-4S] cluster, switching from the four-coordination state with the carboxylate bound to a threecoordination states ready to bind the [2Fe]H precursors. Further work is needed to have more information on the actual mechanism.
The Overall Process
The function of the individual maturases has been the object of extended work, and in the last few years the knowledge of HydG and HydF proteins has significantly progressed. Quite lacking, on the contrary, is the knowledge of the interactions between the different maturases, the sequence of individual steps, and both the stoichiometry and the regulatory mechanisms of the overall process. Due to the multistep nature of the molecular pathway leading to the [FeFe]-hydrogenase maturation described above, a close and coordinate network of protein interactions between several partners must be achieved. The structural features and the dynamic behavior of HydF as scaffold and carrier assign to this protein a key role along the entire [FeFe]-hydrogenase maturation pathway and indicate
The Overall Process
The function of the individual maturases has been the object of extended work, and in the last few years the knowledge of HydG and HydF proteins has significantly progressed. Quite lacking, on the contrary, is the knowledge of the interactions between the different maturases, the sequence of individual steps, and both the stoichiometry and the regulatory mechanisms of the overall process. Due to the multistep nature of the molecular pathway leading to the [FeFe]-hydrogenase maturation described above, a close and coordinate network of protein interactions between several partners must be achieved. The structural features and the dynamic behavior of HydF as scaffold and carrier assign to this protein a key role along the entire [FeFe]-hydrogenase maturation pathway and indicate its capability to establish functional interaction with all the players of this process. These binding events were at first inferred from co-purification of HydE and HydG with HydF [36] and then confirmed and quantified in vitro through a combination of Surface Plasmon Resonance and co-purification experiments using recombinant proteins from C. acetobutylicum [66]. The dissociation constants of HydE and HydG interacting with HydF have been determined both in the absence and in the presence of a non-hydrolysable GTP analogue. The study showed that HydE has a ten times higher affinity for HydF than HydG, both with and without GTP, and that HydG cannot interact with HydF if HydE is bound to it. This suggests that the interactions of HydE and HydG with the HydF scaffold are distinct events occurring in a precise functional order and would be fully consistent with the model proposed below. Nevertheless, the dissociation constants are relatively high, implying that the interaction of HydF with the other maturases is not very strong, as expected for a protein that acts as a scaffold for up to three other proteins (HydG, HydE, HydA). The role of GTP binding is less clear: while both proteins have slightly higher affinity for HydF when GTP is present, the difference is less than an order of magnitude. Together with the other assays used in the work, the authors suggested that HydE and HydG bind separately to HydF and not cooperatively, thereby ruling out the possibility of a ternary complex [66]. It is still unknown, however, if there is an interaction between HydG and HydE occurring before either interacts with HydF.
Since no direct evidence of the role of the cavity formed by the dimeric structure of HydF as the protein-protein interaction interface has been obtained so far, we used rigid-body molecular docking to test this hypothesis, which is relevant for the discussion of the overall maturation pathway. To perform the docking procedure, we docked together proteins from different organisms since there is no common organism for which all structures have been obtained. Note that performing the docking simulations of proteins from different organisms is meaningful since it has been shown that mixing maturases and HydA of different species still yields a functional hydrogenase [70]. For HydF, we used the reconstructed structure from T. melaniensis described above. Then, we chose the structures of the maturases for which the primary sequence had the highest homology with HydF from T. melaniensis: for HydE, the structure from T. maritima (pdb.id 3CIW) and for HydG, the structure from T. italicus (pdb.id 4WCX). For HydA, we chose the structure from C. reinhardtii (pdb.id 3LX4), since it has the smallest complete hydrogenase domain of the available structures. The soft rigid-body docking procedure, adapted from [71], was performed using the webservers of both PatchDock [72] (in tandem FireDock [73]) and ZDOCK [74]. The full results of these simulations will be further refined and presented in a future work, we here report the preliminary results of our analysis in Figure 6, where we chose three docking poses that show that indeed HydE, HydG, and HydA are all able to dock in the "cavity" formed by the dimer of HydF. However, these are rigid body docking simulations, dynamics are not accounted for, and some physiologically relevant docking poses, resulting from different conformations of flexible structural elements, might be lost. Additionally, we used the structure of HydF in the absence of GTP, and since the full structural changes induced by GTP binding are not known, the results might be very different in the GTP-bound state.
Based on the evidence gathered so far, we propose two speculative models of the overall process that are schematically shown in Figure 7. These models provide for a central role of HydF that receives all parts of the cluster and assembles them, with separate binding events of HydG and HydE. As shown in Figure 1, the overall maturation of the [2Fe] H cluster implies a stoichiometry for the maturases that depends on the protein: HydG needs four turnovers at the first active site and two at the second site to produce the two synthons necessary for the complete cluster, while only a single turnover is expected to be necessary for the formation of the dithiolate bridge by HydE. It seems unlikely that the reactive dithiolate bridge is delivered to HydF before a synthon is bound, therefore it is reasonable that the first step ( Figure 7A) always involves the binding of HydG to HydF and the delivery of a synthon. The synthon could easily bind to the [4Fe-4S] cluster of HydF via the cyanide ligand like in the complete [2Fe] F structure. It has to be noted that the only access to the inner cavity of HydG is the single channel on top of the cavity, where both substrate and products are thought to pass, and therefore the access to the active site of HydG would be sterically impossible when it is bound to HydF. Then, the synthesis and delivery of the second synthon imply a dissociation and re-association of HydG. Following the binding of the first synthon two possibilities exist: (1) HydE delivers the dithiolate bridge ( Figure 7B) before a second HydG comes to deliver the second synthon to the partially formed [2Fe] F cluster ( Figure 7C); and (2) HydG binds to deliver the second synthon ( Figure 7D) before HydE comes in to clip the two synthons together via the dithiolate bridge ( Figure 7E). In the latter case, the second synthon could either be attached to the to the first one, implying a rearrangement of the ligands, or it could be delivered to the empty [4Fe-4S] cluster of the other HydF monomer and then coupled by HydE. The presence of two individual synthons in the two monomers of HydF is attractive, since it would provide another justification for its homo-dimeric structure besides the presence of the central cavity where the other maturases could dock. In both cases additional regulatory mechanisms, i.e., GTP binding, changes in protein backbone dynamics, and/or a conformational change of the cluster region following the binding of the first synthon, must modulate the binding affinity of the other maturases to HydF.
A further alternative mechanism would provide for HydG and HydE to interact first, assembling the whole [2Fe] sub-cluster possibly making use of the secondary cluster binding motif of HydE. Then, HydE would deliver the complete cluster to HydF. We performed docking simulations on a possible HydG-HydE complex, but the results do not show any preferential binding interface between the two proteins. However, this mechanism cannot be excluded, and experiments on HydE-HydG binding affinity would be needed to verify this hypothesis. Based on the evidence gathered so far, we propose two speculative models of the overall process that are schematically shown in Figure 7. These models provide for a central role of HydF that receives all parts of the cluster and assembles them, with separate binding events of HydG and HydE. As shown in Figure 1, the overall maturation of the [2Fe]H cluster implies a stoichiometry for the maturases that depends on the protein: HydG needs four turnovers at the first active site and two at the second site to produce the two synthons necessary for the complete cluster, while only a single turnover is expected to be necessary for the formation of the dithiolate bridge by HydE. It seems unlikely that the reactive dithiolate bridge is delivered to HydF before a synthon is bound, therefore it is reasonable that the first step ( Figure 7A) always involves the binding of HydG to HydF and the delivery of a synthon. The synthon could easily bind to the [4Fe-4S] cluster of HydF via the cyanide ligand like in the complete [2Fe]F structure. It has to be noted that the only access to the inner cavity of HydG is the single channel on top of the cavity, where both substrate and products are thought to pass, and therefore the access to the active site of HydG would be sterically impossible when it is A further alternative mechanism would provide for HydG and HydE to interact first, assembling the whole [2Fe] sub-cluster possibly making use of the secondary cluster binding motif of HydE. Then, HydE would deliver the complete cluster to HydF. We performed docking simulations on a possible HydG-HydE complex, but the results do not show any preferential binding interface between the two proteins. However, this mechanism cannot be excluded, and experiments on HydE-HydG binding affinity would be needed to verify this hypothesis.
Funding: This research was funded by the PRAT project CPDA149178/14 from University of Padova to P.C., CARIPARO Foundation M3PC project to D.C., and CARIPARO Starting Grants to M.B.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
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2018-11-01T06:46:16.513Z
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2018-10-01T00:00:00.000
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{
"year": 2018,
"sha1": "2aea93460ee24b4e3ef40a3f89060f625299bf95",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.3390/ijms19103118",
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"Biology"
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"Chemistry",
"Medicine"
]
}
|
126114792
|
pes2o/s2orc
|
v3-fos-license
|
Fusion of neutron-rich oxygen nuclei
Measurement of the fusion excitation function for 18O + 12C and 19O + 12C is described. The fusion cross-section is extracted through the direct measurement of evaporation residues resulting from the fusion process. At near barrier energies, the single additional neutron present in 19O results in an enhancement in the fusion cross-section by a factor of over three as compared to 18O.
Introduction
Measuring the fusion excitation function for an isotopic chain of projectile nuclei presents an unique opportunity to examine the character of neutron-rich matter. For a given element, with increasing neutron number the neutron density distribution usually extends further out while the proton distribution remains largely unaffected. Hence, the repulsive Coulomb potential is largely unchanged while the attractive nuclear potential changes. As fusion at near barrier energies is sensitive to the interplay between the repulsive Coulomb and attractive nuclear potentials, the comparison of the fusion excitation function for isotopically related projectiles provides a sensitive probe of the change in the attractive nuclear potential. This change in the attractive potential can be related to changes in both the structure and dynamics of the neutron density distribution as the number of neutrons increases. Presented in Fig. 1 are the neutron density distributions for oxygen isotopes calculated within the context of a relativistic mean field theory [1,2]. As expected, with increasing neutron number the tail of the density distribution extends further out. It is perhaps surprising however that the tail of the neutron density distribution for 22 O is so close to that of the drip-line nucleus 24 O. This observation is particularly noteworthy as beams of 20,21 O are presently available at GANIL and a beam of 22 O is anticipated in the near future. In this paper, we describe the first step towards the systematic measurement of the fusion excitation functions of the oxygen isotopes namely fusion in 18 O+ 12 C and 19 O+ 12 C.
An extended neutron distribution could impact fusion both through its static spatial extent as well as through its dynamics (e.g. polarization effects). The influence of the static contribution can be estimated by using a onedimensional barrier penetration model such as the Sao-EPJ Web of Conferences 163, 00013 (2017) DOI: 10.1051/epjconf/201716300013 FUSION17 Paulo model [3]. For the density distributions presented in Fig. 1, we have calculated the relative fusion crosssections for several oxygen isotopes and present the results in Fig. 2. Evident in the figure is the fact that at all energies as the neutron-richness increases the fusion cross-section increases. At energies well above the barrier (E c.m. ∼ 8 MeV) this enhancement is relatively constant and can be considered geometric. Near and below the barrier however, the enhancement increases rapidly with decreasing incident energy. In this energy domain the enhancement reflects the increased importance of the nuclear potential due to the neutron-skin. It should be emphasized that as this is a purely static calculation, the inclusion of fusion dynamics could result in an enhancement larger than that depicted in Fig. 2.
In order to measure the fusion excitation function for 18 O + 12 C and 19 O + 12 C two separate experiments were performed at the John D. Fox accelerator laboratory at Florida State University. In the initial experiment we measured the fusion excitation function for 18 O + 12 C, extending the measured cross-section down to the sub 1 mb level, a factor of 30 lower than previously measured. Having established the technique, we subsequently measured the fusion excitation function for 19 O + 12 C. The experimental details of both measurements are summarized below.
Experimental setup
The experimental setup for this experiment consisted of two ExB microchannel plate detectors and two annular silicon detectors as depicted in Fig. 3. The beam first passes through a ExB microchannel plate detector, designated MCP US , situated approximately 1.3m upstream of the target position. For each ion traversing this detector a fast timing signal is generated. The beam subsequently encounters a second ExB microchannel plate detector, designated MCP TGT . The 100 µg/cm 2 thick carbon foil of MCP TGT is dual function. Not only does it serve as a secondary emission for the detector, but it also serves as the target for the experiment. The coincidence of these two ExB MCP detectors with the appropriate time-of-flight provided the incident beam count. The typical intensity of the 18 O beam incident on the target was ∼2x10 5 ions/s.
Measuring the fusion products
Fusion of a 18 O nucleus in the beam together with a 12 C nucleus in the target foil results in the production of an excited 30 Si nucleus. For collisions near the Coulomb barrier the excitation of the fusion product is relatively modest, E * ≈ 35 MeV. De-excitation of this fusion product by evaporation of a few neutrons, protons, and α particles results in an evaporation residue (ER). Statistical model calculations [4] indicate that for a 30 Si compound nucleus, the nuclei 29 Si, 28 Si, 28 Al, 27 Al, and 25 Mg account for the bulk of the ERs. Emission of the light particles deflects the ER from the beam direction allowing their detection and identification in two annular silicon detectors, designated T2 and T3, that are situated downstream of the MCP TGT . These detectors subtend the angular range 3.5 • < θ lab < 25 • allowing detection of the majority of the ERs produced [5]. By measuring the time-of-flight of particles between the MCP TGT detector and the silicon detectors [6] together with the energy deposit in the Si detector, evaporation residues are distinguished from scattered beam, as well as emitted light particles. By utilizing the measured energy deposit and time-of-flight, the mass of the ion can be calculated providing clear separation of ERs from the incident beam [7].
Fusion excitation function for 18 O + 12 C
The fusion cross-section is extracted by summing the total number of evaporation residues observed. Comparison of this yield with the number of incident 18 O ions while accounting for the target thickness and the geometric efficiency of the experimental setup yields the absolute fusion cross-section [7]. The measured excitation function is displayed in Fig. 4a together with previously published results [9][10][11]. Vertical error bars on the new data reflect both the statistical uncertainties as well as a 2% systematic error associated with the analysis. Horizontal error bars represent the uncertainty in whether the fusion occurs at the front or back of the target foil. While prior measurements using the direct measurement of evaporation residues only measured the fusion cross-section down to the 25 mb level [9], in this work the fusion cross-section is measured down to the 820 µb level, a factor of approximately 30 lower in cross-section. In the energy region where the present data overlaps with published data, overall agreement of the cross-sections is good, close to the statistical uncertainties. This overall agreement indicates both that our approach in extracting the fusion crosssection is sound and that there are no significant uncertainties in the values of the target thickness or detector efficiency. Closer comparison of the present dataset with the data of Ref. [9] indicates that the presently measured cross-sections are approximately 3-5% lower for E c.m. ≥ 10 MeV. This is within the statistical uncertainties of the data reported by Eyal et al. In addition, the present data are higher in their statistical quality.
We have compared the experimental fusion excitation function with the predictions of a microscopic model. Over the past several years, the density constrained TDHF (DC-TDHF) method for calculating heavy-ion potentials [12] has been employed to calculate heavy-ion fusion cross-sections with remarkable success [13,14]. While most applications have been for systems involving heavy nuclei, recently the theory was used to study above and below barrier fusion cross-sections for lighter systems, specifically for reactions involving various isotopes of O+O and O+C [15,16]. One general characteristic of TDHF and DC-TDHF calculations for light systems is that the fusion cross-section at energies well above the barrier are usually overestimated [17,18], whereas an excellent agreement is found for sub-barrier cross-sections [15]. neling probability for the experimental data as compared to the theoretical calculations. This enhanced tunneling probability can be associated with a narrower, lower barrier. The underlying reason that the barrier determined from the experimental data is weaker than in the model is presently unclear.
Producing and Chararacterizing a 19 O beam
A beam of 18 O ions at an energy of 80.7 MeV was used to bombard a deuterium gas cell at a pressure of 350 torr to produce the 19 O beam. To increase the gas density, the gas cell was cooled to a temperature of 77 K. Ions of 19 O produced via a (d,p) reaction were separated from the incident beam by the electromagnetic spectrometer RESO-LUT [20]. Despite the rejection of most of the unreacted beam by RESOLUT, the beam exiting the spectrometer consisted of both 19 O and 18 O ions. It was therefore necessary to identify each ion incident on the target. By accomplishing this it was possible to simultaneously measure the fusion excitation function for 18 O + 12 C and 19 O + 12 C which provided an important consistency check. Comparison of the 18 O + 12 C with the prior high statistics measurement of 18 O + 12 C demonstrated that there were no systematic differences between the two measurements. This dual measurement thus provided confidence that any observed fusion enhancement was a robust signal.
Experimental setup
Although the experimental setup for the 19 O beam was largely the same as in the prior experiment, a few changes were made to handle the radioactive beam. In order to identify beam particles, the energy deposit (∆E) and timeof-flight (TOF) of each particle was measured prior to the target. After exiting RESOLUT particles traversed a thin foil (0.5 µm thick aluminized mylar) which served as the electron emission foil for an MCP detector. Approximately 3.5 m downstream of this thin foil the oxygen ions passed through a compact ionization detector (CID) depositing an energy, ∆E. CID served two roles: to help identify the ion as well as to reduce its energy. While the production of 19 O is favored at higher energies, the measurement of the fusion cross-section at near barrier energies requires reducing the energy of the radioactive ion after its production. To perform the excitation function measurement, the energy of the incident beam was decreased by adjusting the gas pressure in CID. Upon exiting CID the ions were incident on a 105 µg/cm 2 carbon foil. This carbon foil, as in the prior experiment, served both as a secondary electron emission foil for the target microchannel plate detector (MCP TGT ) and as the target for the fusion experiment [8]. To measure the energy distribution of 19 O and 18 O ions incident on the target, a surface barrier, silicon detector was periodically inserted into the beam path just prior to the target.
The timing signals from both microchannel plate detectors together with the energy deposit in the ionization chamber allowed identification of ions in the beam through measurement of the ∆E-TOF. Ions of 19 Figure 5. Comparison of the measured cross-section for 18 O + 12 C and 19 O + 12 C. The cross-sections for the 18 O reaction have been scaled by a factor of two for clarity.
Fusion excitation function for 19 O + 12 C
The same general trend is observed for both of the excitation functions depicted in Fig. 5a. With decreasing incident energy the cross-section decreases as expected for a barrier controlled process. At essentially all energies measured the 19 O data exhibits a larger fusion cross-section as compared to the 18 O data. 18 O + 12 C 7.66 ± 0.10 7.39 ± 0.11 2.90 ± 0.18 19 O + 12 C 7.73 ± 0.72 8.10 ± 0.47 6.38 ± 1.00 To quantitatively examine the differences in the two excitation functions we have fit the excitation functions with the Wong formalism of penetration of an inverted parabolic barrier [19]. The fit of the high resolution 18 O data is indicated as the solid black line in Fig. 5a. The solid red curve in Fig. 5a depicts the fit of the 19 Table 1. Since the charge density distribution is essentially unchanged, it is unsurprising that the barrier height, V C , remains essentially the same for both of the reactions examined. Moreover, as expected with increasing neutron number an increase in R C is observed. This increase in the radius can be viewed by calculating the quantity R C /A 1/3 where A is the mass number of the compound nucleus. This quantity has a value of 2.38 for the 18 O induced reaction, while it is 2.58 for the 19 O induced reaction. The most significant change in the fit parameters is a substantial increase in the magnitude of ω for the 19 O case corresponding to a narrower barrier, reflecting an increase in the attractive nuclear potential.
Depicted in Fig. 5b as the solid (red) line is the dependence of the measured ratio of σ( 19 O)/σ( 18 O) on E c.m. . At energies well above the barrier σ( 19 O)/σ( 18 O) is essentially flat at a value of ≈ 1.2. As one approaches the barrier it rapidly increases to a value of approximately 3.5. Hence, the addition of a single additional neutron in 19 O as compared to 18 O results in a dramatic enhancement in the fusion cross-section at sub-barrier energies.
Summary
We have measured the fusion excitation functions for 18 O + 12 C and 19 O + 12 C using low intensity beams. Comparison of these excitation functions indicates a significant enhancement at near barrier energies for the neutron-rich projectile. The addition of a single neutron increases the fusion cross-section by more than a factor of three at the lowest energy measured. This enhancement may reflect the increased role of neutron transfer or coupling to collective degrees of freedom. These measurements represent the first step in the measurement of the fusion excitation function for an isotopic chain of oxygen nuclei. Acquiring a systematic, high quality dataset of this type, coupled with microscopic calculations of the fusion process has considerable promise in elucidating the nature of neutron-rich nuclear matter.
Acknowledgements
The support of the staff at Florida State University's John D. Fox accelerator in providing the 18
|
2019-04-22T13:08:23.416Z
|
2017-01-01T00:00:00.000
|
{
"year": 2017,
"sha1": "b3ff98dc5feda796abd5c950738018942488a8d7",
"oa_license": "CCBY",
"oa_url": "https://www.epj-conferences.org/articles/epjconf/pdf/2017/32/epjconf_fusion2017_00013.pdf",
"oa_status": "GOLD",
"pdf_src": "Anansi",
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"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Physics"
]
}
|
22463309
|
pes2o/s2orc
|
v3-fos-license
|
Structural Characterization and Association of Ovine Dickkopf-1 Gene with Wool Production and Quality Traits in Chinese Merino
Dickkopf-1 (DKK1) is an inhibitor of canonical Wnt signaling pathway and regulates hair follicle morphogenesis and cycling. To investigate the potential involvement of DKK1 in wool production and quality traits, we characterized the genomic structure of ovine DKK1, performed polymorphism detection and association analysis of ovine DKK1 with wool production and quality traits in Chinese Merino. Our results showed that ovine DKK1 consists of four exons and three introns, which encodes a protein of 262 amino acids. The coding sequence of ovine DKK1 and its deduced amino acid sequence were highly conserved in mammals. Eleven single nucleotide polymorphisms (SNPs) were identified within the ovine DKK1 genomic region. Gene-wide association analysis showed that SNP5 was significantly associated with mean fiber diameter (MFD) in the B (selected for long wool fiber and high-quality wool), PW (selected for high reproductive capacity, high clean wool yield and high-quality wool) and U (selected for long wool fiber with good uniformity, high wool yield and lower fiber diameter) strains (p < 4.55 × 10−3 = 0.05/11). Single Nucleotide Polymorphisms wide association analysis showed that SNP8 was significantly associated with MFD in A strain and fleece weight in A (selected for large body size), PM (selected for large body size, high reproductive capacity and high meat yield) and SF (selected for mean fiber diameter less than 18 μm and wool fiber length between 5 and 9 cm) strains (p < 0.05), SNP9 was significantly associated with curvature in B and U strains (p < 0.05) and SNP10 was significantly associated with coefficient of variation of fiber diameter in A, PW and PM strains and standard deviation of fiber diameter in A and PM strains (p < 0.05). The haplotypes derived from these 11 identified SNPs were significantly associated with MFD (p < 0.05). In conclusion, our results suggest that DKK1 may be a major gene controlling wool production and quality traits, also the identified SNPs (SNPs5, 8, 9 and 10) might be used as potential molecular markers for improving sheep wool production and quality in sheep breeding.
Introduction
Wool is an important valuable economic product of Merino sheep and plays an important role in textile industry. Wool still is a vital source of income in sheep operation although the proportion of income derived from wool sales has decreased. In Xinjiang Uyghur Autonomous Region where Chinese Merino sheep are raised, wool generally accounts for 21% of gross flock income. The value of wool is determined by its intrinsic quality which includes a number of wool quality traits. From these traits, mean fiber diameter (MFD) is the most important wool characteristics when assessing value. The variation of mean fiber diameter, expressed as the standard deviation of the mean fiber diameter (FDSD) and coefficient of variation of the mean fiber diameter (CVFD), is also an important trait for wool processing. Less variation in fiber diameter is desirable to wool processor because of its better spinning quality. Wool production and quality traits are polygenic and several of genes have been identified to be associated with wool production and quality trait. For example, it has been shown that the Desmoglein 4 (DSG4) gene is associated with wool length and curvature [1], while the Beta-3 adrenergic receptor (ADRB3) gene is associated with wool mean staple strength and yield [2].
Dickkopf-1 (DKK1), a member of the dickkopf family, is a secreted canonical Wnt signaling inhibitor [3]. The canonical Wnt signaling pathway plays important roles in developmental processes, organogenesis, oncogenetic and self-renewal during tissue morphogenesis [4,5]. In the absence of the Wnt ligands, cytoplasmic β-catenin is phosphorylated and constantly degraded by the destruction complex formed by Axin, APC (WNT signaling pathway regulator), casein kinase 1 (CK1) and glycogen synthase kinase 3 beta (GSK-3β). In the presence of Wnt ligands, they bind to the Frizzled/LRP receptor complex at the cell surface. These receptors transduce a signal into the cells. As a consequence, the degradation of β-catenin is inhibited and β-catenin accumulates in the cytoplasm. The accumulated β-catenin travels to the nucleus to form complexes with the T cell factor/lymphoid enhancer binding factor (TCF/LEF) and activates Wnt target gene expression [6]. The canonical Wnt signaling regulates hair follicle morphogenesis [7], cycling [8] and hair follicle stem cell proliferation [9]. It has also been demonstrated that DKK1 regulates hair follicle density [10], terminal hair [11], hair follicle size [12] and hair follicle cycling [13,14] by inhibiting canonical Wnt signaling. The hair follicle size, density and cycling have an effect on the hair or wool length and fiber diameter [15][16][17]. Although it has been demonstrated that DKK1 regulates hair follicle morphogenesis and cycling, its effects on hair production and quality traits are unclear.
The aim of this work was to investigate the potential involvement of DKK1 in wool production and quality traits. We characterized the genomic structure of ovine DKK1 and performed single nucleotide polymorphism screening and association analysis of the single nucleotide polymorphisms (SNPs) and haplotypes of DKK1 with wool production and quality traits in Chinese Merino population.
Animals and Trait Measurements
A total of 743 ewes from Chinese Merino breed population (Xinjiang Junken type) comprising 181 superfine wool sheep (SF), 134 prolific meat sheep (PM), 138 prolific wool sheep (PW), 151 A strain, 103 B strain and 36 U strain were genotyped and phenotyped in this study. These six Chinese Merino strains were selected for different purposes. The SF strain was selected for mean fiber diameter less than 18 µm and wool fiber length between 5 and 9 cm. The PM strain was selected for large body size, high reproductive capacity and high meat yield. Its wool fiber length ranged between 9.30 and 12.11 cm, while mean fiber diameter ranged between 20.72 and 23.65 µm. The PW strain was selected for high reproductive capacity, high clean wool yield and high-quality wool. The reproduction rate of PM and PW was 182.4%, which is more than 60% higher compared to SF. The A strain was selected for large body size, while B strain was selected for long wool fiber and high-quality wool. The U strain was selected for long wool fiber with good uniformity, high wool yield and lower fiber diameter. Its mean fiber diameter ranged between 15.58 µm and 20.79 µm [18]. All animals were fed ad libitum with grazing diet and maintained under the same conditions of environment, feeding and management. Procedures involving animals and their care were conducted in conformity with the guidelines of National Institutes of Health guidelines [19] (NIH Publication No. 85-23, revised 1996) Wool sample were collected from ewes before pregnancy, which occurred in May 2009. All ewes were aged from 1 to 12 years and shorn at the same year. According to the guidelines of the China Fiber Inspection Bureau (CFIB) and International Wool Textile Organization (IWTO), the wool production and quality traits (MFD, FDSD, CVFD, wool fiber length (WFL), curvature and fleece weight (FW)) were measured in this study. The MFD, FDSD and CVFD were measured using an instrument called OFDA2000 (BSC Electronics, Ardross, Australia) which is recognized by the IWTO test method (47 and 57). Wool fiber length was measured in centimeters and reflected the relaxed length of the staple under no tension on the spine, above the last rib. A crimp is defined as the distance from one peak to the next in the wool staple. Curvature was measured by using the OFDA2000 (BSC Electronics). Fleece weight and the total sheared fleece were weighed [20].
Genomic DNA Isolation
Ear notch samples were collected and genomic DNA was extracted using the standard phenol-chloroform method [21], then stored for genotyping. DNA concentration and purity were measured using the NanoDrop 2000 spectrophotometer (Thermo Scientific, Irvine, CA, USA). An absorbance 260/280 ratio between 1.8 and 2.0 AU and an absorbance 260/230 ratio between 2.0 and 2.2 AU represent a high-quality DNA sample. Genomic DNA integrity was determined by 1% agarose gel electrophoresis using the intercalating agent GelRed™ (Biotium, Fremont, CA, USA) 10,000× with the Bromophenol Blue as carrier, then visualized under ultraviolet light (UV) and photographed.
RNA Isolation and Reverse Transcription
The Liver, testis and kidney tissues of the six strains of Chinese Merino sheep were collected (n = 18, three for each strain). The total RNAs from these samples were isolated using TRIzol reagent (Invitrogen, Rockville, MD, USA) according to the manufacturer's instructions. RNA concentration and purity were measured using the Nanodrop 2000 spectrophotometer (Thermo Scientific). Absorbance 260/280 ratio was used to assess the purity of the isolated total RNAs. The absorbance 260/280 ratio between 1.8 and 2.0 indicate good RNA purity. RNA integrity was determined by 1.2% formaldehyde denaturing gel electrophoresis. To eliminate genomic DNA (gDNA) contamination, all isolated RNAs were treated with RNase-free DNase I (Qiagen Inc., Hilden, Germany). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using Promega Improm-II reverse transcription System (Promega, Madison, WI, USA) following the manufacturer's instructions.
PCR Amplification
The synthesized cDNA was used as a template to amplify the entire coding sequence of ovine DKK1 by PCR using gene specific primer DKK1-F1 and DKK1-R1 (Table 1), which were designed according to the predicted cDNA sequence of ovine DKK1 (GenBank accession No. XM_012138945.2). The PCR amplification of DKK1 cDNA was performed in a 50 µL reaction volume containing 1 µL of Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co., Ltd., Nanjing, China), 1 µL cDNA, 25 µL of 2× Phanta Max buffer, 1 µL of 10 mM dNTP Mix, 2 µL of forward primer (10 µM), 2 µL of reverse primer (10 µM) and 18 µL of RNase-free water. The cycling protocol was 95 • C for To obtain the full-length ovine DKK1 genomic sequence, a total of five primer pairs, were designed to cover the entire genomic region of ovine DKK1 based on the caprine DKK1 genomic sequence (GenBank accession No. GQ480837) and the bovine genome sequence from the assembly of chromosome 26 reported in Genbank (accession number NM_001205544) and the Bos taurus genome sequence (http://genome.ucsc.edu). The 3 -terminus of each amplified fragment overlapped with the 5 -terminus of its adjacent amplified fragment. The primer sequences are listed in Table 1. The PCR amplification of the DKK1 genomic sequence was performed in a 50 µL reaction volume containing 1 µL of Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co., Ltd.), 100 ng ovine gDNA, 25 µL of 2× Phanta Max buffer, 1 µL of 10 mM dNTP Mix, 2 µL of forward primer (10 µM), 2 µL of reverse primer (10 µM) and RNase-free water to final volume of 50 µL. The genomic PCR conditions included: 95 • C for 3 min followed by 35 cycles at 95 • C for 15 s and 46.9 • C (52.0, 56.2, 56.2, 58.6 • C) for 15 s, 72 • C for 2 min and a final extension at 72 • C for 5 min. The products were analyzed by 1% agarose gel electrophoresis.
Sequencing and Sequence Analysis
PCR products of DKK1 cDNA and genomic fragments were purified using the Agarose Gel Extraction Kit according to the manufacturer's instructions (TIANGEN, Beijing, China) and cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA). The recombinant plasmids were extracted using PureLinkR Quick Plasmid Miniprep kit (Invitrogen) and sequenced by Invitrogen. The sequences were aligned using the Align X function of Vector NTI program (Informax, Rockville, MD, USA). The homologous DKK1 mRNA sequences of 16 different animal species used in this study were obtained from National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) database (Table S1). Homology analysis was performed using the Align Sequences Nucleotide BLAST utility at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Translation of the nucleotide sequences into the amino acids was performed using the DNAMAN program (Lynnon Corp., Quebec, Canada). Exon-intron boundaries were identified by alignment of the acquired Merino DKK1 genomic DNA sequence (GenBank accession No. JQ348893.1) and the acquired Merino DKK1 cDNA sequence using the Align Sequences Nucleotide BLAST at NCBI. The signal peptide was analyzed using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/). The conserved domains were analyzed using a conserved domain database (CDD) (http://www.ncbi.nlm.nih.gov/cdd/). Transcription factor binding sites (TFBS) were predicted using Mulan (https://mulan.dcode.org/). Core promoter was predicted using Promoter SCAN (http://www-bimas.cit.nih.gov/molbio/proscan/). The neighbor-joining phylogenetic tree was constructed using the Phydit program version 3.0 [22] based on genetic distances calculated with Kimura's two-parameter method [23].
Identification of Polymorphisms of the Ovine DKK1
To screen for DKK1 polymorphisms, a total of five primer pairs (DKK1-F2/R2-DKK1-F6/R6) were used to amplify DKK1 genomic region (Table 1) using the pooled gDNA as the template, which were from 60 Chinese Merino individuals as previously described [24]. The PCR products obtained from pooled genomic DNA sample were directly sequenced by Sanger sequencing and a SNP was ascertained by the presence of a double peak at the level of a single base in the chromatograms of sequencing of pooled PCR products [25].
Genotyping of the Ovine DKK1
A multiplexed SNP single base extension (SBE) assay was used for SNP genotyping. It was performed using a 384 well plate format on the Sequenom Mass ARRAY platform (Bioyong Technologies Inc., Beijing, China). The Mass ARRAY Assay Design 3.1 software (Sequenom, San Diego, CA, USA) was used to design amplification and allele-specific extension primers. The raw data files generated by Mass Array (Sequenom) were analyzed for the intensity peaks of calibrant to ascertain the quality of the data as previous described [26,27]. An overall call rate of greater than 95% was maintained. For every 96 samples (a quadrant of the Sequenom chip), four samples were duplicated and the call rates were checked for concordance. The calls in the negative control (no DNA) were also monitored in all the runs. The reproducibility of this study was 100%.
Statistical Analysis
Data are summarized as mean values for each parameter measured in each group. Correlation analysis between wool production and quality traits was subjected to the Pearson procedure of SPSS 22.0 (IBM, Armonk, NY, USA). Genotype and allelic frequencies at each SNP site were calculated, with the allele frequencies in subjects for each SNP evaluated for deviation from Hardy-Weinberg equilibrium and differences between groups using the χ 2 test using Statistical Analysis System (SAS Inst. Inc., Cary, NC, USA).
Haplotypes for each individual were obtained in SAS/GENETICS using the PROC HAPLOTYPE procedure. This procedure uses the Expectation Maximization (EM) algorithm to generate maximum likelihood estimates of the haplotype frequencies. Before analyzing the association between the identified SNPs and wool production and quality traits, we performed the data preprocessing: if the number of one genotype was fewer than 5% × the total number of samples, we removed the data for this genotype.
According to the characteristics of the Chinese Merino population, associations of DKK1 SNPs, haplotypes or allele substitution effect with wool production and quality traits were analyzed using mixed linear model procedure in SAS (SAS Inst. Inc.). The model was as below: where Y is the phenotypic value for each individual, µ is the population mean, A is the continuous effect of the age, S is the random effect of the sire, L is the fixed effect of the line, G [L] is the effect of genotype nested within line and e is the random error; Data were subjected to the John's Macintosh Program 7.0 (JMP, SAS Inst. Inc.) which was used to examine the correlation between genotypes and haplotypes and continuous traits (MFD, FDSD, CVFD, WFL, curvature and FW) and to evaluate the least squares means. The genetic and phenotypic correlations between the wool production and quality traits were estimated using ASREML software [28], with line treated as a fixed effect. The bivariate model was used to calculate the genetic and phenotypic correlations.
The standard errors (SE) of correlations between genotypes and wool production and quality traits were approximated as described in the study of Falconer and Mackay [29]. The genetic model used for parameter estimations is described as follows: in which y is an n-dimensional vector of observed values for the traits, X is an n × p matrix of the fixed effects, β is a p-dimensional vector of the fixed effects, Z is an n × q matrix of the random effects, u is a q-dimensional vector of the random genetic effects and e is an n-dimensional vector of the random residual effects.
The random effects u and e were assumed to follow the normal distributions with mean 0, that is, Expectation [y] = Xβ. The variances of u and e were assumed to be Var(u) = Ag and Var(e) = Ir, respectively, in which A is the numerator relationship matrix of all animals in the sire file, g is the additive genetic variance for the single-variate and the additive genetic variance-covariance matrix between traits for the bivariate model analysis, I is the identity matrix of order equal to the number of animals with phenotypes and r is the residual variance for the single-variate and the variance-covariance matrix between residuals on the same animal when performing the bivariate model analysis, where residual covariance equal to 0 [30]. Significance was evaluated based on an SNP-wide and gene-wide type I error rate of 0.05. The values were considered significant at p < 0.05 based on SNP-wide and threshold gene-wide which is p < 4.55 × 10 −3 = 0.05/11 using Bonferroni correction.
Structural Characterization of Ovine DKK1
To investigate the potential involvement of the DKK1 in wool production and quality traits in Chinese Merino sheep, we first determined the full-length coding sequence and genomic structure of ovine DKK1. The cDNA sequence of ovine DKK1 was amplified by Real Time PCR (RT-PCR) from the pooled total RNA of liver, testis and kidney using the primer pair (DKK1-F1 and DKK1-R1) ( Table 1). Sequencing results showed that the amplified DKK1 cDNA fragment is 955 bp long. Further sequence analysis showed that this fragment contained the full-length coding region sequence (789 bp) of ovine DKK1 which encodes a protein of 262 amino acids. The entire coding sequence of DKK1 of Chinese Merino shared 100% nucleotide sequence identity with the recently published Texel sheep DKK1 mRNA sequence (GenBank accession No. XM_012138945.2). The coding sequence of the ovine DKK1 showed 98.10%, 93.61% and 83.96% nucleotide identity with those of caprine, bovine and human DKK1, respectively. The DKK1 deduced amino acid sequence alignment and protein domain analysis are shown in Figure 1. Analysis of the deduced amino acid sequence of ovine DKK1 using SignalP3.0 (http://www.cbs.dtu.dk/services/SignalP) identified a potential signal peptide sequence at its amino terminus positions 1-23, consistent with its role as a secreted protein. The conserved domain analysis showed that ovine DKK1 protein contained an N-terminal cysteine-rich domain and a C-terminal cysteine-rich domain at amino acid positions 87-136 and 188-252 respectively. These two cysteine-rich domains were conserved in six different animal species analyzed. The deduced ovine DKK1 protein showed high sequence similarity to caprine (98.85%), bovine (92.08%) and human (83.90%) DKK1 proteins (Table S2). The phylogenetic analysis based on amino acid sequence of DKK1 protein showed that that Merino sheep was closely clustered with goat and cattle while Human, Chimpanzee and Rhesus monkey formed another closely related group. In contrast, the Atlantic salmon was a distinct group compared with other species (Figure 2).
The genomic sequence of ovine DKK1 was amplified by PCR from the gDNA of Chinese Merino sheep using five different pairs of primer (Table 1) and 1966 bp (chr22:6663304-6665269. All PCR products were sequenced and assembled into the ovine DKK1 genomic sequence. The acquired ovine DKK1 genomic sequence was 7326 bp in length and has been deposited in GenBank (accession No. JQ348893).
Alignment of the acquired Merino DKK1 genomic sequence and its cDNA sequence revealed that Merino DKK1 is composed of three introns and four exons (Figure 3). The consensus sequences at the exon/intron boundaries were identified and all the boundaries conformed to the GT-AG rule. A putative polyA signal sequence (AATAAA), was found at 361 to 366 bp downstream of the termination codon (TAA). Sequence alignment analysis showed that our acquired Merino DKK1 genomic sequence displayed high similarity to the recently published Texel sheep DKK1 genomic sequence (GenBank accession No. NC_019479) (97.59%) and bovine DKK1 genomic sequence (94.38%). Comparison of the Texel sheep and Merino DKK1 genomic sequence showed that Merino DKK1 genomic region had a 5 bp insertion in its 5 flanking region and a 105 bp deletion in 3 flanking region. Comparison of the ovine and caprine DKK1 genomic structure showed that ovine and caprine DKK1 shared the same numbers and sizes of exons and introns and their exon and intron sequences were highly similar to each other. The DKK1 genomic sequence from the start codon ATG to the terminal codon TAA was 95.77% similar between sheep and goat. were highly similar to each other. The DKK1 genomic sequence from the start codon ATG to the terminal codon TAA was 95.77% similar between sheep and goat. Table S1. Table S1.
Bioinformatics Analysis of Ovine DKK1 Promoter
Alignment of the acquired ovine DKK1 genomic sequence (GenBank accession No. JQ348893) and its cDNA sequence (GenBank accession No. XM_012138945.2) identified a 2752 bp genomic sequence upstream of the initiation start codon (ATG) of ovine DKK1. To gain insight into the transcriptional regulation of ovine DKK1, we analyzed this 2752 bp upstream genomic sequence by using promoter prediction software Promoter SCAN (http://www-bimas.cit.nih.gov/molbio/proscan/) and Mulan (https://mulan.dcode.org/). The Promoter SCAN analysis showed that the transcriptional initiation site of ovine DKK1 was at an A residue 515 bp (−515 bp) upstream of the initiation start codon (ATG), its promoter contained a canonical TATA box and a GC box at nucleotide −23 to −30 and −108 to −95 relative to its predicted transcriptional initiation site as revealed by using Promoter SCAN and Mulan software respectively. The TATA box and GC box were conserved in ovine, bovine and caprine DKK1 promoters. Sequence alignment of the 2752 bp genomic sequence and caprine genomic sequence (GenBank accession No. GQ480837) identified a 251 bp sequence containing
Bioinformatics Analysis of Ovine DKK1 Promoter
Alignment of the acquired ovine DKK1 genomic sequence (GenBank accession No. JQ348893) and its cDNA sequence (GenBank accession No. XM_012138945.2) identified a 2752 bp genomic sequence upstream of the initiation start codon (ATG) of ovine DKK1. To gain insight into the transcriptional regulation of ovine DKK1, we analyzed this 2752 bp upstream genomic sequence by using promoter prediction software Promoter SCAN (http://www-bimas.cit.nih.gov/molbio/proscan/) and Mulan (https://mulan.dcode.org/). The Promoter SCAN analysis showed that the transcriptional initiation site of ovine DKK1 was at an A residue 515 bp (−515 bp) upstream of the initiation start codon (ATG), its promoter contained a canonical TATA box and a GC box at nucleotide −23 to −30 and −108 to −95 relative to its predicted transcriptional initiation site as revealed by using Promoter SCAN and Mulan software respectively. The TATA box and GC box were conserved in ovine, bovine and caprine DKK1 promoters. Sequence alignment of the 2752 bp genomic sequence and caprine genomic sequence (GenBank accession No. GQ480837) identified a 251 bp sequence containing 94.27% nucleotide identity. This conserved 251 bp region was located at nucleotides −263 to −13 relative to the predicted transcriptional initiation site of ovine DKK1. A number of binding sites for transcription factors including RAR-related orphan receptor A isoform 1 (RORA1), signal transducers and activators of transcription 1 (STAT1), OCT4 (also called POU domain, class 5, transcription factor-1) were predicted at this conserved region, further analysis showed that RORA1, STAT1 and OCT4 binding sites were conserved among sheep, goat, cattle, pig, chimpanzee, human, rhesus monkey, rabbit, house mouse and Norway rat DKKl promoters. In addition, Mulan program predicted a conserved p53 binding sites in sheep, cattle, rabbit and mouse DKK1 promoters; and one conserved TCF/LEF binding site in sheep, cattle, pig, human, rhesus monkey, rabbit, house mouse and Norway rat DKK1 promoters; and nine SP1 in sheep and cattle DKK1 promoters. The high degree of conservation of these transcription factor binding sites in DKK1 promoters between these different animals indicates that the transcriptional regulation of DKK1 may be similar in these animals.
Identification of SNP in DKK1
By PCR and sequencing, we detected SNPs in the DKK1 genomic region from the pooled gDNA sample. A total of 11 SNPs were identified and named as SNPs1 to 11. The detailed SNP information is summarized in Table 2. Of these 11 SNPs, SNPs1 to 5 were located in intron 2, SNP6 in intron 3, SNP7 in exon 4 which is a silent mutation, SNPs8 and 9 in the 3' UTR and SNPs10 and 11 in the 3 flanking region of ovine DKK1 (Table 2).
Allele, Genotype and Haplotype Frequencies of Ovine DKK1
A total of 743 individuals of the six Chinese Merino strains (SF, PW, PM, A, B and U) were genotyped for the 11 identified SNPs using the SBE assay. For SNPs1 to 8, the frequency of the alleles (G of SNP1, C of SNP2, D of SNP3, G of SNP4, G of SNP5, G of SNP6, G of SNP7 and T of SNP8) is predominantly higher than that of the alternative alleles (A of SNP1, A of SNP2, I of SNP3, C of SNP4, T of SNP5, A of SNP6, A of SNP7 and C of SNP8) in all 6 tested Chinese Merino strains (Table S3). The minor allele frequency of these 11 identified SNPs varied from 15.9% to 49.0% and the SNP1 and SNPs6 to 9 were in Hardy-Weinberg equilibrium ( Table 2; p > 0.05). The frequencies of the alleles and genotypes are shown in Table S3. The χ 2 test results showed that the allele frequencies for these 11 identified SNPs were significantly different among the six strains studied (p < 0.01). Among three genotypes of these 11 identified SNPs in six Chinese Merino sheep strains, only the heterozygous genotype TC of SNP11 was not identified in the U strain. There were 12 haplotypes based on the identified SNPs in all tested individuals. The haplotype frequencies of Ovine DKK1 differed among the Merino strains tested (Table S4).
Phenotypic and Genetic Correlations of the Wool Production and Quality Traits
Phenotypic and Genetic correlation coefficients were calculated for each tested trait. The results showed that in our studied population, there were moderate negative phenotypic correlations between curvature and MFD and FDSD (−0.41 for both) (Table S5; p < 0.05) and low negative phenotypic correlations between curvature and CVFD, WFL (−0.21 and −0.20, respectively) (Table S5). There was high positive phenotypic correlation between CVFD and FDSD (0.78), moderate positive phenotypic correlation between MFD and FDSD (0.57) (Table S5; p < 0.05) and low positive phenotypic correlation between WFL and FW (0.27) (Table S5; p < 0.05).
Genetic correlations between the traits ranged from −0.79 for curvature-MFD to 0.83 for MFD-FDSD (Table S5). There were high negative genetic correlations between curvature and MFD and FDSD (−0.79 for both) and moderate negative genetic correlation between curvature and CVFD (−0.43) (Table S5). There were high positive genetic correlations between MFD and FDSD (0.83), between WFL and FW (0.70) and between CVFD and FDSD (0.73) (Table S5; p < 0.01) and low positive genetic correlations between CVFD and MFD, FL (0.27 for both).
Association of the Identified SNPs with Wool Production and Quality in Sheep
Association analysis using JMP 7.0 (SAS Inst. Inc.) showed that, of these 11 identified SNPs, SNP5 was significantly associated with MFD in B, PW and U strains (Table 3; p < 4.55 × 10 −3 = 0.05/11). The Genotype GT of SNP5 was significantly associated with lower MFD than genotype GG in B and U strains and the Genotype GG of SNP5 was significantly associated with lower MFD than genotype GT in PW strains. (Table 3; p < 4.55 × 10 −3 = 0.05/11). SNP-wide association analysis showed that SNP8 was markedly associated with MFD in A strain and it was also associated with FW in A, PM and SF strains (Table 3; p < 0.05). The Genotype CC of SNP8 was significantly associated with lower MFD and higher FW compared to genotypes TC and TT in A strain (Table 3; p < 0.05). The Genotype CC of SNP8 was significantly associated with higher FW than genotype TT in PM strain and the Genotype TT of SNP 8 was significantly associated with higher FW than genotype TC in SF strain. SNP9 was significantly associated with curvature in B and U strains (Table 3; p < 0.05). The Genotype TC of SNP9 was significantly associated with lower curvature than genotype CC in B strain; and the Genotype TT of SNP9 was significantly associated with lower curvature than genotype CC in U strain. SNP10 was significantly associated with CVFD in A, PW and PM strains and FDSD in A and PM strains (Table 3; p < 0.05). The Genotype TT of SNP10 was significantly associated with lower CVFD than genotype CC in A and PW strains and the Genotype TC of SNP10 was significantly associated with lower CVFD than genotype CC in PM strain. In contrast, SNPs1 to 4, 6, 7, 11 were not found to be associated with the wool production and quality traits in the tested Chinese Merino (Table 3; p > 0.05).
Association of DKK1 Haplotypes with Wool Quality Traits
We also performed the haplotype association analysis using the same model used for the SNP association analysis. There was a total of 12 haplotypes based on the identified SNPs in all tested individuals. Among these haplotypes, haplotype H5 (27.2%) had the highest proportion. The haplotype association analysis results are summarized in Table 4. Sheep with haplotype H2 had a significantly lower MFD than the sheep with haplotype H5 in B strain. Sheep with haplotype H1 had a significantly lower MFD than the sheep with either haplotype H2 in PW strain or haplotype H5 in PM strain (Table 4; p < 0.05). The complete trait data are only included for the traits associated with the identified SNPs; 1 Least square means within columns that do not share a lower-case superscript letter (a, b, c, d, e, f, g) are different, p < 0.05; MFD, means mean fiber diameter; FW, fleece weight; FDSD, standard deviation of the mean fiber diameter; CVFD, coefficient of variation of the mean fiber diameter; 2 p value was evaluated based on threshold gene-wide (p < 4.55 × 10 −3 = 0.05/11) using Bonferroni correction; NE stands for not estimable.
Discussion
In the present study, we characterized the full-length coding sequence and genomic structure of ovine DKK1 and identified a total of 11 SNPs. The association analysis showed that DKK1 polymorphisms were associated with MFD, FDSD, CVFD, FW and curvature in the tested population (p < 0.05).
The ovine DKK1 genomic structure was found to be identical to that of another mammalian DKK1. The nucleotide and amino acid sequence analysis revealed that DKK1 gene was conserved in mammals. Protein domain analysis showed that the C-terminal cysteine-rich region was conserved in mammalian DKK1 homologs, suggesting that the conserved C-terminal cysteine-rich region is essential for the function of DKK1. It has been reported that the C-terminal cysteine-rich region of DKK1 is involved in binding to low-density lipoprotein receptor-related proteins (LRPs), which act as Wnt coreceptors and inhibits Wnt signaling [31].
The promoter analysis revealed that a GC box and a canonical TATA box were present upstream of ovine DKK1, suggesting ovine DKK1 promoter is a classical promoter. In addition, we observed multiple conserved transcription factor binding sites (RORA1, STAT1, POU5F1, TCF/LEF1, p53 and SP1) in the ovine DKK1 promoter region. Consistently, it has been reported that TCF/LEF-1 [32], p53 [33] and SP1 [34] transcriptionally regulate human DKK1. Determining whether these transcription factors directly regulate ovine DKK1 would lead to a better understanding of the role of ovine DKK1 in hair follicle morphogenesis, cycling and wool production.
In the present study, a total of 11 SNPs were identified in ovine DKK1 genomic region. From these 11 identified SNPs, six SNPs (SNPs2 to 5, 10, 11) were not in the Hardy-Weinberg equilibrium, which may be explained by the following reasons: (1) the alleles may be the predominant alleles during genetic evolution, thus being more conserved and more common than other alleles in this population [35]; (2) The number of sheep examined in each strain was not large enough that genetic drift makes a significant force [36]; (3) The alleles may be tightly linked with an advantageous allele; (4) The economically favorable traits were artificially selected [35].
The positive genetic correlations of MFD with WFL were consistent with the previous studies [37,38], although the genetic correlations were lower than those reported in the previous Merino studies. The previous Merino studies showed that the average genetic correlations of MFD-WFL were 0.19 and 0.29 [33,34]. In Targhee sheep, the genetic correlation of MFD-WFL was 0.30 [39]. The genetic correlation of MFD-FDSD (0.83) in our tested population was higher than that reported by Safari et al. in Merino study [40]. The genetic correlation between FDSD and FDCV (0.73) in this study agreed with the estimated value of 0.76 by Safari et al. [33] but the genetic correlation between MFD and FDCV (0.27) disagreed with the estimate value of −0.16 by Safari et al. [37]. This discrepancy may be due to the differences in sheep age and gender, genetic background and environment.
Our association analysis showed SNPs (SNPs 5, 8, 9 and 10) were significantly associated with wool quality traits in several Chinese Merino strains. The improvement of wool production and quality traits such as MFD, FDSD, CVFD, is one of the important goals in sheep breeding programs. We presume that the beneficial alleles of these four identified SNPs might be used for genetic improvement of wool quality traits in Chinese Merino population, which need to be verified in large sheep populations. Wnt pathway plays an important role in hair follicle morphogenesis and cycling [41,42]. DKK1 has been shown to be associated with hair follicle development [14,43]. Wool is the product of hair follicles, the hair follicle size, cycle and density affect hair or wool length, diameter, etc. [15][16][17]. Consistent with this view, our association analysis indicated that four DKK1 SNPs (SNPs5, 8, 9 and 10) were associated with MFD, curvature, FDSD, CVFD and FW in several of our tested strains.
From the four DKK1 SNPs (SNPs5, 8, 9 and 10) associated with wool quality traits, SNP5 was located in intron 2, SNP8 and SNP9 were located in the DKK1 3 UTR, while SNP10 was located in the 3 flanking region. It has been demonstrated that some SNPs, which are located in introns, 3 UTR and flanking regions, can affect gene expression [44][45][46][47][48][49][50][51]. We cannot exclude the possibility that these four identified SNPs (SNPs5, 8, 9 and 10) are functional and affect DKK1 expression, causing changes in wool production and quality traits. It is worthwhile to further explore whether these SNPs affect DKK1 expression in the future.
Conclusions
In the present study, we cloned DKK1 genomic and coding sequences of Merino sheep, an old and influential wool sheep which produce the finest and softest wool. Our results added new insight into ovine DKK1 structure and the identified SNPs might be used as genetic molecular marker for genetic improvement of wool sheep.
Supplementary Materials: The following are available online at www.mdpi.com/2073-4425/8/12/400/s1. Table S1: Information for DKK1 from 16 different animal species, Table S2: The similarities of the deduced amino acids sequence of DKK1 protein in 16 different animal species, Table S3: Genotype and allele frequencies of the SNPs of DKK1 in Chinese Merino, Table S4: Haplotype frequencies of DKK1 in Chinese Merino, Table S5: Genetic (below diagonal) and phenotypic correlation (above diagonal) coefficient between wool production and quality traits, Table S6: A summary of the raw data (phenotypes), Table S7: the least square means of the line effects of SNP in DKK1, Table S8: The allele substitution effect of DKK1 on wool production and quality traits in Chinese Merino, Figure S1: The corresponding quantile-quantile (Q-Q) plots for the association of SNPs of DKK1.
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2018-01-01T18:24:37.960Z
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Interplay between Rab27a effectors in pancreatic β -cells
The small GTPase Rab27a is a member of the Rab family that is involved in membrane trafficking in various kinds of cells. Rab27a has GTP- and GDP-bound forms, and their interconversion regulates intracellular signaling pathways. Typically, only a GTP-bound GTPase binds its specific effectors with the resulting downstream signals controlling specific cellular functions. We previously identified novel Rab27a-interacting proteins. Surprisingly, some of these proteins interacted with GDP-bound Rab27a. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in the secretory process. In pancreatic β -cells, GTP-bound Rab27a regulates insulin secretion at the pre-exocytotic stages via its GTP-specific effectors such as Exophilin8/Slac2-c/MyRIP and Slp4/Granuphilin. Glucose stimulation causes insulin exocytosis. Glucose stimulation also converts Rab27a from its GTP- to its GDP-bound form. GDP-bound Rab27a interacts with GDP-specific effectors and controls endocytosis of the secretory membrane. Thus, Rab27a cycling between GTP- and GDP-bound forms synchronizes with the recycling of secretory membrane to re-use the membrane and keep the β -cell volume constant.
INTRODUCTION
Diabetes mellitus is defined as chronic hyperglycemia due to relative insulin deficiency. Impairment of secretory activity in pancreatic β-cells plays an important role in the pathogenesis of this disease. In particular, decreased output in the early phase of glucose-induced insulin release precedes the onset of type 2 diabetes mellitus [1,2] . Insulin secretion from pancreatic β-cells is finely tuned for glucose homeostasis by multiple physiological factors such as nutrients, hormones and neurotransmitters. Some of these factors induce or enhance insulin release whereas others decrease it, thereby enabling sophisticated glucose regulation. One characteristic of this regulation in pancreatic β-cells is the control of secretion by several nutrients. Glucose, the most important insulin secretagogue, stimulates insulin secretion mainly by the generation of ATP via glucose metabolism, although there appear to be other signaling pathways involved that have not been fully identified.
The insulin secretory process comprises insulin synthesis and its packaging into secretory granules, granule transport in the cytoplasm, interaction with the cell membrane, exocytosis as a result of an increase in cytoplasmic Ca 2+ , endocytosis and retrograde transport. The present study reviews recent progress in our understanding of the roles of the small GTPase Rab27a and its effectors in the secretory process.
SMALL GTPASE CYCLE
Small GTPases are GTP-binding proteins with molecular masses ranging from 20 to 30 kDa. These proteins, comprising Ras, Rho, Rab, Ran and Sar/Arf, are expressed in almost all eukaryotic cells and participate in a wide variety of cellular functions including proliferation, cytoskeletal rearrangement and intracellular transport [3] . Small GTPases have GTP-and GDP-bound forms, and their interconversion regulates intracellular signaling pathways ( Figure 1). Typically, only the GTP-bound small GTPase binds its specific effectors and the resulting downstream signals control specific cellular functions [4] . Therefore, GTP-and GDP-bound small GTPases are considered as active and inactive forms, respectively. The activation of small GTPases is modulated by guanine nucleotide exchange factors (GEFs), GTPaseactivating proteins (GAPs), and GDP-dissociation inhibitors (GDIs). Small GTPases localize in the cytosol as a GDP-bound form under unstimulated conditions. Cell stimulation recruits GDP-bound small GTPases to the vicinity of the plasma membrane and converts them to the GTP-bound form through the action of GEFs [4,5] . These GTP-bound forms interact with their specific effectors and transduce signals. GAPs promote the intrinsic GTPase activity of small GTPases and induce the conversion of the GTP-to the GDP-bound form [4,6] .
GDIs form a complex with GDP-bound small GTPases and induce their intracellular redistribution from the plasma membrane to the cytosol [7][8][9] .
RAB27A
The Rab family, which consists of more than 60 members, regulates membrane trafficking [10][11][12] . Rab27 is a Rab-family member that controls vesicle transport in various kinds of cells [13] . There are two isoforms of Rab27; Rab27a and Rab27b. Mutations in Rab27a have been reported to cause Griscelli syndrome [14] . This disorder results in pigment dilution in the skin and the hair. The same symptoms are observed in ashen mice with a natural mutation in Rab27a [15] . To date, three forms of Griscelli syndrome have been reported. Mutations in Myosin Va [14,[16][17][18] , Rab27a [14,15] , and its effector Slac2-a/melanophilin [19,20] cause type 1 (GS1), type 2 (GS2), and type 3 (GS3) Griscelli syndrome, respectively. These proteins play an important role in melanosome transport in melanocytes. Peripheral melanosome distribution is regulated by the molecular motor protein myosin Va. GTP-bound Rab27a localizes on the surface of melanosomes where it interacts with Slac2-a/melanophilin. Since Slac2-a/melanophilin binds myosin Va, this complex functions as a linker protein between the melanosome and the motor protein [10,21,22] . GTP-bound Rab27 also interacts with another effector protein Slp2-a, thereby regulating docking of the melanosome to the plasma membrane [23] . Griscelli syndrome also results in immunodeficiency [14,17] . Exocytosis of granzyme-A-containing granules is decreased in ashen mice cytotoxic T lymphocytes [24,25] . Although both Rab7 and Rab11 are also present on the surface of these granzyme-A-containing granules, Rab27a is the main regulator of granule exocytosis [26] . Indeed, GTP-bound Rab27a interacts with phosphatidylinositol 4,5-bisphosphate (PIP2) via its effector protein Munc13-4 and regulates the docking of the granules to the synapse. These results suggest that Rab27a regulates the secretion of lytic granules in cytotoxic T lymphocytes.
Rab27b, a closely related isoform of Rab27a, also participates in intracellular transport and secretion. Rab27b and its effector proteins including Slac2-c and Slp4-a are expressed in parotid acinar cells [27] . In these cells, GTP-bound Rab27a interacts with both Slac2-c and Slp4-a. Amylase release was inhibited when streptolysin O-permeabilized cells were treated with anti-Slac2-c antibody [28] . Amylase release was also inhibited when the Rab27b/Slp4-a complex was dissociated with a GST-Slp4-a-linker [29] . Isoproterenol (IPR) stimulation promoted intracellular redistribution of Slac2-c from the cytosol to a luminal site [30] . Since Slac2-c potentially interacts with Myosin Va, it is possible that the Rab27b/ Slac2-c complex regulates F-actin-dependent intracellular transport of secretory vesicles. In contrast, the intracellular distribution of Slp4-a was not changed in the presence or absence of IPR stimulation [30] . The Rab27b/ Slp4-a complex may regulate docking of the secretory vesicles to the membrane [31] . Amylase is also released from pancreatic acinar Yamaoka M et al . Rab27a effectors in pancreatic β-cells cells in which Rab27b is localized on the surface of zymogen granules [32] . Rab27b-Q78L, a constitutively active Rab27b mutant, enhances amylase release. In contrast, Rab27b-N133I, a dominant negative mutant, inhibits amylase release. These results suggest that Rab27b regulates amylase release in pancreatic acinar cells. Exophilin7/Slp1/JFC1 is a GTP-bound Rab27a interacting protein. The number of zymogen granules was increased in the pancreatic acinar cells of fasted Exophilin7/Slp1/JFC1-KO mice [33] . Moreover, amylase release was promoted in pancreatic acinar cells from Exophilin7/Slp1/JFC1-KO mice that were treated with carbamylcholine chloride or cholecystokinin-8, which mimic fed conditions. Thus, Rab27b may be involved in amylase release via Exophilin7/Slp1/JFC1 in pancreatic acinar cells.
Rab3a, which has the highest homology to Rab27, is highly expressed in synaptic vesicles [27] . In PC12 cells, Rab27a and Rab3a are co-localized on the surface of dense-core granules [34] . Silencing of both Rabs caused a significantly greater decrease in the number of these granules docked to the plasma membrane compared to silencing of either Rab alone. Since, Rab27a and Rab3a interact with the same effector proteins, these Rabs must cooperatively regulate the docking step of densecore granules. In contrast, each of these Rabs has a specific function in pancreatic β-cells [35] . Rab3a-GAP inhibited the dot-like distribution of Rab3a in the insulinsecreting β-cell line MIN6. In contrast, the distribution of Rab27a was not changed in Rab27a-GAP expressing cells. Furthermore, whereas Rab3a is localized on insulin granules as a GTP-bound form, the Rab27a on these granules is present as both GTP-and GDP-bound forms. Moreover, these Rabs direct unique kinetic and functional properties of the exocytic pathway.
RAB27A IN PANCREATIC β-CELLS
Rab27a is highly expressed in pancreatic β-cells and is involved in insulin secretion [36] . In ashen mice, blood glucose levels after a glucose load were higher, and insulin secretion in response to high glucose was lower than that in control mice. Interestingly, the decrease in insulin secretion was specific to glucose stimulation. These data suggest that Rab27a signaling in pancreatic β-cells is glucose-specific.
Insulin granules that are synthesized in pancreatic β-cells are eventually released by exocytosis via a series of stages ( Figure 2). Granules that are transported in the cytoplasm attach to the inner surface of the cell membrane (docking). The contents of both docked and undocked granules are eventually released following elevation of intracellular Ca 2+ levels. The granules are categorized into three types [37,38] . Residents are granules that are pre-docked to the plasma membrane before fusion. Passengers are granules that fused without stably docking. Visitors are granules that remain near the plasma membrane for some time before fusion. GTP-bound Rab27a regulates insulin secretion by modulating the transport and docking steps of insulin granules via its effector proteins (Table 1) [39][40][41] . To date, Exophilin8/Slac2-c/MyRIP, Slp4/Granuphilin, Exophilin7/ Slp1/JFC1 and Exophilin1/Rabphilin3A are known to act as GTP-dependent Rab27a effectors in pancreatic β-cells. Exophilin8/Slac2-c/MyRIP possesses a potential 510 April 15, 2015|Volume 6|Issue 3| WJD|www.wjgnet.com pancreatic β-cells.
RAB27A
We have identified novel Rab27a-interacting proteins. Surprisingly, some of these proteins interacted with GDP-bound Rab27a (Table 2) [52,53] . Since GDP-bound GTPases have been considered to be an inactive form, these proteins might be suspected to be regulators of Rab27a GTP/GDP cycles. Indeed, protrudin, which was identified as a GDP-bound small GTPase interacting protein, forms a complex with GDP-bound Rab11 via its GDI consensus sequence and regulates neurite formation [54] . Therefore, protrudin is thought to be a GDI that regulates GTPase cycles. In contrast, the GDPbound Rab27a interacting proteins that we identified do not contain any GDI consensus sequences. Moreover, these proteins interact with GDP-bound Rab27a and transduce downstream signals in a similar manner to classical GTP-dependent effectors of small GTPases. We consider that these proteins are GDP-dependent effectors of Rab27a [40,41] .
To date, more than ten coronin subfamilies have been reported. Coronin 3 is a ubiquitously expressed coronin that shares a central domain with other coronin family members that contains five WD40 repeats, which are known to form β-propeller structures and to mediate protein-protein interactions (Figure 3). Initially, these repeats were thought to form a five-bladed β-propeller structure. However, recent studies of coronin structure demonstrated that the N-terminal region of coronin forms two additional blades (not identified from the sequence). Therefore, coronin 3 is now considered to possess a seven-bladed β-propeller structure [56,57] . It was noted that this propeller structure of coronin 3 is a GDP-bound Rab27a binding site [52] . Oligomerization of coronins and their interaction with F-actin are mediated by the C-terminal 30-40 amino acids, which form a coiled-coil structure. These interactions modulate actin assembly and participate in various cellular functions. Human coronin 3 also modulates F-actin through binding to Arp2/3 [57] , a key protein in the formation of a branched actin filament network [57][58][59] . These direct and indirect modulations of F-actin must play a crucial role in the cellular function of coronins, because they are conserved in eukaryote cells [60] . Both F-actin-binding and -bundling activities of coronin 3 were promoted by its interaction with GDP-bound Rab27a [61] . In contrast, GDP-bound Rab27a did not affect the interaction between coronin 3 and Arp2/3. GDP-bound Rab27a regulates F-actin bundling by modulating a direct effect of coronin 3 on F-actin (Figure 3). myosin binding site. Although Exophilin8/Slac2-c/MyRIP functions as a linker protein between GTP-bound Rab27a and Myosin Va in melanocytes [42,43] , this effector may form a different complex in pancreatic β-cells. Indeed, some reports suggest that there are several conditions under which Exophilin8/Slac2-c/MyRIP does not interact with Myosin Va [44] . Slp4/Granuphilin and Exophilin1/Rabphilin3A interact with Myosin Va in pancreatic β-cells [45] . Moreover, they are linked to a different subset of insulin granules. Further studies are required to investigate the regulation of the transport of insulin granules to the plasma membrane.
Slp4/Granuphilin was identified as a molecule that associates with insulin granules in pancreatic β-cells [46] . This molecule forms a complex with Syntaxin1a and Munc18-1 and regulates the docking step of insulin granules in the insulin secretion pathway [47] . The number of insulin granules docked to the plasma membrane was decreased in pancreatic β-cells from Slp4/Granuphilin-KO mice [48] . Interestingly, insulin secretion was promoted in these mice. These results suggest that Slp4/Granuphilin regulates the docking state and inhibits the fusion of the insulin granule membrane and the plasma membrane in unstimulated pancreatic β-cells (Figure 2). Exophilin7/ Slp1/JFC1 also tethers insulin granules to the plasma membrane [49] . There seem to be multiple docking states of insulin granules in pancreatic β-cells.
Noc2 is a potential Rab27a effector. Noc2 displays 78% similarity to the N-terminus of Exophilin1/ Rabphilin3A [50] . Ca 2+ triggered insulin secretion from pancreatic islets of Noc2-KO mice was impaired, but was restored by treatment with the G-protein inhibitor pertussis toxin [51] . Although Noc2 is involved in insulin secretion through G-protein Gi/o signaling, its role in pancreatic β cells has not been identified. A yeast twohybrid experiment identified zyxin as a Noc2-interacting protein. Because zyxin has been reported to bind the actin-binding protein α-actinin in fibroblasts [50] , the interaction between Noc2 and zyxin may regulate insulin secretion by modulating actin dynamics in 511 April 15, 2015|Volume 6|Issue 3| WJD|www.wjgnet.com Coronins regulate membrane internalization in some types of cells through F-actin remodeling. In pancreatic β-cells, silencing of coronin 3 inhibited the internalization of both FM4-64 and phogrin [52] . Internalization of these molecules was also inhibited when Rab27a-coronin 3 binding was inhibited by a dominant negative mutant of coronin 3. Moreover, the inhibition of F-actin binding to coronin 3 had the same effect [61] . Thus, coronin 3 regulates the endocytosis of secretory membranes via the modulation of actin assembly in pancreatic β-cells.
Endocytosis is a complex process that involves cargo sorting, membrane invagination, vesicle scission and vesicle targeting (Figure 4). The uptake of an antibody against the extracellular domain of phogrin was inhibited and phogrin was located near the plasma membrane in MIN6 cells expressing a dominant negative mutant of coronin 3 [62] . Interestingly, phogrin staining near the plasma membrane remained when the cells were treated with acid wash, suggesting that the anti-phogrin antibody near the plasma membrane is separated from the plasma membrane. Thus the formation of GDPbound Rab27a-coronin 3 complexes regulates the retrograde transport of internalized secretory membrane, at a stage after scission from the plasma membrane.
Insulin secretagogue glucose stimulation induced the intracellular redistribution of both Rab27a and coronin 3 from the cytosol to the plasma membrane [62] . These redistributions were inhibited in Rab27a-silenced and Rab27a-Q78L-expressing MIN6 cells. Glucose-induced translocation of coronin 3 is due to its interaction with GDP-bound Rab27a. It has been reported that coronin 3 forms a closed conformation through an intramolecular interaction between its C-and N-termini [59] . Therefore, the glucose-dependent binding of its N-terminus to GDP-Rab27a may shift coronin 3 to the open conformation, which enables this molecule to interact with F-actin [41] .
In pancreatic β-cells, IQGAP1 interacts with vesicletethering exocysts under basal conditions. Moreover, this IQGAP-exocyst complex is dissociated by GTPbound Cdc42 [71] . GTP-bound Cdc42 regulates insulin secretion by modulating F-actin bundling [72,73] . Active Cdc42 also interacts with SNARE proteins such as VAMP2 and Syntaxin1a, and promotes the fusion step in insulin secretion [74] . Since glucose converts Cdc42 from the GDP-to the GTP-bound form [75] , the IQGAP1vesicle tethering exocyst complex is dissociated by glucose. It has been reported that vesicle-tethering to the plasma membrane is not a prerequisite for, but instead temporarily inhibits glucose-induced membrane fusion [37] . This finding raises the possibility that the IQGAP1-exocyst complex may inhibit subsequent fusion events. IQGAP1 also binds A kinase anchoring protein 79 and functions as a scaffold protein [76] . IQGAP1 binds GDP-bound Rab27a through its RasGAP related domain (Figure 3) [53] . This domain lacks GAP activity and forms part of the GTP-bound Cdc42 and Rac1 interacting site [63,70,77] . Moreover, IQGAP1 interacts with GDP-bound Rab27a when it forms a complex with GTP-bound Cdc42 [53] . IQGAP1 is distributed in the vicinity of the plasma membrane in both glucose-stimulated and -unstimulated cells. In contrast, glucose-induced redistribution of Rab27a and its binding protein coronin 3 was inhibited in both IQGAP1-silenced cells and in cells expressing the dominant negative mutant Cdc42-T17N. Since endocytosis of secretory membrane was also inhibited in these cells, these data indicate that activated Cdc42bound IQGAP1, to which GDP-bound Rab27a binds, 512 April 15, 2015|Volume 6|Issue 3| WJD|www.wjgnet.com recruits endocytic machinery including coronin 3 and regulates endocytosis of secretory membrane. In summary, IQGAP1 functions at pre-exocytotic stages via interaction with the exocyst complex [71] and this complex is dissociated by GTP-bound Cdc42. Our results indicate that IQGAP1 also plays a crucial role in the control of endocytosis via interaction with GTPbound Cdc42 and GDP-bound Rab27a [53] . Based on the combined data, we propose a model where IQGAP1 functions as a scaffold protein and is a key molecule for membrane recycling.
IQGAP1 also interacts with GTP-bound Rac1 [63,77] . Interestingly, IQGAP1 interacts with GDP-bound Rab27a when it forms a complex with GTP-bound Rac1 [53] . Moreover, endocytosis of secretory membrane was inhibited in MIN6 cells expressing the dominant negative Rac1-T17N mutant. These results suggest that Rac1 also recruits endocytic machinery and regulates endocytosis of secretory membrane. Cdc42 and Rac1 display some different characteristics in pancreatic β-cells. The most important difference is the timing of the glucose-induced conversion from the GDP-to the GTP-bound form. Glucose stimulation causes a shift from GDP-bound Cdc42 to GTP-bound Cdc42 within 3 min. In contrast, the glucose induced conversion of GDP-to GTP-bound Rac1 requires 20 min [73,75] . These findings raises the possibility that Cdc42 regulates rapid endocytosis whereas Rac1 regulates subsequent, prolonged endocytosis, a pattern that may be associated with the biphasic release of insulin in response to glucose.
CONCLUSION
In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytotic stages via its GTP-specific effectors ( Figure 5). Glucose stimulation causes insulin exocytosis. Glucose stimulation also converts Rab27a from its GTP-to its GDP-bound form, which interacts with IQGAP1, recruits coronin 3, and controls endocytosis of the secretory granule membrane. A long-term overexpression of a dominant negative coronin 3 mutant caused β-cell death (unpublished data), suggesting that the membrane recycling system controlled by Rab27a may be necessary for β-cell survival. Thus, we consider that Rab27a GTP/GDP cycling synchronizes with the recycling of secretory membrane to re-use the membrane and to keep the β-cell volume constant. It raises the possibility that a pharmacological agent that modulates the recycling system may become a new therapeutic option for the treatment of β-cell dysfunction in diabetes. Further studies are required to investigate whether Rab27a is involved in the pathogeneses of diabetes mellitus.
Typically, small GTPases are predominantly present in the GDP-bound form under unstimulated conditions and are converted to the GTP-bound form by stimulation. In contrast, glucose stimulation causes a shift of Rab27a in pancreatic β-cells from its GTP-to its GDP-bound form [52] . The same conversion also occurs in thrombin stimulated platelets [78] . These findings suggest that specific Rab27a-GAPs are activated by these stimulations. Two candidate Rab27a-GAPs, EPI64A and EPI64B, have been reported [79] . In melanocytes, EPI64A has the higher GAP activity and functions as the main Rab27a-GAP. In pancreatic acinar cells, EPI64B regulates amylase secretion by modulating Rab27a GTP/GDP cycles [80] . Further studies are needed to identify and characterize Rab27a-GAPs in pancreatic β-cells.
ACKNOWLEDGMENTS
We thank all members of our laboratory for helpful suggestions. Figure 5 Rab27a GTP/GDP cycling synchronizes with the recycling of secretory membrane. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytotic stages via its GTP-specific effectors. Glucose stimulation causes insulin exocytosis. Glucose stimulation also converts Rab27a from its GTP-to its GDP-bound form. GDP-bound Rab27a interacts with IQGAP1, recruits coronin 3, and controls endocytosis of the secretory granule membrane. GAPs: GTPase-activating proteins.
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2018-04-03T02:08:30.420Z
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2015-04-15T00:00:00.000
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Stability for intersecting families in PGL ( 2 , q )
We consider the action of the 2-dimensional projective general linear group PGL(2, q) on the projective line PG(1, q). A subset S of PGL(2, q) is said to be an intersecting family if for every g1, g2 ∈ S, there exists α ∈ PG(1, q) such that αg1 = αg2 . It was proved by Meagher and Spiga that the intersecting families of maximum size in PGL(2, q) are precisely the cosets of point stabilizers. We prove that if an intersecting family S ⊂ PGL(2, q) has size close to the maximum then it must be “close” in structure to a coset of a point stabilizer. This phenomenon is known as stability. We use this stability result proved here to show that if the size of S is close enough to the maximum then S must be contained in a coset of a point stabilizer.
Introduction
The Erdős-Ko-Rado (EKR) theorem is a classical result in extremal set theory.It states that if k < n/2, an intersecting family of k-subsets of [n] = {1, 2, . . ., n} has size at most n−1 k−1 ; equality holds if and only if the family consists of all k-subsets containing a fixed element from [n].Intersecting families of maximum size are called extremal families.In [11], Frankl proved that these extremal families are not only unique, but also stable: Any intersecting family of size close to the maximum is "close" in structure to an extremal family.In this paper, we focus on an analogue of these results for permutations groups, in particular, to the natural right action of P GL(2, q) on the projective points of P G (1, q), where q is a prime power.
Let Ω be a finite set and G a finite group acting on Ω.A subset S of G is said to be an intersecting family if for every g 1 , g 2 ∈ S there exists an element α ∈ Ω such that α g 1 = α g 2 .Like in the original EKR-problem, we call intersecting families of maximum size extremal families.Moreover, intersecting families whose sizes are close to the maximum are called almost extremal families.
the electronic journal of combinatorics 22(4) (2015), #P4.41 The following problems about intersecting families in G are considered to be the basic problems in EKR theory.I (Upper Bound) What is the maximum size of an intersecting family?II (Uniqueness) What is the structure of extremal families?III (Stability) Are almost extremal families similar in structure to the extremal ones?
The above three problems were solved for the symmetric group S n .Indeed, Deza and Frankl [10] proved that the maximum size of an intersecting family in S n is (n − 1)!.Moreover, they conjectured that the cosets of points stabilizers are the only extremal families.This conjecture turned out to be rather harder to prove than one might expect.It was first proved by Cameron and Ku [3], and independently by Larose and Malvenuto [15].Finally, the stability of extremal families in S n was settled by Ellis [6], who proved that for any > 0 and n > N ( ), any intersecting family of size at least (1−1/e+ )(n−1)!must be strictly contained in an extremal family.
In [17], Meagher and Spiga studied Problems I and II for the group G q := P GL(2, q) acting on the set of points of the projective line P G (1, q).These authors proved that the maximum size of an intersecting family in G q is q(q − 1).Furthermore, they also solved the uniqueness problem: Every extremal family in G q is a coset of a point stabilizer.In this paper, we prove that extremal families in G q are also stable, like their counterparts in the symmetric group.That is, an almost extremal family in G q must be close in structure to a coset of a point stabilizer.We make this statement explicit in the following theorem.
Theorem 1.There exists an absolute constant C 0 such that the following holds.Let S ⊂ G q be an intersecting family with |S| = (1 − δ)q(q − 1), where 0 δ 1/2.Then there exists a coset of a point stabilizer T ⊂ G q such that where is the symmetric difference of sets.
Using Theorem 1 and some properties of intersecting families in G q we get the following stronger result on almost extremal families in G q .Theorem 2. There exists an absolute constant δ 0 > 0 such that the following holds.If S ⊂ G q is an intersecting family with |S| (1 − δ 0 )q(q − 1), then S is contained within a coset of a point stabilizer in G q .
Theorem 2 is a direct analogue of the Cameron-Ku conjecture proved by Ellis in [6].The main tools in this paper are the eigenvalue method and analysis of Boolean functions on G q .The eigenvalue method was introduced by Lóvasz [16] as a new way to prove for the EKR-theorem.Since then, it has been used several times to prove analogues of the EKR theorem [7,13,17,19].The analysis of Boolean functions on finite groups has been the electronic journal of combinatorics 22(4) (2015), #P4.41 an active research area especially in computer science.A lot of work has been done in recent years to characterize Boolean functions whose Fourier transforms are highly concentrated on some irreducible representations.Friedgut, Kalai and Naor [12] proved that a Boolean function on Z n 2 whose Fourier transform is close to being concentrated on the first two levels, must be close to a dictatorship (a function determined by just one coordinate).Furthermore, similar results have been obtained for other abelian groups [1,14].Recently, Ellis, Filmus and Friedgut [8] showed that similar results can be obtained for the symmetric group S n .Specifically, they proved that if the Fourier transform of a Boolean function f is highly concentrated on the first two irreducible representations of S n and ) then f must be close to a union of cosets of points stabilizers.The proof of Theorem 1 is divided into two parts.First, we prove that the Fourier transform of the characteristic function of the almost extremal families are highly concentrated on two irreducible representations of G q .Second, we use this Fourier characterization of almost extremal families to get structural information.In particular, we note that most of the ideas used in [8], can be used to characterize Boolean functions on G q whose Fourier transforms are highly concentrated on the trivial and standard representations of G q .This partially answers a question of Ellis, Filmus and Friedgut in [9].These authors asked if there were others groups (besides S n ) for which there is an elegant characterization of Boolean functions whose Fourier support is concentrated on certain irreducible representations.Actually, in Section 4, we explain that 3-transitive groups satisfying certain extra conditions have a similar characterization.
The proof of Theorem 2 follows from Theorem 1 and some basic properties of intersecting families in G q .
The rest of the paper is organized as follows.In Section 2 we provide some notation, definitions and basic results.In Section 3 we characterize the Fourier transforms of the characteristic functions of almost extremal families.In Section 4 we prove our main theorems.Finally, in Section 5 we conclude with some remarks and open problems.
Fourier Analysis
Let G be a finite group.We denote by C[G] the vector space of all complex valued functions on G. Definition 3. Let R be a complete set of non-isomorphic irreducible matrix representations of G.The Fourier transform of f ∈ C[G] is a matrix-valued function on irreducible representations.Its value at the irreducible representation ρ ∈ R is We apply the Fourier transform to decompose the vector space C[G] into a direct sum of subspaces indexed by the irreducible representations of G.For every ρ ∈ R, we denote by V ρ the subspace of C[G] consisting of all functions whose Fourier transform is supported only on ρ, more precisely, Since the Fourier transform is an invertible linear transformation, we can write By abuse of notation, we will sometimes use V χρ to denote V ρ where χ ρ is the irreducible character afforded by ρ.
Moreover, we can make C[G] an inner product space.For any f, g ∈ C[G] we define We denote by f the euclidean norm induced by the inner product Let U be any subspace of We denote by U ⊥ the orthogonal complement of U and by P U (f ) the projection of f onto U .Thus, we can write Let Ω = {1, . . ., n} be a set and C[Ω] be the vector space of all C-valued functions defined on Ω.For every i ∈ Ω, we define e i as the function on Ω which takes the value 1 at i and 0 elsewhere.Let G be a group acting on Ω on the right.This action turns C[Ω] into a representation of G of degree n.Indeed, this representation is produced by a linear extension of the (left) action defined by g(e i ) = e ig −1 for all g ∈ G and i ∈ Ω.The vector subspace V std spanned by the vectors { n i=1 x i e i : x i = 0} is a subrepresentation of C[Ω] of degree n − 1, known as the standard representation of G.We denote by χ std the character afforded by the standard representation (we will refer to χ std as the standard character of G).It follows by definition that for every g ∈ G, the value χ std (g) corresponds to the number of elements in Ω fixed by g minus one.
Let X be an inverse-closed subset of G.The Cayley graph on G generated by X is the graph with vertex set G such that there is an edge between g 1 , g 2 ∈ G if and only if g 1 g −1 2 ∈ X.We denote this graph by Cay(G, X).The following lemma says that under certain conditions on X, the subspaces V ρ are eigenspaces of Cay(G, X).
conjugation invariant, and let Cay(G, X) be the Cayley graph on G with generating set X.For every ρ ∈ R, the vector subspace V ρ is an eigenspace of Cay(G, X) with eigenvalue where χ ρ is the irreducible character of ρ.Besides, if λ is an eigenvalue of Cay(G, X) corresponding to the irreducible representations {ρ 1 , . . ., ρ s } ⊂ R then the dimension of the λ-eigenspace is s i=1 χ ρ i (1) 2 .
P GL(2, q)
Let F q be the finite field of size q and F q 2 its unique quadratic extension.We denote by F * q and F * q 2 the multiplicative groups of F q and F q 2 , respectively.Let V be a 2-dimensional vector space over F q then GL(V ) denotes the group of all invertible linear transformations on V .The subgroup of all invertible linear transformations on V with determinant 1 is known as the special general linear group SL(V ).We denote by Z(GL(V )) and Z(SL(V )) the centers of the groups GL(V ) and SL(V ), respectively.
The projective general linear group of V is defined as P GL(V ) = GL(V )/Z(GL(V )), and the projective special linear group of V is defined as P SL(V ) = SL(V )/Z(SL(V )).
We denote by P G(1, q) the set of 1-dimensional subspaces of V .Thus, P G(1, q) is a projective line over F q and its elements are called projective points.An easy computation shows that P G(1, q) has cardinality q + 1.From the above definitions, it is clear that the groups P GL(2, q) and P SL(2, q) define a natural right action on P G(1, q).Moreover, the action of P GL(2, q) on P G(1, q) is sharply 3-transitive.
We briefly describe the character table of G q := P GL(2, q).We refer the reader to [18] for a complete study of the complex irreducible characters of G q .We start by describing its conjugacy classes.By abuse of notation we will denote the elements of G q by 2 by 2 matrices with entries from F q .We choose the following representatives for the conjugacy classes of G q : where the label x runs through all the elements of F * q of order greater than 1 up to inversion, and the label r runs through all the elements of F * q 2 /F * q of order greater than 1 up to inversion.
The complex irreducible characters of G q are described in Table 1.The trivial character is denoted by λ 1 .The character ψ 1 corresponds to the standard character which is an irreducible character of G q .Thus, for every g ∈ G q , the value of ψ 1 (g) is equal to the number of projective points fixed by g in P G(1, q) minus 1.The label β in Table 1 runs through all homomorphism β : F * q 2 /F * q → C of order greater than 2 up to inversion.Therefore, the number of irreducible characters {η β } β is q/2 if q is even and (q − 1)/2 if q is odd.The label γ in Table 1 runs through all the homomorphism γ : F * q → C of order greater than 2 up to inversion.Therefore, the number of irreducible characters {ν γ } γ is q/2 − 1 if q is even and (q − 3)/2 if q is odd.
If q is odd then G q has two more irreducible characters denoted by λ −1 and ψ −1 in Table 1.The values of these characters depend on the function δ.We define δ(x) = 1 if d x ∈ P SL(2, q) and δ(x) = −1 otherwise.Similarly, δ(r) = 1 if v r ∈ P SL(2, q) and δ(r) = −1 otherwise.
Using the notation introduced in the above paragraphs we can write when q is even, and when q is odd.
The eigenvalue method
As was remarked in the introduction, the eigenvalue method has been used several times to get upper bounds on the size of intersecting families for EKR-type problems.In this section, we apply the eigenvalue method to show that the characteristic function of every extremal family of G q has a Fourier transform supported on just two irreducible representations of G q .In the next section, we will show that almost extremal families have a similar Fourier characterization.The first step of the method is to reformulate the problem in graph theory terminology.Indeed, the problem of finding the maximum size of an intersecting family in G q is equivalent to the problem of finding the maximum size of an independent set in a certain graph.Then, we can apply Hoffman's bound to get an upper bound on the size of an independent set.The following variant of Hoffman's theorem will be enough for our purposes.
Theorem 5. (Hoffman's bound) Let Γ be a k-regular, n-vertex graph.Let A be the adjacency matrix of Γ and let λ min be the minimum eigenvalue of A.
If equality holds then the characteristic function 1 S of S satisfies: where V 1 is the vector space spanned by the all-ones vector and V λ min is the λ min -eigenspace.
Recall that an element g ∈ G q is a derangement if for any α ∈ P G(1, q) we have that α = α g .Denote by D q the set of derangements in G q .We define Γ as the Cayley graph on G q with generating set D q .This graph is known as the derangement graph of G q .Note that every independent set in Γ corresponds to an intersecting family in G q .Hence, an upper bound on the size of independent sets in Γ is also an upper bound on the size of intersecting families in G q .
To apply Hoffman's bound, we need to compute the eigenvalues of Γ.Note that the set D q is a union of conjugacy classes and inverse-closed.Therefore, Γ satisfies the conditions of Lemma 4. Thus, to compute the eigenvalues of Γ we just need to evaluate the character sum 1 χ(1) x∈Dq χ(x) for every irreducible character χ of G q .Now, using the character table of G q (Table 1) and Lemma 4, Meagher and Spiga [17] computed the eigenvalues of Γ for every q: Then, applying Hoffman's bound, they proved that the maximum size of an intersecting family in G q is q(q − 1).Therefore, the cosets of point stabilizers in G q are extremal families.For every α, β ∈ P G(1, q), we denote by T α,β the coset of a point stabilizer sending α to β.
Furthermore, Hoffman's bound also provides information about the characteristic function of an extremal family.Indeed, if S is an intersecting family of maximum size then its characteristic function 1 S is contained in V λ 1 ⊕ V ψ 1 when q is even, and in V λ 1 ⊕ V ψ 1 ⊕ V λ −1 when q is odd.In the next lemma, we show that it is possible to improve this result in the case when q is odd.Lemma 6.Let q be odd.Let S ⊂ G q be an intersecting family of size q(q − 1) and denote by 1 S its characteristic function.Then To prove Lemma 6, we will need the following result proved by Meagher and Spiga in [17].
Lemma 7. Consider the natural right action of P SL(2, q) on the projective points of P G(1, q).Let X be the set of derangements of P SL(2, q).An independent set of maximum size in Cay(P SL(2, q), X) has size q(q − 1)/2.
Proof of Lemma 6.We already know that 1 Recall that P SL(2, q) is a subgroup of G q .The irreducible character λ −1 is a function on G q such that λ −1 (g) = 1 if g ∈ P SL(2, q) and −1, otherwise.Therefore, 1 S , λ −1 = 0 if and only if exactly half of the elements in S are in P SL (2, q).From Lemma 7 it follows that he maximum size of an intersecting family in P SL(2, q) is q(q − 1)/2.Therefore, at most q(q − 1)/2 elements of S are contained in P SL(2, q).
Since P SL(2, q) is a subgroup of index 2, there exists g ∈ G q such that G q = g P SL(2, q) ∪ P SL(2, q).Assume to the contrary, that more than q(q − 1)/2 elements of S are contained in g P SL (2, q).If we multiply each of these elements by g then we get an intersecting family in P SL (2, q).This is a contradiction because the maximum size of an intersecting family in P SL(2, q) is q(q − 1)/2.Therefore, exactly half of the elements in S are contained in P SL(2, q).
Fourier characterization
Let S be an intersecting family of maximum size in G q .It follows from Section 2 that the Fourier transform of 1 S is supported only on the irreducible representations affording the characters λ 1 and ψ 1 .In this section, we prove that the characteristic functions of almost extremal families in G q have Fourier transforms highly concentrated on the irreducible representations affording the characters λ 1 and ψ 1 .To do this we apply a stability version of Hoffman's bound (this term was coined by Ellis in [5]).The next two lemmas show that if an intersecting family S ⊂ G q satisfies that |S| is very close to q(q − 1) then 1 S must be close to Lemma 8. Let S be an intersecting family in G q .If q is a power of 2 then, the electronic journal of combinatorics 22(4) (2015), #P4.41 Proof.Let A be the adjacency matrix of the graph Γ = Cay(G q , D q ).Let {x 1 , . . ., x N } ⊂ C[G q ] be an orthonormal basis of real eigenvectors for A (recall that A is symmetric).Let θ i be the eigenvalue of A such that Ax i = θ i x i , for 1 i N .Note that, • 1 S = N i=1 i x i where i = 1 S , x i for every i = 1, . . ., N .
Let x 1 be the all 1 s vector with eigenvalue q 2 (q − 1)/2.Since every intersecting family corresponds to an independent set in the graph Γ we get where λ min = −q(q − 1)/2.Recall that the second smallest eigenvalue of Γ is zero.Therefore, from equation ( 1) we obtain the following inequality By definition we have Combining ( 2) and (3) we get The next lemma deals with the case q odd.The proof is a little more complicated because in that case the minimum eigenvalue of Γ is afforded by two distinct irreducible characters, ψ 1 and λ −1 .
Lemma 9. Let S be an intersecting family in G q such that |S| = (1 − δ)q(q − 1), δ > 0. If q is an odd prime power then the electronic journal of combinatorics 22(4) (2015), #P4.41 Proof.Using the notation introduced in the proof of Lemma 8 we get Recall that the vector space V λ −1 is one dimensional.Hence, we denote by x λ −1 the only eigenvector in the set (δ/(q + 1)) 2 .Note that x λ −1 is the irreducible character λ −1 .Hence, x λ −1 is a function on G q such that x λ −1 (g) = 1 if g ∈ P SL(2, q) and −1, otherwise.Besides, note that S ∩ P SL(2, q) and S ∩ (G q \ P SL(2, q)) have size at most q(q − 1)/2 because the maximum size of an intersecting family in P SL(2, q) is q(q − 1)/2.Putting all the above remarks together By definition we have Combininig ( 4), ( 5) and ( 6) we get
Structural Characterization
In this section we give a characterization of the structure of Boolean functions on G q whose Fourier transform is highly concentrated on U .The technique used to prove this result is from [8].In that paper, Ellis, Filmus and Friedgut proved that if a Boolean function on S n has Fourier transform that is highly concentrated on the first two irreducible representations of S n (which correspond to the trivial and standard representation) then it must be close to a union of cosets of points stabilizers.Their proof is only based on the fact that the action of S n on [n] is 3-transitive.
Let G be a group acting 3-transitively on a set Ω.It is well-known (and easy to show) that the standard representation is irreducible for any 2-transitive group.Also, recall that V 1 and V χ std are the vector subspaces of complex-valued functions on G whose Fourier transform has support on the trivial and the standard representation, respectively.The following proposition is a generalization of Theorem 1 from [8] 1 .
1 Actually, Proposition 10 is a generalization of a special case of Theorem 1 from [8].To fully generalize that theorem we need to consider S ⊂ G with |S| = c|G|/n, where c = o(n).
the electronic journal of combinatorics 22(4) (2015), #P4.41 Proposition 10.There exist absolute constants C 1 , 1 > 0 such that the following holds.Let G be a finite group acting 3-transitively on a set Ω of size n.
, where 1 , then there exists T ⊂ G such that T is a coset of the stabilizer of an element of Ω, and The proof of this proposition is exactly the same as the proof of Theorem 1 in [8].Since the action of G q on P G(1, q) is 3-transitive, Proposition 10 can be used to characterize Boolean functions on G q whose Fourier transform is highly concentrated on U .Recall that U is the vector subspace of all of complex-valued functions on G q whose Fourier transform has support on the trivial and the standard representation.
Now we are ready to prove Theorem 1.
Proof of Theorem 1.We choose C 0 = max( 4 where C 1 and 1 are the absolute constants from Corollary 11.With this choice of C 0 , if 1 /2 δ 1/2 then the statement of the theorem holds trivially with any choice of a coset of a point stabilizer T .Now, we consider the case where δ < 1 /2.By assumption we know that |S| = (1 − δ)q(q − 1).Thus, it follows from Lemmas 8 and 9 that P U ⊥ (1 S ) 2 δ 1 S 2 when q is even and P U ⊥ (1 S ) 2 2δ 1 S 2 when q is odd.This implies that the characteristic function 1 S is highly concentrated on U .Hence, we can apply Corollary 11 to conclude that where T is some coset of a point stabilizer.
Theorem 1 implies that almost extremal families are almost contained in a coset of a point stabilizer.Furthermore, we can refine this result to conclude that almost extremal families are fully contained in a coset of a point stabilizer.
Hence, if S is an intersecting family of G q with |S| (1 − δ 1 )q(q − 1) then |S| = q(q − 1).Therefore, by the characterization of intersecting families of maximum size in G q given in [17], we conclude that S must be equal to a coset of the stabilizer of a point.
If h ∈ S ∩ T α,α then g −1 h contains at least one fixed point (recall that S is an intersecting family).Hence, the elements h ∈ T α,α such that g −1 h is a derangement must be contained in T α,α \ S.
Thus, we get a contradiction.Finally, we choose the universal constant δ 0 to be equal to min(δ 1 , δ 2 ).
Conclusions and Open Problems
In this paper we prove that extremal families in G q are not only unique, but also stable: any intersecting family in G q of size close to q(q − 1) must be close in structure to a coset of a point stabilizer.Actually, Theorem 2 implies that for q sufficiently large the cosets of point stablizers are the only extremal families in G q .This result was already proven by Meagher and Spiga [17] using different methods.It is possible to apply the ideas used in this paper to prove similar results for some 3-transitive groups.Let G be a finite group acting 3-transitively on a finite set Ω. Suppose that this action satisfies the following conditions: 1.The maximum size of an intersecting family in G is |G|/|Ω| (note that this number is equal to the size of a coset of a point stabilizer in G).
2. Let D be the set of derangements of G with respect to its action on Ω.The standard character is the unique irreducible character affording the minimum eigenvalue of the derangement graph Cay(G, D) (recall that since D is inverse-closed and conjugationinvariant there is a correspondence, given by Lemma 4, between the eigenvalues of Cay(G, D) and the irreducible characters of G).
Thus, applying Hoffman's bound it follows that the characteristic vector of any intersecting family of maximum size lies in the vector subspace Recall that V 1 and V χ std are the vector subspaces of complex-valued functions on G whose Fourier transform has support on the trivial and the standard representation, respectively.Now, let S ⊂ G be an intersecting family.If the size of S is close to |G|/|Ω| and the size of the gap between the smallest and the second-smallest eigenvalue of Cay(G, D) is big enough then we can use analogues of Lemmas 8 and 9 to conclude that the characteristic function 1 S is close to V .Moreover, as was remarked in Section 4, the result of Ellis, Filmus and Friedgut in [8], for Boolean functions on S n , can be generalized to any 3transitive action of a finite group on a finite set.Thus, if 1 S is close to the vector space V then it must be close in structure to some coset of a point stabilizer in G. Therefore, we can use these ideas to prove that extremal families in G are unique and stable.
Consider the action of P SL(2, q) on the points of P G(1, q) for q an odd prime power.This action is 2-transitive.Using the eigenvalue method it is easy to prove that the maximum size of an intersecting family in P SL(2, q) is q(q − 1)/2.Furthermore, the characteristic vector of any extremal family in P SL(2, q) lies in V = V 1 ⊕ V χ std (recall that the standard character is irreducible for 2-transitive actions).However, the argument used here cannot be applied in a straightforward way to solve the uniqueness or stability problems because the action of P SL(2, q) on P G(1, q) is not 3-transitive.It was conjectured by Meagher and Spiga [17] that the cosets of points stabilizers are the only extremal families in P SL (2, q).Here, we extend their conjecture: the extremal families in P SL(2, q) are not only unique but also stable.
Conjecture 12. Let S be an intersecting family in P SL(2, q) with q an odd prime power.Then 1. Uniqueness: If |S| = q(q−1) 2 then S is a coset of a point stabilizer.
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2016-03-22T00:56:01.885Z
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2015-12-23T00:00:00.000
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265653568
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v3-fos-license
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Hydrochemical Characteristics and Water Quality Assessment of Irkutsk Reservoir (Baikal Region, Russia)
: The Irkutsk Reservoir, belonging to the largest unified freshwater Baikal–Angara system, is an important source of drinking water in the region. Therefore, studies of its hydrochemical characteristics are of prime importance in deciding on the role of anthropogenic activity in water quality. The water samples were collected across the reservoir in 2007, 2012, and 2021 and then were analyzed for major ions and trace elements. The data revealed that the distribution of HCO 3 − , SO 42 − , Cl − , Ca 2+ , Mg 2+ , Na + and K + is stable across the reservoir. Trace element concentrations varied from 1.13 to 15.39 µ g L − 1 for Al, from <DL to 0.39 µ g L − 1 for Cr, from 0.39 to 23.12 µ g L − 1 for Mn, from 1.25 to 53.22 µ g L − 1 for Fe, from 0.005 to 0.100 µ g L − 1 for Co, from 0.20 to 1.98 µ g L − 1 for Cu, from <DL to 13.40 µ g L − 1 for Zn, from 0.25 to 0.48 µ g L − 1 for As, from 0.004 to 0.127 µ g L − 1 for Cd, from <DL to 0.195 µ g L − 1 for Sn, from <DL to 0.0277 µ g L − 1 for Cs, from <DL to 1.13 µ g L − 1 for Pb, from <DL to 0.0202 µ g L − 1 for Th, and from 0.27 to 0.75 µ g L − 1 for U. The concentrations of all major ions and trace elements in water were below the drinking water standards. CF values showed considerable and high contamination of samples with Al, Mn, Fe, Co, Cu, Cd, Sn, Pb, and Th. PLI values classified the majority of water samples as water with baseline levels of pollutants, and part of the samples was classified as either polluted or highly polluted.
Introduction
Surface water bodies, providing drinking water and water for economic use, are a vital resource and are of crucial importance for economic and social development [1,2].Freshwater resources in these water bodies and their availability can significantly increase the industrial-agricultural and recreational potential of an area, leading to a higher rate of urbanization on the coast.The growing activity of the population, growth in agricultural areas, and industrial development in the reservoir basin led to the influx of pollutants, therefore leading to negative transformations in the quality of water resources [3][4][5].At present, numerous studies are devoted to the ecological state of surface water bodies, including the identification of water pollution sources, spatial-temporal dynamics of water hydrochemical composition, and assessment of its quality [6][7][8]).
One of the main industries of human economic activity, which is developed at large surface water bodies is hydropower.Despite certain economic advantages, dams give rise to various environmental transformations: changes in the amount of regional precipitation, river discharge from different locations along the river, biodiversity, etc. [9][10][11].The water hydrochemical composition of reservoirs, including the concentrations of major ions and trace elements, relies on natural (weathering processes, lithology of the basin, the composition of water in tributaries, etc.), and anthropogenic factors [12,13].The anthropogenic enrichment of water chemical composition is determined both by the creation of the reservoir [14], and the entry of these elements with discharge water from different industries and agriculture, surface runoff, and atmospheric transport from urban areas.Irrespective of Water 2023, 15, 4142 2 of 20 the origin, the accumulation of pollutants, in particular heavy metals, is a serious ecological threat [15].First of all, it is a problem of deterioration in the quality of water used for drinking.Bioaccumulation and biomagnification of potentially toxic elements in hydrobionts of different trophic levels [16] and further consumption of biological resources from such a reservoir may negatively affect the health of the local population [17,18].
The water resources of the Baikal region, with Lake Baikal located in its center, are characterized by fresh and ultra-fresh surface and groundwater [19].Lake Baikal itself is the largest freshwater reservoir on the planet.In terms of the concentrations of 58 trace elements, the lake is classified as the cleanest lake of the biosphere [20].The only channel of the surface runoff of Lake Baikal is the Angara River, which carries about 61 km 3 annually.The anthropogenic transformation of the hydrological and hydrochemical regimes of the river is related to the construction of a series of water reservoirs, the so-called cascade of the Angara River reservoirs (Irkutsk, Bratsk, Ust-Ilimsk, and Boguchany).Water resources of the Irkutsk Reservoir, which is the first reservoir in the Angara cascade, are used for supplying drinking water to the population of large cities (Irkutsk and Shelekhov) and a number of smaller settlements of the Irkutsk district.The gathered resources of the Irkutsk Reservoir are also used for generating hydroelectric power and navigation, as well as for fisheries and a number of recreational activities.
The major ion composition and the components of the water trophic status were studied at different stages of the Irkutsk Reservoir operation [21,22].However, the data on water trace element composition of the Irkutsk Reservoir are still scarce.The studies by [23] were conducted to assess average total Ni, Zn, Cu, Pb, V, Co, Mn, Al, and Cr concentrations and to compare them with the trace element contents in other reservoirs of the Angara cascade.The present study focuses on: (a) concentrations of major ions (HCO 3 − , SO 4 2− , Cl − , Ca 2+ , Mg 2+ , Na + and K + ) and trace elements (Al, Cr, Mn, Fe, Co, Cu, Zn, As, Cd, Sn, Cs, Pb, Th, and U) in surface and near-bottom waters of the Irkutsk Reservoir; (b) spatial-temporal dynamics in the concentrations of major ions and trace elements; (c) main natural and anthropogenic factors affecting the hydrochemical composition; (d) assessment of water quality in the Irkutsk Reservoir in terms of trace element concentrations using the single factor pollution index (CF) and the pollution load index (PLI).The studies are of significant practical interest for assessing the water quality of the large unified Baikal-Angara freshwater system and therefore contribute to preserving it as the key source of clean drinking water.
Study Site
The creation of the Irkutsk Reservoir (Figure 1), formed by the dam of the Irkutsk Hydroelectric Power Plant in 1956, resulted in flooding the valley of the Angara River between the dam (Irkutsk city) and the river source (Listvyanka settlement).As a result of the filling of the Irkutsk Reservoir, the water level of Lake Baikal increased by approximately 1 m on average.The Irkutsk Reservoir has a water surface area of 154 km 2 at normal water level (457 m a.s.l.), a volume of 2.1 km 3 , a length of 55 km, and a width ranging from 0.5 to 3.5 km.The maximum depth of the reservoir occurring close to the dam is 35 m.Water level fluctuations in the reservoir are determined, to a greater extent, by the annual water level changes in Lake Baikal and the regime of the Irkutsk Hydroelectric Power Plant.The amplitude of the Irkutsk Reservoir level variations can reach as high as 3 m; however, during most time of its operation, the reservoir is lowered by 0.14-1.4m than the normal water level [24].Since 2001, the water levels in the Irkutsk Reservoir have been limited to the range between 456 m a.s.l. and 457 m a.s.l. in order to minimize the impact of the retention management on Lake Baikal ecosystem [25].As a result of filling the reservoir, 40 bays were formed.The largest among them are Ershi, Kurma, Kartakoi, Elovyi, and Uladova.The northwestern part of the Irkutsk Reservoir lies within the Irkutsk-Cheremk plain.At the southeast end of the basin, there are western spurs of the Primorsky R The northwestern part of the basin is characterized by a hilly, gently rolling lands with incised valleys and vast flat watersheds.In the eastern part, there are groups o hills and low ridges with intrefluves lying hypsometrically higher.The left shore is posed of bedrock, represented by the Jurassic sandstones, argillites, and siltstones right shore is made up of the Quaternary diluvial-alluvial sediments, containing l like loam and sandy loam as well as sand and pebbles, which were subject to abr along almost the entire shore [24].
The dam of the Irkutsk Hydroelectric Power Plant is located in Irkutsk city (F 1), the largest city in the Baikal region, whose leading industries are aircraft building, alwork, and electric power generating.There are also metallurgy, forestry, food, pulp paper, and woodworking industries.All major industrial enterprises are located b the dam of the Irkutsk Hydroelectric Power Plant.The left shore of the reservoir is steep and therefore, less accessible and untapped.On the shores of such bays as Ku Kartakoi, Ershi, and Mel'nichnaya Pad', there are many settlements and tourist ca However, from Kurma Bay to the source of the Angara River, settlements and to camps are less numerous.At the source of the Angara River, there is an abandoned The northwestern part of the Irkutsk Reservoir lies within the Irkutsk-Cheremkhovo plain.At the southeast end of the basin, there are western spurs of the Primorsky Ridge.The northwestern part of the basin is characterized by a hilly, gently rolling landscape with incised valleys and vast flat watersheds.In the eastern part, there are groups of low hills and low ridges with intrefluves lying hypsometrically higher.The left shore is composed of bedrock, represented by the Jurassic sandstones, argillites, and siltstones.The right shore is made up of the Quaternary diluvial-alluvial sediments, containing loess-like loam and sandy loam as well as sand and pebbles, which were subject to abrasion along almost the entire shore [24].
The dam of the Irkutsk Hydroelectric Power Plant is located in Irkutsk city (Figure 1), the largest city in the Baikal region, whose leading industries are aircraft building, metalwork, and electric power generating.There are also metallurgy, forestry, food, pulp and paper, and woodworking industries.All major industrial enterprises are located below the dam of the Irkutsk Hydroelectric Power Plant.The left shore of the reservoir is very steep and therefore, less accessible and untapped.On the shores of such bays as Kurma, Kartakoi, Ershi, and Mel'nichnaya Pad', there are many settlements and tourist camps.However, from Kurma Bay to the source of the Angara River, settlements and tourist camps are less numerous.At the source of the Angara River, there is an abandoned shipyard that has been in operation since the 1890s.There is also an operating cargo and passenger port (Port Baikal) here.Along the right shore of the Irkutsk Reservoir, you can find a number of settlements, camps, and agricultural fields.The highway runs close to the reservoir connecting Irkutsk and Listvyanka settlements.The tourism industry is particularly developed in Listvyanka settlement, lying on the northeast shore of Lake Baikal, at the source of the Angara River.It should be noted that recreational attractiveness, in turn, entails an anthropogenic load on the environment.Around Listvyanka settlement and adjacent areas, the coastal zone is constantly developing from year to year.At the same time, the lack of a centralized wastewater treatment system in the settlement leads to the increasing anthropogenic load.The creation of the Irkutsk Reservoir provided good conditions for shipping from the dam to Lake Baikal.Currently, water transport for personal use is becoming more popular.
Sampling and Analytical Methods
There were five sampling campaigns to collect water samples from the Irkutsk Reservoir, conducted in July 2007, 2012, and in May (2021 (M)), July (2021 (J)), and September (2021 (S)) 2021.Sampling sites from S1 to S11 were distributed across the entire water area of the reservoir and were localized in the channel part and bays (Figure 1, Table S1).At the sampling sites with a water depth of over 2 m, the samples were collected from both the surface water layer (0.5 m) and the bottom one (in 1 m layer from the bottom).In 2021, the water samples were additionally collected along the right (S1-r, S11-r) and the left (S1-l, S11-l) shores of the reservoir, in the vicinity of Irkutsk city and Listvyanka settlement.
At each sampling site, three parallel water samples were collected.Replicate samples from the same site were taken within about 100 m of one another and then were well mixed.The mixed water samples were filtered through a 0.45 µm Millipore membrane filter and placed into polyethylene bottles which were prewashed with 3% nitric acid.The primary filtration part was discarded to clean the membrane.For the trace element analysis, the water samples were immediately acidified by addition of HNO 3 (ultrapure «Merk», Darmstandt, Germany).Prior to the analysis, the water samples were stored in the refrigerator.
The chemical analysis of water samples was accomplished at the Center for Collective Use «Isotope-Geochemical Research» located at the IGC SB RAS (Irkutsk, Russia).In the present study, SO 4 2− , Cl − , Ca 2+ and Mg 2+ , Na + , K + were analyzed by the method of capillary electrophoresis using devices of the "Drops" series (Lumex Ltd., St. Petersburg, Russia) and HCO 3 − was determined by the titrimetric method.The detection limits for elements were as follows: 6.1 mg L −1 for HCO 3 − ; 0.5 mg L −1 for SO 4 2− ; 0.5 mg L −1 for Cl − ; 0.5 mg L −1 for Ca 2+ ; 0.3 mg L −1 for Mg 2+ ; 0.5 mg L −1 for K + ; 0.5 mg L −1 for Na + .Inductively coupled plasma mass spectrometry (ICP-MS) via a high-resolution doublefocusing mass spectrometer ELEMENT-2 (Thermo Finnigan, Bremen, Germany) was used to analyze the total concentrations of trace elements.Multi-element standard samples such as ICP Multi-Element Standard Solution-Sol X CertiPUR for Surface Water Testing, Sol XII CertiPUR (MERCK, Darmstandt, Germany), and Combined Quality Control standard IQC-026 (NIST, North Kingstown, RI, USA) were applied to ensure the reliability of the analytical measurements.Water purified by the Millipore-ELIX-3 system (Millipore SA, Molsheim, France) was used to prepare washing, blank, calibration, and analyzed solutions.Three repetitions of the analysis yielded a relative standard deviation of <5%.Otherwise, the measurements were repeated until all the data reached the standard.The following metals were tested in water samples 27 Al, 52 Cr, 55 Mn, 56 Fe, 59 Co, 63 Cu, 66 Zn, 75 As, 111 Cd, 120 Sn, 133 Cs, 208 Pb, 232 Th and 238 U.The detection limits (DL) for the elements were as follows: 0.81 µg L −1 for Al; 0.05 µg L −1 for Cr; 0.37 µg L −1 for Mn; 0.75 µg L −1 for Fe, 0.003 µg L −1 for Co; 0.03 µg L −1 for Cu; 0.64 µg L −1 for Zn; 0.12 µg L −1 for As; 0.002 µg L −1 for Cd; 0.010 µg L −1 for Sn; 0.0003 µg L −1 for Cs, 0.013 µg L −1 for Pb, 0.0003 µg L −1 for Th; 0.001 µg L −1 for U.
The statistical data processing was performed using SPSS statistical software (IBM, Armonk, NY, USA, v. 20.0).
Pollution Indices
The following generally accepted pollution indices were used to calculate the water contamination in the Irkutsk Reservoir: 1.
The single-factor pollution index (CF) was used to determine only one element in a sample [26]: where C i is the concentration of the analyzed element and, C 0 is the concentration of metal in the control material.The CF value is divided into categories: CF < 1-low contamination, 1 The pollution load index (PLI) was used to calculate the total contamination in each sample [27]: where CF is an individual element pollution index.The PLI is divided into categories: PLI < 0-non-polluted, 0 < PLI ≤ 1-baseline levels of pollutants, 1 < PLI ≤ 10-polluted, 10 < PLI ≤ 100-highly polluted, PLI > 100-progressive deterioration of the environment.
Major Ion Composition
In samples collected in 2007, 2012, and 2021, the pH levels varied from 6.4 to 8.6, with an average value of 7.7.Table S2 presents the limits and mean concentrations of major ions and total mineralization (TDS) in 2007, 2012, May, June, and September 2021 in the Irkutsk Reservoir waters.Table 1 shows the data summarized for all sampling campaigns.The data available from monitoring studies of water in the Irkutsk Reservoir (Tables 1 and S2) showed insignificant spatial and temporal variations in major ion composition.The concentrations of the majority of main ions in the water samples collected during all sampling campaigns were found to lie within ±2 standard deviation (SD) of their mean.Only, the concentrations of SO 4 2− in three samples, Ca 2+ in two samples, and Mg 2+ in one sample, were found to fall within ±3 SD of the mean.In water samples taken from the Irkutsk Reservoir, the cations were dominated by Ca 2+ (from 11.7 to 17.7 mg L −1 , with a mean value of 14.9 mg L −1 ) and the anions were dominated by HCO 3 − (from 50.4 to 73.2 mg L −1 , 64.1 mg L −1 as a mean value).The water type was characterized as HCO 3 -Ca.Notes: * Above the line-minimum-maximum value, below the line-mean value ± standard deviation.
In the study area, the water samples collected during all sampling campaigns demonstrated low TDS levels: from 78.5 to 104.0 mg L −1 .According to our determinations, the average TDS value in the Irkutsk Reservoir (92.5 mg L −1 ) was less than the world river water median (127 mg L −1 ) [32].This value was close to the average TDS level in Lake Baikal (~96 mg L −1 ) [28] and the source of the Angara River (95.6 mg L −1 ) [29] (Table 1).The main natural sources of dissolved salts, which change the composition of water over a wide range, include cyclic salts and weathering of minerals [33][34][35].Similar average concentrations of major ions and TDS values in water columns of Lake Baikal, the Angara River source (S1), and the Irkutsk HPP headwater (S11) (Tables 1 and S2) suggest that the main source of dissolved substances entering the reservoir is Lake Baikal runoff.Weather-ing processes and cyclic salts were found to insignificantly influence the major ion water chemistry in the reservoir.Higher concentrations of the major ions in the water column of the Bratsk and Ust-Ilimsk Reservoirs (Table 1), located downstream on the Angara River, suggest a lower contribution from the lake's runoff and a higher contribution from natural and anthropogenic sources.
As shown by monitoring studies conducted since the beginning of the 20th century, in the pelagic part of Lake Baikal major ion composition exhibits weak seasonal and interannual variability within the accuracy of methods [36,37]).In the Irkutsk Reservoir, in particular at the Angara River source, the levels of main ions, including HCO 3 − , Cl − , and SO 4 2− were higher than those in Lake Baikal (Table 1).A similar distribution pattern of main ions was found in the Angara River source during the period of 1997-2003 [38]).Based on the results of long-term monthly investigations, it was shown that HCO 3 − concentration at the river's source could be affected by the fluctuations of water level, whose maxima correlate with minimum ion levels, while SO 4 2− concentrations were influenced by large seismic events.The long-term dynamics of the water level in the Irkutsk Reservoir are recorded as the alternation of maximum and minimum cycles.Thus, in the period from 2004 to 2018, the water level in the reservoir was close to the normal water level [39].Larger water level variations were observed in 2021.Especially pronounced was the drop in the water level to 456.20 m in the first half of May resulting from both the increased discharges of the Irkutsk HPP, and contrast weather conditions of the spring of 2021, with frequent colder weather, extended snowmelt leading to losses of water by evaporation and, as a result, a slow increase in water inflow into the reservoir.In May, HCO 3 − exhibited the greatest variations.Moreover, its level was minimal throughout all sampling campaigns (Table S2).From June to September, there was a steady increase in the water level (up to 457.22 m) due to a decrease in discharge flows of the Irkutsk HPP and an increase in the water level of Lake Baikal.
In addition to natural factors, anthropogenic factors are also responsible for the water ionic composition in reservoirs.The anthropogenic impact may lead to increased concentrations of ions, mainly of Cl − and SO 4 2− [5,40].These ions were ascribed to the main pollutants in the southern part of Lake Baikal, whose runoff was found to significantly affect the chemical water composition at the source of the Angara River and in the Irkutsk Reservoir.For several decades, the sulfate concentrations in the water column of the South Baikal and in the Angara River source had been significantly affected by the wastewater from the Baikalsk Pulp and Paper Mill (BPPM) closed in 2013 [41].In addition to SO 4 2− , large amounts of Cl − enter (entered) the South Baikal from different sources: wastewater of BPPM, 7.3 ± 0.2 ton/year; wastewater of Ulan-Ude city-5274 ± 648 ton/year; polluted water of Selenga River (Lake Baikal largest tributary) 70.65 ± 9.91 tons/year [42].A number of suburban settlements along the shores of the Irkutsk Reservoir were likely another source of Cl − in the water of the reservoir.The potassium chloride used as a component of agricultural fertilizers is known to increase Cl − ion concentration in the aquatic system [43].With the data available, the contribution of fertilizers cannot be quantified.However, in the Irkutsk Reservoir, K + levels vary insignificantly over the entire monitoring period (0.91-0.96 mg L −1 as an average; Table S2); there is no correlation between the ions of chlorine and potassium.Therefore, this study revealed no contribution from agricultural fertilizers to the Cl − concentration at all sampling localities within the Irkutsk Reservoir.A larger influence on Cl − concentrations is likely to be recorded in the water of the bays as the areas adjacent to the bays are more developed.A large collection of data on trace element geochemistry in surface waters of Africa Europe, Asia, and North and South America are presented in [44].The average concentrations of Al, Mn, Fe, Co, Cu, As, Cd, Cs, and Th in the Irkutsk Reservoir were markedly lower than the average world value; Cr levels were lower than their minimum values and the concentrations of Zn, Pb, and U exceeded the corresponding world average but were below the worldwide maximum values (Table 2).A large collection of data on trace element geochemistry in surface waters of Africa, Europe, Asia, and North and South America are presented in [44].The average concentrations of Al, Mn, Fe, Co, Cu, As, Cd, Cs, and Th in the Irkutsk Reservoir were markedly lower than the average world value; Cr levels were lower than their minimum values and the concentrations of Zn, Pb, and U exceeded the corresponding world average but were below the worldwide maximum values (Table 2).When studying the trace element water chemistry of the Irkutsk Reservoir, it is reasonable to compare the trace element characteristics of the reservoir with those of Lake Baikal (Table 2).The data on hydrochemistry of Lake Baikal showed that like the ionic, the trace element composition was stable at all depths in the pelagic zone of the lake [46].At the same time, its hydrochemical characteristics were influenced by natural (multi-component flows of more than three hundred rivers of its catchment area, hot and cold springs [49], and anthropogenic factors as well as by seismic activity [50], etc.).The effect of anthropogenic factors was demonstrated by the chemical composition of snow cover from the lake's water area near the settlements [51].The results revealed that the snow chemical composition was characterized by higher concentrations of Mn, Al, Pb, Cu, Fe, and Zn in the southern part, increased Mn, Al, Pb, Cr, Fe, and Zn levels in the middle part, and higher Mn, Al, Pb, Cu, Cr, and Fe contents in the northern part of Lake Baikal.Long-term monitoring of concentrations of major ions [38] and mercury [50] in the Angara River source indicates that hydrochemical characteristics of water from the river's source characterize the average water composition of the entire Lake Baikal.Therefore, when studying trace element water composition in the Irkutsk Reservoir, the Angara River source is considered separately.
In different periods of studies [20,46,52], the concentrations of the vast majority of trace elements were in good agreement with each other, except for Zn and Th (Table 2).In the water of Lake Baikal, the Zn concentration measured by Sklyarova [46] (0.56 µg L −1 ) is much lower than its abundance determined by Falkner et al. [52] (2.9 µg L −1 ) and Vetrrov et al. [20] (4.3 µg L −1 ).For thorium, this situation was quite different: Th levels given in [20] (0.004 µg L −1 ) were an order of magnitude higher than its concentrations measured by Sklyarova [46] (0.0006 µg L −1 ).In the water of the Angara River source, Zn concentrations were in line with its abundances in Lake Baikal obtained by Vetrov et al. [20], while Th levels were in good agreement with Th contents in Lake Baikal measured by Sklyarova [46].The concentrations of As and U measured in the Angara source are well comparable with their levels in the water from the pelagic part of the lake (Table 2, Figure 3).The levels of Al, Cr, Mn, Fe, Co, Cu, Cd, Sn, Cs, and Pb in the water of the river source were higher than those in the lake's water.The results of this study are in line with the data on trace element compositions obtained from 3-year (2006-2008) monthly water monitoring of the Angara River source [45].
At the source of the Angara River in different sampling campaigns of 2021, higher Al, Mn, Fe, Co, Cu, As, Cd, Sn, Cs, Pb, Th concentrations were found along the left shore of the river as compared with its center, while increased levels of Cr, Mn, Fe, Co, Cu, Zn, As, Cd, Sn, and U were observed along the right shore (Figure 3).In June, the water of the middle part was characterized by higher Al, Cr, Fe, Cu, and U contents as compared with the shores.The anthropogenic impact on this part of the reservoir is primarily related to the activity of the cargo and passenger port, whose ships and ferries run between the left and right banks all year round.Antifouling paints, metals, and steel alloys, as well as petroleum products, are an important source of trace elements in the water bodies near the shipyards and yacht clubs [53][54][55].The area close to the Listvyanka settlement with a well-developed tourist infrastructure may also be a main source of trace elements in this part of the Irkutsk Reservoir.The study of surface and groundwater in the vicinity of the settlement revealed that the anthropogenic impact resulted in the pollution of primarily groundwater, whose subaqual discharge led to higher Mn, Zn, and Pb concentrations in the near-bottom water of the lake's coastal zone [56].The Baikal under-ice water taken close to the Listvyanka settlement was characterized by higher Mn, Cu, Co, Al, Fe, and Zn contents as compared with the deep-seated water [57].The changes in the chemical composition of coastal waters are thought to be related to the dissolution of rocks and soils, mechanical transport of the detrital material from the coastline, and atmospheric transport.
Water 2023, 15, x FOR PEER REVIEW 10 of 21 the near-bottom water of the lake's coastal zone [56].The Baikal under-ice water taken close to the Listvyanka settlement was characterized by higher Mn, Cu, Co, Al, Fe, and Zn contents as compared with the deep-seated water [57].The changes in the chemical composition of coastal waters are thought to be related to the dissolution of rocks and soils, mechanical transport of the detrital material from the coastline, and atmospheric transport.(1-May, 2-June, 3-September).a -concentrations of trace elements, except for Th, in Lake Baikal water are given from [20], Th concentrations-are taken from [46].
Irkutsk Reservoir
As opposed to major ions, the trace element concentrations showed a more significant spatial-temporal variability (Tables 1 and 2).In the vast majority of water samples, the concentrations of trace elements were found to lie within mean ± 2SD.We calculated the median, which is unlike the mean and is not sensitive to the outlier measured values.Table 3 illustrates sampling sites, where the trace element concentrations were above 2SD of the median.In some samples, trace element concentrations were found to exceed the median + 2SD: Al-7 samples; Cr-6 samples; Mn-8 samples, Fe-8 samples, Co-6 samples, Cu-7 samples; Zn-12 samples; Cd-3 samples; Sn-12 samples, Cs-7 samples; Pb-11 samples; Th-4 samples.(1-May, 2-June, 3-September).a -concentrations of trace elements, except for Th, in Lake Baikal water are given from [20], Th concentrations-are taken from [46].
Irkutsk Reservoir
As opposed to major ions, the trace element concentrations showed a more significant spatial-temporal variability (Tables 1 and 2).In the vast majority of water samples, the concentrations of trace elements were found to lie within mean ± 2SD.We calculated the median, which is unlike the mean and is not sensitive to the outlier measured values.Table 3 illustrates sampling sites, where the trace element concentrations were above 2SD of the median.In some samples, trace element concentrations were found to exceed the median + 2SD: Al-7 samples; Cr-6 samples; Mn-8 samples, Fe-8 samples, Co-6 samples, Cu-7 samples; Zn-12 samples; Cd-3 samples; Sn-12 samples, Cs-7 samples; Pb-11 samples; Th-4 samples.There are several features that characterize the influence of Lake Baikal runoff on the water trace element composition in the Irkutsk Reservoir.Firstly, during all sampling campaigns, median concentrations of Al, Cr, Mn, Co, As, Th, and U across the reservoir were close and the median Cu, Zn, Cd, Sn, Cs, and Pb contents were lower than those measured at the source of the Angara River.Secondly, the concentrations of Zn and U in the water of both the Irkutsk Reservoir and Lake Baikal exceeded the average value for the world's surface water (Table 2).Higher U concentrations (similar to maximum), as compared with the average world values, and increased Zn levels (similar to mean ones) in the water of Lake Baikal characterize its geochemical background in the area surrounding the lake [46].Thirdly, the concentrations of trace elements in the water of the Irkutsk Reservoir decreased in the following order Fe > Mn > Al, Zn > Cu, U > As > Pb > Cr > Cd, Sn > Co > Cs > Th, which is similar to the pattern found for the water at the Angara River source.
When comparing the channel part and the bays of the reservoir (Table S3), the number of elements showing higher concentrations was larger in the surface water of the bays.It is, in particular, true for the Kurma Bay (S4), whose water area is widely used for fishery, Burduguz (S3), and Uladova (S5) Bays (Table 3).In the channel part, higher concentrations of trace elements are primarily found opposite the Mel'nichnaya Pad' settlement (S9).Water transport, which is rapidly developing at present, was another anthropogenic factor influencing the water hydrochemistry of both the Irkutsk Reservoir and the Angara River source.In addition, a number of suburban settlements, which are not connected to the urban sewer system or septic tanks for treating household waste, are located along the coastline of the bays in the immediate vicinity of the water.Untreated waste can significantly affect the water quality in waterways connected to the reservoir [58].In the absence of wastewater treatment facilities, the entry of pollutants into the Irkutsk Reservoir with both surface (meltwater and rainwater flowing from coastal settlements) and underground runoffs can be significantly higher.The elements, whose concentrations were higher at the majority of sampling sites included Mn, Fe, Al, Zn, Sn, Pb, Cu, and Cs (Table 3).Giri and Singh [59] related the enhanced Mn, Pb, and Zn levels in the waters of the Subarnarekha River (India) to industrial wastes and transport pollution.In coastal waters of China, Pb and Zn abundances were found to be closely associated with motor transport [60].The Pb and Zn levels in the Aibi Lake are mainly influenced by agricultural fertilizers, emissions from traffic, and/or urban construction in its basin [61].
The results from this study indicate that enhanced Al, Mn, Fe, Cu, Zn, As, and Cd levels were observed along the right and left banks of the Irkutsk Reservoir, close to Irkutsk city (Figure 4).In July and September, Pb concentrations significantly increased in relation to May.A variety of anthropogenic activities in urban areas led to the accumulation of a wide scope of trace elements, including Cd, Cu, Pb, and Zn [62] in the upper soil layer.From the area near Irkutsk, trace elements of anthropogenic origin enter the reservoir with the surface runoff.Therefore, the trace element concentrations can increase, primarily in the aquatic environment along the shores.In the vicinity of Irkutsk, the anthropogenic impact on the left shore is mainly due to the city beach, while on the right shore, this impact is related to a passenger port and parking for private water transport (about 200 small vessels and yachts).Herewith, near Listvyanka settlement, the navigation takes place all year round, but near the HPP dam, water transport activity and the growing number of tourists are observed in the warmer season (July-August).
Another source of trace elements in the reservoir, in particular major elements of the Earth's crust, is the abrasion of shores.The creation of the reservoir facilitated the processes of shore erosion, both in the reservoir itself and on the shores of Lake Baikal.Within the Irkutsk Reservoir, the shores, which are not exposed to erosion, lie close to the source of the Angara River, while the abrasive shores formed in the Jurassic sandstones and Quaternary sediments make up about 54% of the reservoir's shoreline [63].The composition of the bottom sediments in the reservoir reflects the geochemical specifics of rocks, which compose the shores exposed to impacts from processes of erosion.Along the banks composed of the Jurassic sandstones, the bottom sediments showed the dominance of Mn, Cr, V, Zn, Cu, while on the banks composed of the Quaternary diluvial loess loams the bottom sediments contained higher abundances of Mn, Co [64].It was found that, with the material from the coastal erosion, the reservoir receives 4700 tons of Fe, 154 tons of Mn, and about 220 tons of other trace elements each year [64].The terrigenous material, entering the bottom of the reservoir due to erosion processes, is able to suspend in the water column as particles of different fractions for a long time leading to higher Al, Mn, Fe, Co, as well as Cr, Zn, Cu levels in the water of the Irkutsk Reservoir.
The retrospective impact of human activity on the reservoir can be assessed by studying the composition of bottom sediments, which are active accumulators of elements of anthropogenic origin [65,66].In the Bratsk Reservoir, the decrease in anthropogenic impact led to better water quality characteristics [47].However, the results of layer-by-layer sampling of its bottom sediments showed the accumulation of the vast majority of trace elements in the middle layers of the bottom sediments formed during the period of the greatest anthropogenic impact [67].In this regard, the bottom sediments of the Irkutsk Reservoir have not been sufficiently studied.The most complete information on the chemical composition (30 elements) of its bottom sediments is given in Jagus et al. [68].In four sectors of the reservoir (Tal'tsy, Patrony, Novogrudinina, and Mel'nichnaya Pad' settlements) we analyzed 10 samples of bottom sediments.Amongst the elements discussed in this study, Co, Pb, and Cr are worthwhile and noteworthy: Co and Pb demonstrate different spatial distribution patterns and Cr had higher concentrations as compared to its levels in other dammed reservoirs worldwide.At the same time, in Cartagena Bay, the bottom sediments taken at stations related to the repair and maintenance of ships were characterized by high concentrations of Cr, Cu, As, and Cd while sediments from stations receiving inputs from petroleum plants displayed high Pb levels [69].Therefore, it can be suggested that higher concentrations of trace elements in near-bottom water layers of the Irkutsk Reservoir, particularly close to the dam (Table 3), may be a result of their release from bottom sediments.
Water 2023, 15, x FOR PEER REVIEW 13 of 21 els in other dammed reservoirs worldwide.At the same time, in Cartagena Bay, the bottom sediments taken at stations related to the repair and maintenance of ships were characterized by high concentrations of Cr, Cu, As, and Cd while sediments from stations receiving inputs from petroleum plants displayed high Pb levels [69].Therefore, it can be suggested that higher concentrations of trace elements in near-bottom water layers of the Irkutsk Reservoir, particularly close to the dam (Table 3), may be a result of their release from bottom sediments.
Correlation Analysis
The Pearson correlation coefficient is widely used to determine general sources of elements' entry [70,71].A number of significant positive and significant negative correlations were obtained using the information from the study of trace element composition of water in the Irkutsk Reservoir (Table 4).It was found that correlation relationships between elements were not stable across sampling campaigns in different years.At the same time, there were pairs of elements preserving significant correlation during 3 or 4 sampling campaigns.Amongst major ions, these pairs included HCO3 − vs. Са 2+ , SO4 2− vs. Mg 2+ .The positive correlation between HCO3 − vs. Ca 2+ in the Irkutsk Reservoir, found also in the Boguchany Reservoir, being the Angara Cascade Reservoir as well [72]), is characteristic of freshwater calcium bicarbonate composition [73].Of the trace elements, the pairs with a positive correlation included: Al-Mn, Al-Fe, Al-Co, Al-Th, Mn-Fe, Co-Fe, and Pb-Cr.Aluminum, which is the most abundant metal in the Earth's crust, is noteworthy [74]).
Correlation Analysis
The Pearson correlation coefficient is widely used to determine general sources elements' entry [70,71].A number of significant positive and significant negative correlations were obtained using the information from the study of trace element composition of water in the Irkutsk Reservoir (Table 4).It was found that correlation relationships between elements were not stable across sampling campaigns in different years.At the same time, there were pairs of elements preserving significant correlation during 3 or 4 sampling campaigns.Amongst major ions, these pairs included HCO 3 − vs. Ca 2+ , SO 4 2− vs. Mg 2+ .The positive correlation between HCO 3 − vs. Ca 2+ in the Irkutsk Reservoir, found also in the Boguchany Reservoir, being the Angara Cascade Reservoir as well [72]), is characteristic of freshwater calcium bicarbonate composition [73].Of the trace elements, the pairs with a positive correlation included: Al-Mn, Al-Fe, Al-Co, Al-Th, Mn-Fe, Co-Fe, and Pb-Cr.Aluminum, which is the most abundant metal in the Earth's crust, is noteworthy [74]).The positive correlation of Al with Fe, Mn, Co, as well as of Fe with Mn, Co, Mg 2+ over several sampling campaigns showed that these elements can enter the water environment of the Irkutsk Reservoir as a result of weathering of the parent material, pedogenesis, and erosion activity.The data concerning Th concentrations in rocks, composing the drainage area of the Irkutsk Reservoir, are still lacking.At the same time, maximum Th levels in the bottom sediments of the Irkutsk Reservoir (12.9 ppm) exceeded the values of the geochemical background of sedimentary rocks in the region [68].The significant positive correlation between Th and Al over three sampling campaigns might indicate that bedrock, composing the shores of the reservoir, is probably the main source of thorium entering the reservoir.The positive correlation between Pb and Cr, determining the relationships between these two elements, reflects that the combustion of petroleum or gasoline used for water transport is probably the main mechanism by which these trace elements enter the water reservoir.
Trace Element Pollution and Analysis of Water Quality
Water supply to the population in Irkutsk and smaller settlements of the Irkutsk district is provided from the water intake, located in the Ershi Bay.Residents of suburban settlements living on the shores of the reservoir, in the spring, summer, and autumn use submersible pumps for pumping water from nearby bays.Currently, in Russia, the requirements for the quality of drinking water are based on maximum permissible concentrations (MPC) of trace elements and are regulated by the Sanitary Rules and Norms for Drinking Water [48].Concentrations of all the elements under consideration in the water of both the Irkutsk Reservoir and Lake Baikal are substantially below the standards for drinking water (Table 2).
An important tool that helps to assess surface water pollution is the choice of a criterion that should be used as a control material (geochemical background) [75].At the same time, such a criterion is to be chosen on both local and global scales [71].As the Irkutsk Reservoir is a part of the unified Baikal-Angara water system, the summarized hydrochemical characteristics of Lake Baikal which include the average concentrations of trace elements at the source of the Angara River, can be used as a control material.
suggest that the water of the Irkutsk Reservoir still copes with the anthropogenic load.The impact of anthropogenic activity could likely be more significant in the coastal areas of the reservoir.The lack of water treatment facilities makes the source of drinking water supply not protected from the anthropogenic impact.Human activity in the reservoir and on its coasts may affect the unique physical and chemical properties of the water, as well as the aquatic flora and fauna inhabiting the reservoir.All of the above underlines the need to take environmental protection measures aimed at preventing irreversible changes affecting the water quality and aquatic organisms, primarily endemic Lake Baikal species, poorly tolerant to habitat transformation.
Figure 1 .
Figure 1.(а) Location of the Irkutsk Reservoir.(b) Schematic map with indicated sampling sit
Figure 1 .
Figure 1.(a) Location of the Irkutsk Reservoir.(b) Schematic map with indicated sampling sites.
Water samples of the Irkutsk Reservoir were classified as low contaminated: in 2007 by the concentrations of Cd, Sn, Zn, and Cs; in 2012, May 2021-by the concentration of Cs, and in September 2021-by the concentration of Th.Water samples were classified as low and moderately contaminated: in 2007 by concentration of Cr, Mn, Co, Cu, As, Pb, and U, in 2012-by concentration of Cr, Cu, Zn, As, Cd, Pb, Th, and U, in May 2021-by concentration of Cr, Zn, As, Cd, Sn, Pb, Th, and U, in July 2021-by concentration of Cu, Zn, As, Pb, Th, and U, in September 2021-by concentration of Cr, Co, Cu, Zn, As, Cd, Sn, Cs, and U. Part of the water samples revealed considerable and very high contamination: in 2007 by concentrations of Al, Fe, Th, in 2012-by concentrations of Al, Mn, Fe, Co, and Sn, in May 2021-by concentrations of Al, Mn, Fe, Co, and Cu, in July 2021-by concentration of Al, Cr, Mn, Fe, Co, Cd, Sn, and Cs, in September 2021-by concentrations of Al, Mn, Fe, and Pb.
Table 1 .
Comparison of major ion concentrations (mg L −1 ) in water of the Irkutsk Reservoir and other ponds of the Baikal-Angara water system.
Table S3 and
Figure 2 present statistics of trace element concentrations in 2007, 2012, May, June, and September 2021 in the Irkutsk Reservoir waters.Table 2 shows the data summarized for all sampling campaigns.
Table 2 .
Comparison of trace elements concentrations (µg L −1 ) in Irkutsk Reservoir and other ponds of the Baikal-Angara water system.
Table 3 .
Sampling sites, where trace element concentrations in water exceed median + 2SD.
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2023-12-05T16:53:15.758Z
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2023-11-29T00:00:00.000
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Geometric metasurface enabling polarization independent beam splitting
A polarization independent holographic beam splitter that generates equal-intensity beams based on geometric metasurface is demonstrated. Although conventional geometric metasurfaces have the advantages of working over a broad frequency range and having intuitive design principles, geometric metasurfaces have the limitation that they only work for circular polarization. In this work, Fourier holography is used to overcome this limitation. A perfect overlap resulting from the origin-symmetry of the encoded image enables polarization independent operation of geometric metasurfaces. The designed metasurface beam splitter is experimentally demonstrated by using hydrogenated amorphous silicon, and the device performs consistent beam splitting regardless of incident polarizations as well as wavelengths. Our device can be applied to generate equal-intensity beams for entangled photon light sources in quantum optics, and the design approach provides a way to develop ultra-thin broadband polarization independent components for modern optics.
incident beam can be separated after passing through the metasurface as in typical Wollaston prisms. In this case, power ratio of diffracted beams is not identical, i.e. it depends on the incident polarization state. The Rochon prism which leaves ordinary ray undeviated can also be realized by controlling both propagation and geometric phase of each unit structure 35 , but its working frequency is strictly limited to a narrow band.
In modern optics such as quantum communication, equal-intensity coherent beams are usually required to generate entangled photon light sources [36][37][38][39] . Some of related works on metasurfaces to generate equal-intensity beams at NIR wavelengths regardless of incident polarization are reported 40,41 , but their working frequencies are limited to NIR region. In addition, the number of generated beams is not controllable, i.e. they always generate two diffracted beams. By attaching two equivalent metasurfaces with opposite linear phase gradient, equal-intensity beams can also be generated 42 . However, its functionality is highly affected by alignment of light source and the metasurface.
Here we propose and experimentally demonstrate GEM-based holographic beam splitter that always generates equal-intensity beams regardless of incident polarization at visible wavelengths. In principle, the number of generated beams which have equal-intensity from our device is controllable. Our device achieves both broadband characteristics and the polarization-independence by using Fourier holography on GEMs. The term of 'broadband' in this work does not mean consistent efficiency. Instead, it means that our device functionality can be maintained in a certain wavelength range because phase distribution of our metasurface does not change by incident wavelengths. We notice that a reversed conjugate image of Fourier hologram appears at the same image plane as the original image 43 . If the original image has origin-symmetry, the original and conjugate images can overlap exactly. Right circular polarization (RCP) and left circular polarization (LCP) generate the conjugate images each other from GEMs encoded by Fourier hologram, so we can realize GEMs that produce exactly the same outputs in any kind of polarization states by encoding an origin-symmetric image. The proposed device is experimentally demonstrated using hydrogenated amorphous silicon (a-Si:H), and diffracted beam power of the outgoing optical wave is symmetric regardless of polarization. The efficiency of our device can be further improved by using low loss materials in visible region such as crystalline silicon (c-Si) [44][45][46] , titanium dioxide (TiO 2 ) 47,48 and gallium nitride (GaN) 49-51 without changing our fundamental design principle. Our device and design approach can be applied to interferometry in classical optics as well as generation of entangled photon light sources in quantum optics.
Results
Our device belongs to the GEM; nevertheless, it exhibits consistent functionality regardless of the incident polarization ( Fig. 1a,b). Conventional beam splitters based on the GEM has been designed by linear phase gradient which leads to separation depending on the circular polarization direction 31,32,34 . Rotating antennas of GEMs generate opposite phase delays against a pair of orthogonal circular polarizations, i.e. RCP and LCP, so the optical response of the conventional GEM varies depending on the incident polarizations. Therefore, the power ratio between diffraction beams varies depending on the incident polarization states. To remove the polarization-dependence, Fourier holography is employed to exploit the conjugate functionality of RCP and LCP. The conjugate image of Fourier hologram appears in reverse at the same position as the original image. Nanoantennas based on the geometric phase generate opposite phase variation against RCP and LCP. When circularly polarized plane wave is incident to the GEM, complex amplitudes of transmitted cross-polarized light which are RCP and LCP immediately after the metasurface are given by LCP j x y ( , ) where x and y represent the coordinate of the metasurface. A(x, y) is the amplitude of the transmitted cross-polarized light, and φ(x, y) indicates the phase distribution of the metasurface. Amplitude variation by the metasurface can be ignored because all the nanoantennas in it are designed to induce the same amplitude change.
The only difference between the complex amplitude of RCP and LCP is the sign of the imaginary part, so they have a conjugate relation as
RCP L CP
Intensity distributions at the image plane can be expressed as where x′ and y′ indicate the coordinate at the image plane, and F{•} denotes the two-dimensional Fourier transformation operator. The conjugate symmetry of the Fourier transformation yields Finally, the intensity distributions of RCP and LCP are related as Therefore, if the encoded image is origin-symmetric, the generated images by RCP and LCP exactly overlap each other.
The origin-symmetric image of two white spots with a black background is encoded in our metasurface based on computer-generated phase-only Fourier holography (Fig. 1c). The original image has a size of 800 × 200 pixels, which is expanded to 1250 × 1250 pixels by using zero padding to increase the image fidelity. We use the Gerchberg-Saxton (GS) algorithm 52 to derive a desired phase distribution from the enlarged image by two hundred iterative processes combined with the initial random phase mask. The random phase mask is employed to the original image for easy convergence of the algorithm. It is usually attached to the object image to spread the energy over a wide spectrum to ignore the amplitude of a complex Fourier hologram 53 . Otherwise, most of the energy concentrates in the zeroth order beam and the effective dynamic range is reduced. The Fourier hologram can be represented in the complex form of H(x, y) = A(x, y)exp [−jϕ(x, y)]. If we can ignore the amplitude A(x, y), a gray-tone image can be generated by controlling ϕ(x, y) only, which refers to the phase-only hologram. The derived phase distribution is discretized into eight uniform phase steps from 0 to 2π. The arrangement of the metasurface elements according to the hologram corresponds to the discrete phase distribution from the GS algorithm. Eight uniform phase steps match eight uniform rotation steps of nanoantennas from 0 to π in our metasurface.
The phase profile of our metasurface is not equivalent of two diffraction grating placed next to each other. The phase profile retrieved from the GS algorithm is not origin symmetric because the random phase mask is attached to the original origin symmetric image of two spots. The value of each pixel of the image with the random phase mask becomes complex number which is no more origin symmetric. The phase profile of our metasurface looks linear, but one can easily notice that it is not exactly linear ( Supplementary Fig. S1). Orientation of the nanorods varies not only horizontally, but also vertically. The vertical variation results from the GS algorithm with the random phase mask. If our device has origin symmetric phase profile, the orientation of the nanorods should not be changed in vertical direction. The Fourier hologram uses a Fourier-transformed image to encode the hologram, so the outgoing optical wave from the hologram should be Fourier transformed again to recover the original image. A convenient way to conduct Fourier transformation of optical waves is to let the wave propagate to an infinitely-distant plane; this process works the same as a thin optical lens. Although an infinitely-distant plane does not exist in the real world, our metasurface is small enough, i.e. 300 μm × 300 μm, so an image plane a few centimeters away from the metasurface can be regarded as infinitely distant without causing significant error. The generated holographic image only experiences magnification without change of overall configuration along the distance from the hologram consistently maintaining two circular spots; hence, it works as a beam splitter generating equal-intensity beams.
The most attractive advantage of our device is that it can generate two or more equal-intensity beams simply by changing the encoded image. In this work, we use two point image with origin symmetry for the hologram encoding. If we use four point image with origin symmetry, we can obtain four equal-intensity beams. Theoretically, any even numbers of equal-intensity beams are possible while odd numbers are impossible due to the origin symmetry condition. The metasurface beam splitters based on one dimensional binary phase profile can also increase the number of beams by arranging two dimensional binary phase profile, but controllability is not as high as our approach because our device can use eight or more phase steps.
The a-Si:H as structuring material for our metasurface achieves a broadband functionality in visible wavelengths (Fig. 2a,b). The a-Si:H refers to amorphous silicon which has hydrogen impurities. The impurities decrease the defect density of amorphous silicon, so a-Si:H has a much lower extinction coefficient than typical amorphous silicon in visible wavelengths 54 (Fig. 2c). We also calculate the cross-polarization transmittance (CPT) of the unit cell made of the a-Si:H (Fig. 2d). The CPT is defined as the ratio of the power of the transmitted cross-polarized light to the total incident optical power, and the calculated CPT reaches up to 50% at the wavelength λ = 550 nm. The size and pitch of nanoantennas are determined based on the calculated CPT. The conversion efficiency increases as the CPT increases, so they are designed to maximize the CPT minimizing the reflectance (Supplementary Fig. S2). If we exploit other materials such as c-Si, TiO 2 and GaN whose extinction coefficient is lower than a-Si:H, the CPT of the metasurface can be further improved. The fabricated metasurface shows the consistent beam-splitting regardless of the incident polarizations (Fig. 3). The diffraction angle increases as incident λ increases, but is not affected by the incident polarization. The powers of diffracted beams are characterized by diffraction orders, incident polarizations and wavelengths. As a result, the power of each diffraction order of m = +1 and m = −1 is always equal for RCP and LCP as well as wavelengths. Arbitrary polarization states can be decomposed into the combination of RCP and LCP, so our device functionality is valid for the arbitrary polarization states. The normalized intensity difference is originated from the instability of the laser power; nevertheless, measured values by each polarization can be supposed to be equal within the error range. Total efficiency which is defined by the ratio of the sum of diffraction beam powers (m = +1, −1) to the incident beam power is 18.3% at λ = 532 nm and 9.1% at λ = 635 nm. This difference agrees with the fact that the CPT is higher at λ = 532 nm than at λ = 635 nm wavelength. Diffraction efficiency which is defined by the ratio of the sum of diffraction beam powers to the total transmitted beam power at λ = 532 nm is 10 times higher than at λ = 635 nm. The difference of the diffraction efficiency mainly comes from the incident beam size and shape. In the case of 635 nm, we use a laser diode which has elliptical beam shape (Fig. 4). If we use this source to cover the square shaped metasurface, considerable amount of incident light does not pass the metasurface resulting diffraction efficiency decrease. It causes the difference as well as the discrepancy with the simulation. However, it does not affect our device functionality because our device still generates same powered diffraction beams.
The intensity distributions of each diffracted beam are also measured using a charge-coupled device (CCD) (Fig. 4). Regarding of the saturation, a power meter is used to measure the beam powers while the CCD is used to capture the intensity profiles. The CCD is saturated to clearly show the beam profiles and sizes, but the power meter is not saturated because the incident beam power is less than the measurement limit of the power meter. Optical power is limited to a certain boundary, but randomly distributed due to the appearance of laser speckles. The strong coherence of the laser source causes the speckles, and this disturbance can be reduced using a diffuser 48 or Dammann grating 9,55 .
The broadband polarization independent beam splitting can be realized by using circular pillar based metasurfaces which does not belong to the GEM 56 . However, our approach as a GEM has advantages of robustness to the fabrication error as well as intuitive design process to achieve broadband functionality. Phase variation from the circular pillar based metasurfaces is strongly affected by the size of structures which always has discrepancy with the design due to the perturbation in fabrication conditions. In contrast, phase variation from the GEM is determined by the orientation of the nanorods which is less sensitive to the fabrication error. This is a general advantage of GEMs. In addition, by using GEM we can achieve broadband characteristics without any further algorithm.
In summary, we investigate and experimentally demonstrate a holographic beam splitter that generates equal-intensity beams with incidence of any polarization at visible wavelengths. In contrast to conventional metasurface beam splitters of linear phase gradient, Fourier holography is exploited to maintain the consistent functionality for incident polarization. A perfect overlap of an original and a conjugate image of Fourier hologram is a key concept of our device principle. Theoretically, the number of the generated beams can be controlled by replacing the two point image into others, e.g. four point and eight point images. The fabricated metasurface based on a-Si:H performs consistent beam splitting regardless of incident polarizations as well as wavelengths, and the efficiency can be improved by using low loss materials. Our device can be exploited to generate two or more entangled photon light sources in quantum optics, and design approach provides a platform for broadband polarization independent optical components of modern optics. where A is the strength of the absorption peak, B is the broadening term, E 0 is the energy position of the absorption peak, and E G is the optical band gap energy. The fitting parameters for our material were A = 93.974 eV, B = 2.339 eV, E 0 = 4.128 eV, and E G = 1.271 eV. The mean square error between the fitting curve and the measured data was 11.123. Diffracted beams have random distributions because of laser speckles, but are located within a certain boundary that can be regarded as the beam size. The image plane is located 14 cm behind the metasurface, and the beam diameters can be estimated as ~3 mm for λ = 532 nm and ~5 mm for λ = 635 nm. Corresponding beam diverging angles are calculated from the estimated beam diameters as 2.5° and 4.1°, respectively. Zerothorder distributions show that incident beam sizes are bigger than our metasurface, so some of incident light go directly to the laser power meter without any interaction with the metasurface. Therefore, the measured diffraction efficiencies become lower than the simulation results.
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2018-06-22T13:24:59.063Z
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2018-06-21T00:00:00.000
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Beyond the Provisional Nature: Towards a Radial Concept of Practice
Practice is one of the key concepts of our time. During the last 30 years, the Practice-Based Studies have spread all over the world. The “rediscovery” of practice has inspired many reflections and empirical researches, contributing to the recent development of the organizational and management studies. This article aims, on the one hand, to reconstruct the debate around the concept of practice and, on the other hand, discuss its meaning, as defined within the PBS. In this paper, it will be argued that practice should be conceived as a “radial concept”. This idea, coming from the political science, is the key to explore new universes of meaning and to read and compare the practitioners' activities, the functioning of organizations and the collective learning processes. The paper will introduce the phenomenon of the so-called Observatories of Civil Justice. These interprofessional communities represent a unique window on the Italian judicial system. This case study enables to critically analyse the concept of practice and to highlight its subtypes: experimental practice, solipsistic practice, diffused practice, concerted practice, fixed practice, institutionalized practice.
Our parents were accustomed to leaf through the pages of an encyclopaedia; unlike them, if we want to know something or understand a concept, in most cases we rely on a search engine on-line.In particular, Google and Yahoo have become a sort of "prophets" of our time.
Typing on a search engine the word "practice", in less than half a second, we have access to over a billion results, articulated in a confused way in a great number of definitions, contributions and references of any kind.The famous image of the Tower of Babel, narrated in Genesis, in some ways describes the complex "world" of languages, ideas and perspective that can be found in the search of information on the concept of practice.This babel of information inevitably generates confusion, overlaps and distortions.
Despite not having a universally accepted definition, practice is one of the key concepts of our time, a sort of fulcrum of many other expressions, which is used many times in the language of everyday life, by the media, in training activities, within the professions, by the scholars from different disciplines, and especially in the broad panorama of organizational and management studies.
In this framework, the article aims, on the one hand, to reconstruct the debate around the concept of practice and, on the other hand, discuss its meaning as defined within the socalled Practice-Based Studies (PBS).
In this paper, it will be argued that the "practice" should be conceived as a "radial concept".This idea, coming from the political science literature, emphasizes the fact that some concepts may have different subtypes (Ostiguy, 1993;Collier & Mahon, 1993;Schedler, 1999;Lindberg, 2009).The attributes contribute to define the "semantic core" of the primary category.These subtypes are not always clearly distinct and often overlapping each other (Lindberg, 2009).The relationship between the general concept and its attributes is the key to explore new universes of meaning.Defining practice as a "radial concept" has a considerable heuristic value, in order to read and compare the practitioners' activities, the functioning of organizations and the collective learning processes.
The article is divided into seven main sections.The first paragraph will trace the debate's roots, which may be identified in the emergence of a new way of understanding knowledge, understood as something social, situated and procedural (knowing).The second Teoria e Prática em Administração, v. 6, n. 2, 2016 Beyond the Provisional Nature: Towards a Radial Concept of Practice Verzelloni, Luca DOI: http://dx.doi.org/10.21714/2238-104X2016v6i2-31614paragraph will deal with the rediscovery of practice, which developed as a result of the overcoming of the paradigms of the rationalism, cognitivism and functionalism.The third paragraph will discuss the research questions of this paper, which are based on the idea to move beyond the provisional nature of practice.The fourth paragraph will present the phenomenon of the "Observatories of Civil Justice".This case represents a unique window on the Italian judicial system.The fifth paragraph will discuss the empirical data and it will identify some subtypes of practice.The conclusions will highlight some possible research horizons, based on a new "radial concept" of practice.
The paper is based on the results of a long period of empirical research on the Observatories of Civil Justice.The analysis was launched in 2006.It has considered 19 Observatories, operating in various Italian judicial offices (north, center and south): Avellino, Bari, Bologna, Cagliari, Catania, Florence, Genoa, Messina, Milan, Modena, Naples, Reggio Calabria, Rome, Salerno, Turin, Trento-Rovereto, Trieste, Udine and Verona.From a methodological point of view, the research has used qualitative methods, such as semistructured interviews, participatory observations and documental analysis.The article will refer only to civil sector and will not consider the Italian criminal justice.
From "knowledge" to "knowing"
The overcoming of the paradigms of the rationalism, cognitivism and functionalism marked an epochal change in the organizational and management studies (Bacharach, Gagliardi, & Mundell, 1995;Czarniawska & Joerges, 1995).The idea of an organization as a "machine", which decides in an efficient and effective way, considering all the variables of the situation, has been replaced with the image of a real organization that works by trials and errors (March & Simon, 1958).
In order to understand the complexity of an organizational context is not enough to analyse its formal structure, but it is necessary to investigate the processes of construction and sharing of knowledge.Knowledge cannot be conceived as a variable kept in the people's minds or in the organizational charts or in the databases, but it is something social, situated and procedural (Gherardi, 2000).The discourse about the nature of knowledge dates back to the origin of philosophy and during its evolution has involved numerous authors, from different theoretical perspectives.Far from wanting to trace the entire development of this wide-ranging debate, it is possible to highlight some fundamental steps.
In this sense, Polanyi is undoubtedly a reference point.The author's work is based on a critique of the objectivity, which is prevalent in many fields of knowledge (Polanyi, 1958(Polanyi, , 1967)).Since every human act involves an evaluation, it is necessary to overcome the idea of the existence of an objective knowledge.Knowledge crystallizes itself in the practical activities.The touch of a pianist, for example, cannot be explained in a manual or in a mathematical formula; acquire the "right touch" is something that can be transmitted to an apprentice only through the master's example.Knowledge has a tacit, aesthetic, intuitive and sensory component, which cannot be explained (Polanyi, 1958;Strati, 1999) -quoting the famous Polanyi's sentence: "we can know more than we can tell" (1967: 4).The same existence of the apprenticeship indicates very clearly that alongside to the abstract, explicit and codified knowledge, there is a tacit, implicit and sensory knowledge, which with the experience becomes part of eyes, ears and hands of the expert practitioner (Goodwin, 1994(Goodwin, , 2003;;Freidson, 2001).
Secondly, Bourdieu's idea of habitus is an important turning point in the studies on the knowledge (1972)."Knowing" is not only an individual process, but also a social activity.
People develop a specific "sens pratique" (Bourdieu, 1980).Like a chess player, every person adapts her/his role to the real-life circumstances.In this sense, knowledge is a "symbolic capital" founded on social interaction.
Thirdly, it is necessary to remember also the Suchman's concept of "situated action" (1987).Knowledge cannot be considered as something abstract and decontextualized, but it has a situated dimension.Each action course depends on the development of the socialmaterial circumstances.Knowledge is continuously created and recreated in a complex microcosm of actors, technologies and socio-material artefacts (Suchman, 1987).
Finally, in the literature, it is possible to identify five types of knowledge: embodied, embedded, embrained, encultured and encoded (Collins, 1993).Blackler has proposed to switch from the study of "knowledge" to the study of "knowing" (1995).The author has outlined five characteristics of this process: knowing as mediated (by language, technology, collaboration and control); knowing as situated (in a specific time and space); knowing as provisional (constantly changing); knowing as pragmatic (purposive and object-oriented); knowing as contested (in conflicts between individuals).
Knowledge is created and re-created through the processes of individual and collective learning (Argyris & Schön, 1978).Knowledge, in fact, is intrinsically linked to the practical action (knowing-in-practice) (Gherardi, 2000(Gherardi, , 2006) ) and individuals develop a specific "competence-to-act" through their mutual interactions (Gherardi & Nicolini, 2002).
The Rediscovery of Practice
In the light of these considerations, during the last 30 years, the so-called "Practice-Based Studies" (henceforth also PBS) have spread all over the world.These approaches -in the plural form -have become a sort of "bandwagon" (Fujimura, 1988;Corradi et al., 2010).
The common meaning of "practice" has a plurality of acceptations (Corradi et al., 2010, p.
279):
a learning method; an occupation or field of activity; the way something is done.
In the academic debate, the notion of practice was used simultaneously as an "empirical object" and as a "way of seeing" the processes of organizational learning (Corradi et al., 2010).
Practice is both the field where observe the everyday activities of practitioners, that an epistemology (Raelin, 2007) -as explained effectively by Cohen (1996), who introduced the distinction between "theories of action" and "theories of praxis" -to explore the "social orderliness of local settings" (Garfinkel, 1967;Rawls, 2000: 547;Fox, 2006).
In general, Gherardi has defined the concept of practice as: "a mode, relatively stable in time and socially recognized, of ordering heterogeneous items into a coherent set " (2006: 34).
In this sense, there are no distinctions among doing, thinking, deciding, knowing, organizing, learning, etc.People learn-by-doing, in the mistakes, by repetition, developing and testing new ways of practicing.
Through the processes of legitimate participation (Lave, Wenger, 1991), the newcomers are socialized to practice and they become part of the social group in which operate (Becker, 1953).Every organizational context is a "constellation of interconnected practices" (Gherardi & Nicolini, 2002).Despite the criticism, the concept of "community of practice" (CoP) has influenced the entire spectrum of studies on organizational learning (Lave, Wenger, 1991;Wenger, 1998;Brown, Duguid, 2001;Swan, Scarbrough, & Robertson 2002;Contu & Willmot, 2003;Roberts, 2006;Amin & Roberts, 2008).As defined by Wenger: "communities of practice are groups of people who share a concern or a passion for something they do and learn how to do it better as they interact regularly" (Wenger, 1998, p. 84).In the literature, there are various definitions and applications of the concept of community of practice.Amin and Roberts have identified four basic varieties of CoPs: craft/task based (aesthetic, kinaesthetic and embodied knowledge); professional (specialised knowledge); expert/creative (expert knowledge); virtual (codified and tacit knowledge) (Amin & Roberts 2008, p. 357).
In this sense, the discursive practices are fundamental to analyse human behaviour.The language, in fact, is the basis of learning (Vygotskij, 1934;Wittgenstein, 1953), because "it is not only the expression of social relations; it is also the medium for their creation" (Czarniawska-Joerges, 1991;Gherardi, 2006: 23).
In general, every field of practice is an "actor network", which it is composed by human and non-human actors (Callon, 1987;Latour, 1987), connected together in "ecologies of sociomaterial relations" (Fujimura, 1995).Artefacts, texts, rules and technologies act as "material intermediaries" (Law, 1987).
Beyond the Provisional Nature of Practice
The concept of practice is usually conceived as something provisional and ephemeral.
People build a texture of practices that is ever changing.Practice, as defined within the PBS, is "relatively stable" (Gherardi, 2006), being constantly undermined by the activity of the practitioners.
In this sense, the description of a practice is extremely difficult, because it looks like to take a photo of a moving object, or in other words, to get a freeze-frame shot.Practices are developed by repetition: over time, some practices change, others disappear and being replaced or forgotten.These "circuits of reproduction" involve new and old practices, connecting each other practices, "proto-practices" and "ex-practices" (Pantzar & Shove, 2006).However, each practice is always inserted into a specific cultural-historical context.As highlighted by the Cultural-Historical Activity Theory (Engeström, 1987;Blackler, 1995;Miettinen & Virkkunen, 2005) and by the Soviet Cultural-Historical Psychology (Vygotskij, 1934;Leont'ev, 1981), it is necessary to consider the "system of activities".This system is a social construction, rooted in a particular time and space; it includes objects, subjects, mediating artefacts (as signs and tools), rules, communities and division of labour (Engeström, 1987).
In the light of these considerations, since not all practices have a temporary nature, this article raises some research questions: is the concept of practice able to include both provisional and institutionalized practices?How is it possible to distinguish the practices?And, in general terms, how is it possible to redefine the concept of practice?
The Observatories of Civil Justice
The context: a never-ending season of reforms Over the past 30 years, several factors had a negative impact on the Italian judicial system, such as: the increasing rates of litigation, the lack of human, financial and material resources, the unreasonable duration of the judicial proceedings and the atmosphere of distrust of the citizens (Castelli et al., 2014).
In recent years, European Union, Council of Europe, World Bank and OECD have put great pressure on the Italian government to introduce some judicial reforms to improve efficiency and quality of justice (Piana, 2016).The Bank of Italy has estimated that the overcoming of these difficulties would represent a gain of more than a percentage point of the national GDP.This topic has established itself into the public debate and it has influenced the political agenda.
In the last 20 years, 22 major reforms of the Code of Civil Procedures (c.c.p.) have been introduced by the 13 Governments, which have followed each other in Italy.In particular, from 1995 to 1998, 10 major reforms were adopted.These changes have been so fast, repeated and significant that some authors have spoken of a "tsunami of civil justice reforms" (Costantino, 2005(Costantino, -a: 1167)).Some articles of the Code have been changed several times: for example, the article 420 c.c.p. on the labour proceedings has been rewritten 3 times in the space of 22 months.This never-ending season of reforms has had a direct impact on the legal professionals.
In particular, lawyers and judges were constantly asked to adapt their activities to the changes of the "rules of the game", with obvious repercussions on courts' functioning.
The issue is part of a broader discussion.According to several authors, some of the gravest difficulties of the Italian judicial system derive from organizational problems (Di Federico, 1975, 2008;Zan, 2003Zan, , 2011;;Marchesi, 2003;Biavati et al., 2008;Verzelloni, 2009;Piana, 2016).In particular, the Italian judicial offices often look like a condominium (Zan, 2003), or an island archipelago, populated by various practitioners: judges, clerks, lawyers, etc. -all of them operating independently.In these contexts, there are not collective learning occasions and the legal professionals are comparable to monads, who produce autonomous and solipsistic interpretations of the written rules (Verzelloni, 2009).
Finding common solutions
The repeated changes of the Code of Civil Procedure (Box 1) have pushed -and continue to push -many practitioners (especially judges, lawyers and clerks) to take part in the meetings of the Observatories of Civil Justice (Caponi, 2003;Costantino, 2005-b;Verzelloni, 2009;Berti, 2011).The "Observatories" -as these entities define themselves -are spontaneous groups that have been developed from the bottom-up in various Italian judicial offices (Box 2)."We started in 1994, to discuss the reform of the Code that would take effect in 1995.We wanted to reflect on how the rules would change our work organization.[ The idea of the Observatories appeared in the first part of the 90's.Since 1993 -the year of foundation of the first Observatory of Milan -these experiences have spread in many other courts: currently, there are at least 30 Observatories.The composition is very variable, but always includes lawyers and judges.
These "interprofessional communities of practice" represent a local functional response to the everyday problems faced by the operators.The Observatories have created a space for discussion, in a more or less structured way, in order to find common solutions (Box 3).On the one hand, in fact, the Observatories represent a place where the practitioners can overcome their isolation and share their interpretative and behavioural practices.On the other hand, the Observatories aim to define some "virtuous practices", in order to improve the professional activities and the functioning of courts (Box 4-5).
"The Observatory started with us, judges.[...] We began to discuss with the lawyers and the court clerks in order to find common solutions that would allow us to work".
Box 3. Interview with a judge of the Observatory of Reggio Calabria.
"The premise is that things work out badly, but we can make them work better [...] eliminating our self-reference.[...] We all must be involved in the discussion.We have to accept the idea that we are all part of the same organization"."hearing protocols".This process is central to the life of these communities and, in many cases, justifies their existence.
The hearing protocols
The "hearing protocols" -as they are called by the members of the Observatories -are documents that collect and comment some interpretative and behavioural practices, defined as virtuous by the same operators.These documents are very different from the protocols used in medicine, biochemistry and biology (Lynch, 2002), since they are not binding, not even in terms of professional ethics, and they only refer to the local court in which they were adopted.
At the national level, there are more than 90 hearing protocols.The first document of this kind was made in Salerno in 2002.Some Observatories have developed various protocols.
These texts deal with various issues, some general as, for example: conduct of the hearings (Box 6); priorities of treatment; minutes; attorneys' fees; notifications; telematics communications; structure of acts of defenders and judges.And others highly specialised issues, such as: testimony of minors (Box 7); concessions in cases of conflictual separation; compensation for damage caused by road accidents; executions; bankruptcies; leases; labour and social security.
"Article 4. Both the judge and the defenders will put great care to comply with the time fixed for the beginning of a hearing and for dealing with each proceeding.[...] Article 11.Judges and defenders will take care to come to the hearing with an actual knowledge of the case, so as to: ensure the effective treatment of the issues relevant to the proceedings; that the decision at the hearing is made with due priority to substantive and procedural matters".
Box 6. Excerpt of the hearing protocol of the Observatory of Modena (2007).
"Article 2. The testimony of a minor should be organised in a way that prevents any exasperation of the conflict and, in any case, during a scheduled hearing, to be organised preferably outside school hours, in a suitable environment and behind closed doors.[...] Article 5.The hearing will take place only in the presence of the minor, the titular judge, the possible auxiliary and, if such is appointed, defence attorney of the minor or the minor's curator.In order to avoid constraints, it does not seem appropriate to consider the presence of the parties and of the defence attorneys".
Box 7. Excerpt from the hearing protocol on the subject of minors of the Observatory of Milan (2007).
The drafting of these documents is very often a complex negotiation process.In this sense, the Observatories are a place of mediation, in which the professional divisions are put aside (Box 8).
"We discuss all the time, but in the end we come out with a common solution.This is the spirit of the Observatories".
Box 8. Interview with a lawyer of the Observatory of Naples.
Although not binding, these protocols represent "cultural instruments" with a strong symbolic value.Given their covenantal nature, in some local contexts the protocols are not simple catalogues of rules of "good behaviour", but they are also "accountability tools", even if informal, in order to evaluate and, potentially, to sanction, under the aspect of reputation, those who do not operate in the agreed manner (Box 9).
"With the protocol, we have mainly obtained some «cultural results».We discussed and we understood that the problems were in the management of hearings.[...] Now there is a sort of «social stigma» against judges who run the hearing in a different manner from the one recommended in the protocol".
Box 9. Interview with a judge of the Observatory of Rome.
Over the time, some hearing protocols have assumed a considerable importance at the national level, so as to be recognized by the Supreme Court of Cassation and to inspire some reforms introduced by the Italian Parliament.In some cases, the practices, as fixed in the protocols, have become national laws and thus binding on all operators.In particular, it should be remembered:
Discussion
The Observatories are a multifaceted entity, which lends itself to different interpretations.In light of the research questions of this article, the case study enables to critically analyse the concept of practice, as defined within the PBS.
This experience highlights at least six different subtypes of practice.These dimensions identify some aspects that coexist within the general concept of practice.The six attributes should be considered exclusively as "interpretive tools" to develop comparisons.
Firstly, practice can be "experimental".The long season of judicial reforms has had a negative impact on the Italian legal professionals.In general, any change -not only a new legislation -requires a transition period.During this phase, the operators test new practices, in order to adapt their working activities.These practices are, by definition, provisional and ephemeral.This dimension is particularly evident in the legal context, but it is applicable whenever people have to change their usual ways of doing things.
Secondly, practice can be "solipsistic".As mentioned in the previous pages, one of the main problems of the Italian judicial system is the fact that some courts are comparable to a condominium, where there are no collective learning opportunities.Beyond the Italian case, it is possible to identify another subtype of practice.In every organization, in fact, operators adopt some stable and repeated practices, which are intrinsically individual.On some Teoria e Prática em Administração, v. 6, n. 2, 2016 Beyond the Provisional Nature: Towards a Radial Concept of Practice Verzelloni, Luca DOI: http://dx.doi.org/10.21714/2238-104X2016v6i2-31614occasions, these practices are not even known by the other practitioners and they represent a sort of "personal treasure".Thirdly, practice can be "diffused".The Observatories are "interprofessional communities of practice": places of encounter and exchange, where the operators can overcome their isolation and share their interpretative and behavioural practices.In the organizations, there are always practices of this type, which are transferred from practitioner to practitioner through language and imitation.
Fourthly, practice can be "concerted".The Observatories aim to develop "virtuous practices", in order to overcome the problems faced by courts.The "solutions" are made through complex processes of negotiation among practitioners.These practices are not only "socially recognized" (Gherardi, 2006: 34), but they are conceived as the "appropriate" ways of doing things.In general, concerted practices are result both of explicit and implicit negotiations.These processes can be associated with collective/organizational learning phenomena.
Fifthly, practice can be "fixed".The hearing protocols represent "repertories" of stable practices.These documents are closely linked to the contexts in which they were written.As a result of agreements, the protocols may redefine the boundaries of individual discretion.These practices are not ephemeral and temporary, but conversely they are "internalized" by the operators, are potentially included in manuals and internal procedures, and they are taught to newcomers.Sixthly, practice can be "institutionalized".Some hearing protocols have been transposed into the national legislation.Practices have become binding on all operators, extending their scope beyond the court in which they were defined.In general, these practices overcome organizational boundaries and they spread across society.The practices of this kind turn into "rules" for behaviour.
Figure 1 shows the six subtypes of practice.These attributes are not strictly separated.
Each subtype, in fact, has a common semantic space with other categories.The attributes are represented as a chain around the concept of practice.The subtypes can also be conceived as different stages of the institutionalization of practice (Figure 2).This path is not deterministic and does not follow a predetermined order: for example, a practice can remain "experimental" and be forgotten; another can become "diffused", but never become "concerted"; another can also move from "solipsistic" to "fixed".These phases are not automatic, but depend on a number of factors.This process is cyclical: an institutionalized practice can also turn into a new experimental practice.
Concluding Remarks
The article has discussed the concept of practice, as defined within the Practice-Based Studies.Since not all practices have the same nature, the main conclusion of this paper is that it is necessary to overcome the actual representation of practice, understood only as something provisional and temporary.
The article proposes to consider the practice as a "radial concept".This idea leads to rethink the macro concept of practice.In this sense, the case of the Observatories of Civil Justice highlights six different subtypes of practice: In conclusion, this exploratory paper aims to generate a debate on the "radial concept" of practice.This perspective opens new research horizons in the fields of organizational and management studies.In the first place, it is necessary to conduct comparative studies, both in public and private sectors, to test the validity of the six subtypes of practices.In the second place, it is opportune to evaluate the possibility to introduce some semantic changes, so as to complete the wide spectrum of meanings of the concept of practice.Finally, it is essential to analyse the factors that contribute to the institutionalization of practice.In this sense, the subtypes allow to split the process, in order to consider its effects over time.
Box 4 .
Interview with a judge of the Observatory of Cagliari."The purpose of the Observatory is to seek virtuous practices, in order to do a better work.[...] It is not possible that the same injunction is rejected in a room and accepted next door".Box 5. Interview with a lawyer of the Observatory of Bari.The Observatories deal with many activities, including: organize workshops, archive the local jurisprudence and elaborate questionnaires on different subjects.But the most significant activity -with respect to the objectives of this paper -is the elaboration of the Teoria e Prática em Administração, v. 6, n. 2, 2016 Beyond the Provisional Nature: Towards a Radial Concept of Practice Verzelloni, Luca DOI: http://dx.doi.org/10.21714/2238-104X2016v6i2-31614 Teoria e Prática em Administração, v. 6, n. 2, 2016 Beyond the Provisional Nature: Towards a Radial Concept of Practice Verzelloni, Luca DOI: http://dx.doi.org/10.21714/2238-104X2016v6i2-31614 Beyond the Provisional Nature: Towards a Radial Concept of Practice Verzelloni, Luca DOI: http://dx.doi.org/10.21714/2238-104X2016v6i2-31614 the so-called "tables" for the liquidation of non-pecuniary damage (Observatory of Milan), recognized by the "United sections" of the Italian Supreme Court (judgment 12408/2011 and order 134/2013); the so-called "Office for Trials" (Observatory of Florence), established by article 50 of the Decree-Law 90/2014, converted in Law 114/2014; some provisions related to the "On-Line Civil Trial" (various Observatories), included in Decree-Law 179/2012, converted in Law 221/2012 and Decree-Law 83/2015, converted in Law 132/2015; the so-called "calendar of the trial" (Observatory of Florence), introduced by Law 69/2009 and Decree-Law 138/2011.
Figure 1.The radial concept of practice and its subtypes.
Figure 2 .
Figure 2. The phases of the institutionalization process.
Figure 3 .
Figure 3. Different levels of practice.
institutionalized practice.The subtypes are connected each other and they represent, at the same time, different steps of the institutionalization of practice.The model considers four levels: individual Teoria e Prática em Administração, v. 6, n. 2, 2016 Beyond the Provisional Nature: Towards a Radial Concept of Practice Verzelloni, Luca DOI: http://dx.doi.org/10.21714/2238-104X2016v6i2-31614practitioner,social group, organizations and society.However, this process should not be conceived as something predetermined.Practices, in fact, have different stories and trajectories: not all practices pass from one stage to another and not all practices are institutionalized.
|
2018-12-09T00:33:14.660Z
|
2016-12-27T00:00:00.000
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"oa_url": "https://periodicos.ufpb.br/ojs/index.php/tpa/article/download/31614/16828/",
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239033569
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pes2o/s2orc
|
v3-fos-license
|
COVID-19 Diagnosis Using Capsule Network and Fuzzy C-Means and Mayfly Optimization Algorithm
The COVID-19 epidemic is spreading day by day. Early diagnosis of this disease is essential to provide effective preventive and therapeutic measures. This process can be used by a computer-aided methodology to improve accuracy. In this study, a new and optimal method has been utilized for the diagnosis of COVID-19. Here, a method based on fuzzy C-ordered means (FCOM) along with an improved version of the enhanced capsule network (ECN) has been proposed for this purpose. The proposed ECN method is improved based on mayfly optimization (MFO) algorithm. The suggested technique is then implemented on the chest X-ray COVID-19 images from publicly available datasets. Simulation results are assessed by considering a comparison with some state-of-the-art methods, including FOMPA, MID, and 4S-DT. The results show that the proposed method with 97.08% accuracy and 97.29% precision provides the highest accuracy and reliability compared with the other studied methods. Moreover, the results show that the proposed method with a 97.1% sensitivity rate has the highest ratio. And finally, the proposed method with a 97.47% F1-score rate gives the uppermost value compared to the others.
Introduction
In recent decades, several new diseases have emerged in different geographical areas with pathogens including the Ebola virus, Zika virus, NIPA virus, and coronaviruses. Recently, a new type of pathological infection has emerged in Wuhan, China. The new strain is severe acute respiratory syndrome 2 (SARS-CoV-2), which causes Coronavirus Disease 2019 .
Following the increase in the number of patients, the Chinese public clinical and scientific associations reacted quickly to allow the new virus to be identified promptly, and the viral gene sequence to be identified and distributed to other countries around the world. Following extensive research on January 30, 2020, the World Health Organization (WHO) declared the prevalence of public health emergencies to be an international concern [1].
By increasing the extension of this disease, researchers have worked on different methods for early detection of this case at least for minimizing the outbreak. People with suspected COVID-19 should determine as soon as possible if they are infected [2]. Therefore, they should quarantine themselves, receive medical treatment, and inform and warn their relatives. One of the most popular and less harmful imaging methods for diagnosis of this area is chest X-ray imaging. Chest X-ray images are images that use small doses of ionizing radiation to take pictures of the inside of the body called radiographs [3]. The X-rays can help physicians in different cases such as bone fractures, dislocations, or joint inflammation, abdominal pain, and also some cancer cases [4]. Based on this fact, lots of researchers go through work on using chest X-ray imaging for the diagnosis of COVID-19.
Pereira et al. [5] proposed a method for the identification of COVID-19 in chest X-ray images using hierarchical and flat classification scenarios. They used hierarchical and multiclass learners for disease identification. COVID-19 texture was also explored from the chest X-ray images of pneumonia.
Minaee et al. [6] proposed another method for COVID-19 detection from radiology images. The method was performed on chest X-ray images from publicly available datasets. The images were injected to train four general convolutional neural networks, containing ResNet50, ResNet18, SqueezeNet, and DenseNet-121 for COVID-19 diagnosis in chest X-ray images. The method assessed the models on the residual images, and most of the networks presented high sensitivity and specificity ratios. Even though the efficiency was so hopeful, they presented that more examinations were needed to provide a more consistent estimation.
Rasheed et al. [7] proposed a different methodology for the diagnosis of COVID-19 from chest X-ray images. They used two widely used classifiers including logistic regression (LR) and convolutional neural networks (CNN). They also used the principal component analysis (PCA) to decrease the complexity of the system and to increase the speed of the system. They utilized an online available dataset incorporating GAN to have 500 X-ray images. The final results showed high accuracy for the proposed system.
Elaziz et al. [8] proposed another diagnosis system for the detection of COVID-19 from chest X-ray images. They extracted features from the input images based on fractional multichannel exponent moments (FrMEMs). Then, parallel computing was used for speeding up the system. Then, a modified manta-ray foraging optimization algorithm was used to select the main features. The method was assessed based on the COVID-19 X-ray dataset. The proposed method provided high accuracy for the datasets.
Rehman et al. [9] proposed another computer-aided method for fast detection of COVID-19 based on a convolutional neural network (CNN). The method used the residual neural network (ResNet50) for the purpose. For validating the proposed method, it is performed on a dataset containing X-ray images. Simulation results showed about 98% accuracy for the proposed method.
As can be observed from the literature, different techniques based on chest X-ray images have been proposed for the proper diagnosis of COVID-19. The main objective of this paper is to present another optimal methodology to provide a diagnosis system with higher accuracy. The key purpose is to deliver a new precise computer-aided diagnostic system for COVID-19 diagnosis to help physicians for the detection of COVID-19. Here, we utilized a new optimal machine learning-based approach for the computer-aided diagnosis of COVID-19. This study employs an optimal configuration for proper diagnosis of COVID-19 based on an optimized fuzzy C-ordered means (FCOM) and an improved version of the enhanced capsule network (ECN).
The ECN is modified by using the mayfly optimization (MFO) algorithm. The final results show well results for the proposed method.
Database Preprocessing
The present study proposed a new method based on a clustering approach to provide a proper automatic segmentation system for COVID-19 diagnosis. More specifically, a new optimized fuzzy C-means method (FCM) has been proposed based on a newly developed version of a new metaheuristic algorithm to offer a system with higher efficiency.
However, the traditional clustering methods such as K-means clustering make partitions, wherein each cluster includes just one pattern (a set including accurate and crisp values), fuzzy clustering spreads this technique more to give or associate the present patterns in an image with the data clusters of the image based on a membership function. The fuzzy C-means contains a "soft clustering" methodology and delivers a precise calculation for the cluster membership and is utilized successfully for image clustering applications, especially in medical imaging.
The proposed developed FCM is then used as a new method for segmentation of the chest X-ray of COVID-19. The system classification has been accomplished according to the enhanced capsule network (ECN). The method is based on using deep learning including multiple stages of receipt of raw data supposed as a beginning stage, and the model classification presentation has achieved at the final stage. The graphical abstract of the system is provided in Figure 1. 2 BioMed Research International patients [10]. Figure 2 shows some examples of the chest X-ray images collected from these datasets.
Data Normalization.
Sometimes, the raw data we have for analysis is not suitable for a group of statistical tests and to be able to use this category of statistical tests and to increase the accuracy of the analysis, we have to make changes in the raw data. One of these changes is called data conversion. Data conversion is a mathematical method used to modify variables that do not follow the statistical assumptions of normality, linearity, and uniform scattering, or have patterns with unusual outliers. Among data conversion methods, data normalization has high efficiency. Normalization has different meanings in statistics, the simplest use of which is to normalize data or normalize variables, and is a method that puts data in the same domain when they are not [11]. In other words, a data miner may encounter situations where the properties of the data include values that are in different ranges or domains. These large-value features may have a much greater effect on the cost function than low-value features. This problem will be solved by normalizing the properties so that their values are in the same range [12]. In constructing a metamodel from the data, before model training begins, the data is subdivided into its largest corresponding values to be normalized to values between zero and one scales, to minimize the effect of the absolute scale and have almost all inputs in the same range.
The min-max method is one of the popular and simplest normalization methods in medical imaging [13]. Based on this method, over and above the unifying data scale, the data changing edges will be distributed in the range between 0 and 1. By assuming attribute X, such that it has a mapping from the data set between X min and X max , the min-max normalization ðX norm Þ will be achieved by the following [14]: 2.3. Contrast Enhancement. Another preprocessing step to improve the image quality is contrast enhancement. The image contrast enhancement, especially in medical images, is an important issue that can be improved the accuracy of the system in a sensible term. This process can increase the contrast between different considered objects to simplify the next segmentation steps. In the present study, contrast enhancement has been used on COVID-19 chest X-ray images to highlight the significant areas with keeping the other areas fixed. The study uses a 16-bit lookup table for this purpose that is then stored on a disc. This technique can be mathematically formulated as follows [15]: where Min hist and Max hist represent the lowest and the highest levels of the gray magnitudes of the main image histogram, respectively, and A hist and B hist represent the input and the output images before and after contrast enhancement, respectively. Figure 3 shows some examples of the image processing applied to the input images.
Mayfly Optimization (MFO) Algorithm
Mayfly is a tiny, fragile, and soft-body insect that has more than 3100 types around the world. However, this insect needs almost one year to birth; it dies after a maximum of 1 day of living. The main target of their birthing is mating. Most of them even do not bother themselves with feeding. The mayfly swarms for mating usually include several males from a few to hundreds of individuals, which are about 1 m to 4 m above the ground for about 1.5 hours to 2 hours in the early morning. Formatting and grabbing the females, males do some nuptial dance over a characteristic up-and-down pattern of movement. Afterward, the couples were released to the vegetation for mating. The mayfly optimization algorithm uses this conception for optimization [16]. This algorithm uses a hybrid conception of the particle swarm optimization (PSO) algorithm, the genetic algorithm (GA), and the firefly algorithm (FA). Based on the MFO algorithm, the initial population is divided into two classes of male and female mayflies that are generated randomly. The initial population (candidates) is considered a d-dimensional vector X = ½x 1 , x 2 , ⋯, x d T which has been randomly positioned in the solution space. The mayflies have a velocity equal to V = ½v 1 , v 2 , ⋯, v d T , and their direction relates to both social and individual flying experiences. Then, the candidates tune their position close to their best position (p best ), as well as the best position of the other candidates (g best ).
By considering x i as the present position of the candidate with a step equal t, the updated position is obtained by the following equation [16]: where x i ð0Þ is limited between x min and x max . The movement of mayflies on the top of the water to dance is mathematically modeled as follows: where β describes the visibility coefficient utilized for restraining mayfly visibility to others; pbest i defines the i th best position candidate had ever visited; r p and r g describe the Cartesian distance between x i and pbest i and x i and gbest; x t ij and v ij ðtÞ represent the position and the velocity of the i th candidate in dimension j, respectively; and a 1 and a 2 signify the constants for positive attraction scaling the involvement of the social and cognitive component, respectively.
The best position for personally succeeding in the time step t + 1 is as follows: where f ð:Þ describes the objective function to define the quality of the solution. Then, the global best position (gbest j ) is achieved as follows: where N describes the total number of male candidates in the swarm. The Norm 2 equation has been utilized to determine the r p and r g as follows: where x ij describes the j th component of the candidate i.
For retaining the algorithm with the best candidates, the best mayflies keep to dance and update their velocities by the following equation: where n d signifies the nuptial dance coefficient and δ describes a random value in the range ½−1, 1. However, each male mayfly belongs to a special swarm; the females do not belong to groups. They fly around the males for breeding. With assuming y i ðtÞ as the i th female candidate path, in the solution space, the position has been updated by the following equation: The best male breeds with the best female, the secondbest male with the second-best female, etc. Therefore, the velocity has been considered as follows: where β represents a fixed visibility coefficient, a 2 describes a positive attraction constant, r mf describes the Cartesian distance between male and female candidates, y t ij and v t ij ðtÞ signify the i th female candidate position and velocity in dimension j at time step t, and r w defines a random walk coefficient, and r is a random value in the range ½−1, 1.
The MFO algorithm uses crossover as the mating process between the male and female candidates such that two candidates are first selected as male and female. The way of selecting the parent is similar to the method of female attraction by males. The new generation of the crossover process has been achieved as follows: where ζ describes a random value and male and female describe the parents. And the early velocity of the offspring is set at zero. The main reason for using the mayfly optimization algorithm is that it combines the major advantages of swarm intelligence and evolutionary algorithms, which makes it 5 BioMed Research International stronger in providing a good balance between exploration and exploitation [17].
3.1. Data Segmentation. The next step after preprocessing of the input images is to segment the pattern data. The present research uses mayfly optimization (MFO) algorithm to develop a new version of the fuzzy C-means technique for optimal clustering (MFO-FCM). The MFO-FCM is also a good tool for noise reduction in this application. By assuming partitioning of a set of data based on this algorithm including N points into C clusters, the best results of the fuzzy C-means are achieved by minimizing the following equation [18]: Subject to where κ jk signifies the feature for the j th cluster and the k th the point, m signifies the weight that is set here 2, and a and b describe the effect of the variable on the status of membership and feature, respectively. In the event that a > b, membership provides more effect on the data; otherwise, the data reduces the noise effect and Lðx k , v j Þ describes a value in the range x k to the center of the cluster v j and has been obtained by the following equation: As mentioned in Equation (12), the fuzzy C-means minimization is usually performed by the Lagrange multiplier theorem that is performed based on the following equation: In this study, the mayfly optimization (MFO) algorithm is used to generate the memberships and possibilities along with using typicality to tune the impact of the outlier. Here, the parameters T, V, and U are updated to minimize the considered function during the iterations. This will be terminated if The present study uses within-cluster sum of squares errors (WCSS) as the objective function for the optimization. By considering S = ½s 1 , s 2 , ⋯, s N as clusters and X = ½x 1 , x 2 , ⋯, x N as data points, the WCSS function is formulated by the following equation: where Lðx j , v i Þ determines the distance from v i and x j , and v i represents the center of the cluster s i . This method contains two parts: the first part is to identify the model parameters and the second part is to centroid clustering. Six variables are considered in the MFO-FCM model. By assuming cluster centroids including V = ½v 1 , v 2 , ⋯, v c , the centers of the clusters are defined. The pseudocode of the MFO-FCM model is explicated in the following: (ECN). The present study uses enhanced capsule networks (ECN) to provide a suitable diagnosis system. In ECN, the fragmented pixel set of the X-ray image is labeled as a set of nerve cells regarding the capsule. The system used a pixel vector as an actuation vector encompassed by an active capsule such that it may be a particular category like healthy or COVID-19 for the X-ray image.
Enhanced Capsule Networks
Here, the capsule output and the coupling coefficient have been multiplied by the capsule routing in a layer. The parent capsule resistance for routing defines the value of the coupling coefficient. Based on the routing-by-agreement mechanism, the low-level COVID-19 diagnosis has been determined by the high-level capsule activation [19].
1) Start 2) Initializing parameters of the MFO and the number of clusters
3) Initializing the cluster centers based on MFO 4) Calculating the objective function 5) Updating the candidate's position using the MFO algorithm 6) If the termination criteria are satisfied, go to ((6)) 7) Else go to ((2)).
8) End
Pseudocode 1: The pseudocode of the MFO-FCM 6 BioMed Research International With assuming y i ∈ ½healthy, COVID-19 as the i th output capsule, and we ij as the weight matrix, we havê whereŷ ðj|iÞ signifies the detection vector which identifies the output parent capsule j using capsule i. The value of the weight will be amended if the values will be reduced or the pixels contain probably to the positive class. For going before layer capsules, the softmax function has been utilized and the potential parent capsule as the coefficient is encrypted c ij such that essential logits b ij appears the log past conceivable outcomes of the i th routing capsule within the last layer to the j th capsule within the succeeding layer.
The routing-by-agreement mechanism is mathematically performed by the following equation: And the proceeding layer indicates a critical element in the evaluation of input of the parent capsule j as follows: The squashing function has been utilized to accomplish the pixel vector compression in the range ½0, 1Þ as follows: where ε = 10 −7 . And the capsule in the next layer is formulated by the following equation: The overall classification of the capsules which are considered as individual margin loss (Loss k ) in the categories capsule k for the capsule networks based on the loss is where T k describes the instant attendance in category capsule k and m − , m + , and λ represent hyperparameter assistances. Finally, the ECN has been trained by 700 iterations using Adam optimizer to get the optimal results of hyperparameters. The learning rate is considered an amount of 1e − 6 [20].
Results and Discussions
The proposed model is performed on three datasets including a popular resource collected by the Renmin Hospital of Wuhan University and two affiliated hospitals, Sun Yat-sen Memorial Hospital and the Third Affiliated Hospital of the Sun Yat-sen University in Guangzhou with 12 and 76 patients [10]. The method is programmed in MATLAB 2016b 64-bit version and executed on the computation environment of Intel Core i7 CPU 2.00 GHz, 2.5 GHz, 8 GB RAM, and 64-bit operating system. As before mentioned, the key purpose of this study is to design a new CADbased system for the diagnosis of COVID-19. The model has been analyzed based on four measurement indicators including accuracy, precision, F1-score, and sensitivity.
4.1. Accuracy. The precision determines how nearly the measured esteem is to the genuine (real) value. This indicator is accomplished by the proportion of correct identification where TN and FN describe the true negative and false negative, respectively, and TP and FP represent the true positive and false positive, respectively.
Precision.
Precision defines how near the measured values are to each other. This indicator is accomplished by the proportion of positive identification values to the whole number of identifications. This can be mathematically described as follows: 4.3. Sensitivity. Sensitivity is the extent of positives that are accurately recognized (i.e., the extent of those who have a few conditions (influenced) who are accurately recognized as having the condition). This indicator is accomplished by the proportion of true identification values to the true positive and false negative number of identifications. This can be mathematically described as follows: 4.4. F1-Score. F-score or F-measure could be the degree of a test's exactness. It is achieved based on the precision and sensitivity of the test. The most noteworthy conceivable value of an F-score is 1.0, showing idealized exactness and review, and the least conceivable value is 0, with the chance that either the precision or the sensitivity is zero. The F1 -score is additionally known as the Dice similarity coefficient (DSC). This measure is mathematically defined as follows: The method analysis of the classification based on the offered enhanced capsule networks (ECN) is reported in Tables 1-4. The method has been compared with three other state-of-the-art methods including FOMPA [21], MID [22], and 4S-DT [23] for better clarification.
To offer a better clarification of the efficiency of the proposed method, a bar plot of the results is shown in Figures 4-7. As can be observed from Figure 4, the simulation shows a 97.08% accuracy with 97.29% precision for the suggested methodology compared to the other studied methods. However, FOMPA, MID, and 4S-DT are in the next ranks. Figure 5 shows the precision results for the studied algorithms. Figure 6 shows the bar plot of the sensitivity results for the studied algorithms.
The results show that 500 epochs for all algorithms have been utilized. As can be observed from Figure 6, the proposed classifier offers a higher sensitivity rate to the other state-of-the-art. The designed classifier of the proposed method offers a 97.1% sensitivity rate, whereas FOMPA, MID, and 4S-DT have 95.85%, 93.66%, and 91.68%, respectively, for 500 epochs. Figure 7 illustrates the bar plot of the F1-score results for the studied algorithms.
Based on Figure 7, after 500 epochs for all algorithms, the proposed method presents the best better F1-score rate to the other methods. As can be observed, the suggested method with a 97.47% F1-score rate has the highest value, and the FOMPA, MID, and 4S-DT with 96.39%, 94.18%, and 91.76%, respectively, are in the next ranks.
Conclusions
COVID-19 was formed in late 2019 and is spreading rapidly across the world. Early diagnosis of COVID-19 can be so beneficial to the treatment of the disease and to prevent its outbreak. Due to the probability of human error among the experts in finding COVID-19, the application of 9 BioMed Research International machine learning has been recently increased as an auxiliary tool. The present study proposed a method based on image processing for the diagnosis of COVID-19. This study presented an optimal configuration for proper diagnosis of COVID-19 based on an optimized fuzzy C-ordered means (FCOM) and an improved version of the enhanced capsule network (ECN). The ECN was improved based on the mayfly optimization (MFO) algorithm. The proposed method was then performed on the chest X-ray COVID-19 images from publicly available datasets. The results were analyzed by comparing some other methods, including FOMPA, MID, and 4S-DT, and the results showed the higher effectiveness of the proposed method. As mentioned, the proposed method has good accuracy in terms of theory. However, due to using complicated methods, using it in real-time applications is not feasible. Therefore, in future work, we will work on using a simplified technique of the proposed method to perform on a microprocessor for realtime applications.
Data Availability
The presented study uses three datasets including a popular resource collected by the Renmin Hospital of Wuhan University and two affiliated hospitals, Sun Yat-sen Memorial Hospital and the Third Affiliated Hospital of the Sun Yat-sen University in Guangzhou, that can be achieved by email to the sources.
Conflicts of Interest
The authors declare no conflict of interest.
|
2021-10-21T05:12:33.518Z
|
2021-10-19T00:00:00.000
|
{
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245940785
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pes2o/s2orc
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v3-fos-license
|
Phenotypic and Molecular Characterization of Mango Cultivars in Southwest Nigeria
ABSTRACT Morphological and microsatellites (SSR) markers are efficient tools for determining genetic relatedness among mango cultivars. Seventeen mango cultivars were used for this study. Eight fruit quantitative traits were collected and subjected to mean separation using One-Way ANOVA and correlation using Principal Component Analysis (PCA). Also, molecular analysis was done using PCR-based SSR markers. The resulting binary matrix was analyzed using Numerical Taxonomy and Multivariate Analysis System (NTSYSpc). Significant variation (p ≤ .05) among all the 17 mango cultivars was observed for the eight quantitative traits studied. The PCA showed that the fruits length, width, thickness weights, %pulp and %stone contributed to 98.73% of the variation observed in all the mango cultivars. A total of 21 alleles were detected from the seven polymorphic primers ranging from two to five alleles per locus with an average of 3.0 alleles per locus. The polymorphic information content (PIC) ranged from 0.52 to 0.80 with an average of 0.66. Both the morphological and molecular markers showed that the mango cultivars were diverse except for ‘Saigon’ and ‘Julie’ as well as ‘Harden’ and ‘Lipen’ which though appear morphologically distinct based on the understudied traits but showed strong similarity to each other through molecular analysis. Dendrogram constructed using the Unweighted Pair Group Method with Arithmetic mean (UPGMA) based on SSR markers revealed a similarity coefficient of 48–93% indicating high level of variability and the presence of outbreeding. Results of the morphological and microsatellite (SSR) analyses showed wide diversity among the mango cultivar used in this study.
Introduction
Mango (Mangifera indica L.) which is often referred to as 'the King of fruits' (Singh, 1996) because of its popularity and importance in the tropical world belongs to the genus Mangifera and the family Anacardiaceae. According to the Food and Agriculture Organization, the top mango-producing countries are India, China, Thailand, Indonesia and Mexico, with Nigeria ranking as the third in Africa (FAO, 2019). The flowering process of the crop is a complex process owing to the cool inductive temperatures which induce mango flowering under subtropical and upper-latitude tropical conditions (Ramírez and Davenport, 2010). Mango is heterogenous in nature and has a great diversity in seedling genotypes which have shown wide genetic diversity in terms of shape, color, bearing habits, maturity stage and yield (Serry et al., 2019). Most mango cultivars including some superior clones are hybrids, resulting from natural cross-pollination (Krishna and Singh, 2007). Several factors including selection, mutation, genetic drift and recombination provide sources of genetic diversity. Despite the level of genetic diversity that exists among mango landraces and cultivars, the genome has been reported to be allopolyploid with chromosome counts of 2n = 40, i.e. the diversity occurring in mango may be genomic structural variation (Begum et al., 2016;Krishna and Singh, 2007;Schiessl et al., 2018).
Genetic resources for potential crop improvement are invaluable in mango breeding programs, hence the need for collection, evaluation, characterization and documentation to increase their genetic base. Morphological characterization has been the traditional method of identification of many crops including mango. However, this method is influenced by environmental factors and the results are often biased and unreliable (Matthew and Oziegbe, 2016;Subedi et al., 2009). The ease and inexpensive nature of morphological characterization makes it a paramount technique in plant characterization and taxonomy. Mango varieties have been differentiated based on fruit characteristics that include size, shape, color and nutrient composition (Igbari et al., 2019). Arogundade et al. (2017) characterized some mango cultivars in Nigeria employing physicochemical and morphological properties of the fruits to show the diversity of the collected cultivars. Morphological traits in the characterization of mango cultivars create ambiguities in the identification of closely related cultivars (Begum et al., 2016). Also, the use of morphological markers and isozymes techniques in the characterization of mango cultivars often does not provide an accurate evaluation of diversity among mango cultivars, and this could lead to misidentification or duplication of the crop genotypes (Kumar et al., 2013). In the case of genetic erosion, where there is a need for conservation, a more in-depth classification and study is needed.
Molecular markers are valuable tools for the assessment of genetic variation (Varshney et al., 2005) and therefore assist in selection. Among the available molecular markers, polymerase chain reaction (PCR)-based markers are the most suitable for revealing genetic diversity and include RAPD, SSR, ISSR, AFLP and SNP. Among these, SSRs are receiving attention due to their multiallelic nature, reproducibility, hypervariability, codominant inheritance, comprehensive genome coverage, relative abundance and suitability for high-throughput genotyping (Parida et al., 2009;Rafalski et al., 1996). The use of SSR for genetic characterization was reported to serve two purposes that include the identification of genotypes and estimation of their genetic relatedness (Ravishankar et al., 2000). SSR markers have been effectively utilized for estimating genetic variability in mango (Honsho et al., 2005;Ravishankar et al., 2015;Shamili et al., 2005). Begum et al. (2016) showed that a high genetic variability exists among 90 mango cultivars using simple sequence repeat (SSR) and concluded that there is an increasing genetic erosion in their locality. Also, for clonal selection and improved breeding, there is a need for molecular evaluation. This study, therefore, investigated the genetic variability and relatedness among different mango cultivars using both morphological and SSR markers. These findings will be useful in mango genetic resource conservation and in breeding programs.
Survey and Sampling
An exploratory survey was conducted during the mango fruiting seasons following a selective sampling strategy so that mango cultivars already sampled were not repeated. Mango orchard of the National Horticultural Research Institute (NIHORT), Ibadan, comprising landraces and cultivars and some locations (Ogbomosho, Ikole and Ibadan) in southwest Nigeria were selected for this study. Seventeen mango varieties used for this study include Alfonso, Edward, Harden, John Bull, Julie, Kent, Lipen, Madoe, NG-25, NG-26, NG-27, Ogbomoso, Palmer, Peach, Saigon, Tommy Atkin and Uno.
Morphological Characterization
Morphological characterization was based on tree-ripened fruit characteristics. Fruit sampling was carried out thrice on 15 randomly selected mango fruits during the harvest season. Morphological characters of sampled fruits were recorded following mango descriptors (IPGRI, 2006). The fruit samples were evaluated for eight quantitative traits that include fruit length (cm), fruit width (cm), fruit thickness (cm), fruit weight (g), fiber length (mm), peel (%), pulp (%) and stone (%).
DNA Extraction and Quantification
Genomic DNA was extracted from young leaves using a modified Cetyltrimethylammonium Bromide (CTAB) procedure (Mignouna et al., 1998). The quality and quantity of DNA was assessed by gel electrophoresis using 1% agarose with known concentrations of undigested lambda DNA (Sigma, St. Louis, MO, USA). Quantification of DNA was done using a spectrophotometer (Beckman Coulter DU530) at 260 nm. Extracts were diluted with sterile water to obtain DNA concentrations of 25 ng/µL.
Polymerase Chain Reaction
In this study, a total of 15 simple sequence repeat (SSR) primer pairs were used. PCR for SSR reactions was conducted in a 20 µL volume in a 96-well microliter plate using an automated thermal cycler (model: Peltier Thermal Cycler 200). The reaction volume contained 25 ng of template DNA, 100 µM each of dNTP and 2.5 mM MgCl 2 , 0.5 µM each of forward primer and reverse primer. 1X reaction buffer and 2 units of Taq DNA polymerase (Invitrogen) were carried out in a volume of 10 μL containing 1X reaction Buffer, 0.2 mM MgCl 2 , 0.25 mM dNTP, 0.5 μM Primer, 1 Unit Taq DNA Polymerase, and Template DNA. The PCR for SSR was carried out with conditions, i.e., 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 45 s and 72°C for 45 s, with a final extension step at 72°C for 7 min. PCR products were separated on 2% (w/v) agarose gels using 0.5X TBE Buffer at 90 V and stained with ethidium bromide for visualization under UV light.
Data Analysis
The quantitative data obtained on the mango fruits were subjected to principal component analysis (PCA) using PAST 3. Cluster analyses were carried out using unweighted pair group method arithmetic averages (UPGMA) to construct a dendrogram using Gower's genetic distance, considering Eigenvalues of >0.3 in the PCA of the diversity study. Each genotype for the SSR primers was scored visually on the basis of their presence (1) or absence (0) separately for each cultivar. The sizes of fragments (molecular weight in base pair) were estimated using the DNA hyper-ladder 2 (Bioline) as base pair (bp) ladder that was run along with the amplified products. The scores obtained using all seven polymorphic primers for SSR were used for constructing the matrix. The statistical software NTSYSpc (Rohlf, 2005) and POWER MARKER software were used to construct a UPGMA dendrogram using hierarchical clustering. Using NTSYS software, a dissimilarity matrix was calculated utilizing Jaccard (1908) coefficient. Cluster analysis based on the dissimilarity matrix was performed using UPGMA (Sneath and Sokal, 1973) of the NTSYSpc version 2.2 (Rohlf, 2005).
Morphological Variation in 15 Mango Cultivars
Significant differences (p < .05) were observed in all 17 mango cultivars evaluated for fruit size (length, width and thickness) and weight, percent peel, pulp, stone by weight and fiber length (Table 1). Fruit length varied from 8.4 cm (NG-25) to 18.8 cm . Fruit width ranged from 1.8 cm (NG-25) to 3.5 cm (NG-27). Fruit thickness ranged from 5.3 cm (NG-25) to 10.7 cm (NG-27). Fruit weight ranged from 106.6 g (NG-25) to 893.7 g (Peach). NG-27 had significantly (p ≤ .05) higher fruit length and fruit weight than all the other mango cultivars. NG-27 had the highest fruit length, fruit width, fruit thickness, fruit weight and the lowest % stone. NG-25 had the lowest fruit length, fruit width, fruit thickness and fruit weight. The percent peel varied from 16.3 (Kent) to 23.5 (Saigon). The percent pulp varied from 46.9 (NG-25) to 74.3 (Julie). The percent stone varied from 7.2 (Edward) to 22.9 (NG-25). Fiber length ranged from 26 mm (Saigon) to 115 mm (Julie). The highest % pulp, fiber length and lowest % peel were observed in Julie. NG-25 had the highest % stone and lowest % pulp. Saigon had the highest % peel and lowest fiber length.
Principal Component Analysis
PCA took into account the first two PCA at cumulative variance of 99.91% of the total variance ( Table 2). The first PCA, i.e., PC 1, showed that fruit length (cm), fruit width (cm), fruit thickness (cm), fruit weight (g), %pulp and %stone contributed 98.73% of the total variation in fruit morphology among the mango cultivars evaluated, while the PC 2 took into account fruit length (cm) and %pulp, which contributed 1.18% of the total fruit morphological variation ( Table 2).
The correlation among the mango cultivars and the separation of PC 1 and PC 2 showed dispersion in all the quarters for the morphological characters ( Figure 1). Figure 2 shows the agglomerative hierarchical clustering dendrogram that illustrates the relationship among the cultivars using the unweighted pair group method with arithmetic mean (UPGMA). The cluster analysis classified the 15 cultivars into different clusters with Gower similarity index ranging from 0 to 0.45. At a genetic distance of 0.3, the dendrogram was divided into four branches. At 0.3 similarity coefficient, Julie and Edward were grouped into Cluster I; Kent, Lipen and NG-27 were grouped into Cluster II; and ED-20 and NG-25 were grouped into Cluster III. Cluster IV had the highest number of cultivars (NG-26, Ogbomoso, Alfonso, Uno, Saigon, Madoe, Harden and Palmer) grouped together (Figure 2). Means followed by the same letter(s) are not significantly different at P < .05 using the Duncan's multiple range test.
Genetic Variation in 15 Mango Cultivars
Fifteen (15) SSR primers (Ajayi et al., 2019) were used for generating banding profiles (Figure 3), out of which seven primers (SSR20, EF592206, EF592216, EF59210, EF592198, EF592211 and EF592197) gave clear polymorphic bands for all the cultivars under study. The numbers of alleles detected varied from 2 (EF592206, EF59210 and EF592198) to 5 (EF592211). The average number of alleles per primer pair was 3. The allele size ranged from 120 (EF592206) to 300 bp (SSR 20). All the SSR markers used in the study showed a high level of polymorphism (Table 3). The polymorphism information content (PIC) ranged from 0.5215 to 0.8032, while the gene diversity ranged from 0.5813 to 0.8235. In this study, the highest PIC and gene diversity were recorded for the marker EF59210, while the lowest PIC and gene diversity were recorded for the markers EF592211 and EF592206. The cluster analysis separated almost all the cultivars into separate groups at a similarity coefficient of 0.82 (Figure 4). At 0.93 similarity coefficient, 'Saigon' and 'Julie' were grouped as identical cultivars as well as 'Harden' and 'Lipen' cultivars. 'Edward' was distinctly separated as a different cultivar from the SSR Markers.
Discussion
Understanding the genetic diversity among plant species provides useful information for varietal development, conservation and management (Romero et al., 2009). Determination of genetic relatedness in mango cultivars will provide important information for varietal selection and conservation in mango breeding programs (Ravishankar et al., 2000). Identification of genotypes using molecular markers can help in the selection of desired traits in tree crops; this will also minimize the risk of mixups in orchards (Struss et al., 2001). The mango classification was done using 17 cultivars that include three cultivars, NG-25, NG-26 and NG-27. The results from the morphological and microsatellite characterizations showed that most of the mangoes were diverse. Karihaloo et al. (2003) reported that high diversity occurred among cultivated and wild mangoes studied.
The fruit length and fruit thickness fall within the range reported by Begum et al. (2016) except cultivar NG-27 which also happens to be the longest and thickest of all the studied cultivars. NG-27 could be a new cultivar owing to the diversity in some of the traits studied. Pandey (1998) and Krishna and Singh (2007) reported that the outbreeding ability of mango would have resulted in the genetic diversity within the crop. The morphological traits showed that the fruits length, width, thickness weights, %pulp and %stone contributed to the diversity in the first principal component. The separation of PC values showed that mango cultivars were dispersed in all quarters, indicating a high level of genotypic variation among the mango cultivars in southwestern Nigeria. The dendrogram showed that although NG-25, NG-26, NG-27 and Ogbomoso are all local cultivars, they were genetically diverse. NG-27 belonged to a different clade. Krishna and Singh (2007) reported that agroclimatic conditions contributed to the high diversity in mango cultivars. This report was later corroborated by the study of Begum et al. (2016) which showed that there is high genetic erosion among the local mango cultivars and each cultivar possesses its distinct characters which contributed to both economic and cultural values of the crop. Fruit length, width, thickness, weight, %pulp and % stone showed significant variation suitable for morphological characterization of mango fruit using PCA as shown by the study.
A morphology-based method is sufficient for plant characterization (Patwardhan et al., 2014); however, the use of molecular markers in conjunction with the classical phylogenetic approach is more reliable. Microsatellites have become the markers of choice for use in the characterization of many plant species due to their codominant, highly polymorphic and reproducible nature (Gupta and Varshney, 2000). All the seven markers used in this study were polymorphic, showing high allelic variations in the DNA of the 15 mango cultivars, with marker EF59210 being the most polymorphic possessing a PIC value of 0.80 and gene diversity of 0.82. These results confirm the effectiveness of microsatellites when used in genetic diversity studies. Filho et al. (2010) and Parthiban et al. (2018) reported that such discriminating power and high polymorphism primers can be used effectively in constructing genetic linkage maps. UPGMA cluster analysis in this study revealed significant genetic diversity as evident from the Jaccard's similarity coefficient ranging from 0.48 to 0.93 for SSR markers. This is similar to the work of Shamili et al. (2005) who found 35-100% genetic similarity among 41 mango cultivars using 16 SSR markers. Kumar and Narayanaswamy (2001) reported Jaccard's similarity in the range of 61-95% in 50 Indian cultivars, and Fitmawati et al. (2010) observed 69-98% similarity in 82 cultivars of mango from Indonesia.
Conclusion
The morphological and microsatellite (SSR) results obtained for the mango cultivars showed valuable diversity among the mango cultivars studied, which may be due to the outcrossing ability of the mango. This study will provide information on interesting traits unique to the mango cultivars studied that will suit diverse consumer preferences and industrial uses.
Disclosure Statement
No potential conflict of interest was reported by the author(s).
|
2022-01-15T16:07:47.578Z
|
2022-01-12T00:00:00.000
|
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257176788
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pes2o/s2orc
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v3-fos-license
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Changing Parental Attitudes Towards Rotavirus Vaccine
Background: Rotavirus is known to be one of the most common infections, usually associated with severe diarrhea. Despite the existence of two licensed vaccines, many countries, including Turkey, have not included rotavirus vaccination in their nationally funded vaccination program. This article explores what factors influence parents' decisions about whether to have their children vaccinated against rotavirus and which factors changed from 2010 through 2016. Materials and Methods: Data were collected over two periods via questionnaires. The first period was from January 2009 through March 2010, and data were gathered from a semi-private pediatric outpatient clinic in Kocaeli, Turkey. The second period was from August 2015 through May 2016, and data were collected from parents during their pediatric outpatient clinic visits. Two questionnaires were designed to find out the rotavirus vaccination status of the children, socio-demographic factors, and reasons for excluding/accepting the rotavirus vaccine. The level of knowledge about the rotavirus vaccine was investigated. Parents indicated their level of agreement with each statement using a five-point Likert scale. Results: While only 3.8% of the parents accepted the rotavirus in 2009-2010, it increased to 69.5% in 2015-2016. Significant factors influencing parents’ decision to vaccinate their children for rotavirus were advice from a pediatrician, a lack of correct and timely rotavirus information, and the cost of the vaccine. Conclusions: The acceptance of the rotavirus vaccine depends on parental perceptions, which may be influenced by accurate and timely information, the advice of their healthcare provider, and inclusion in the nationally funded vaccination program. In contrast to other studies reported, the education level of the mothers and fathers and their job types appear to be important. It was also found that parents’ attitudes and perceptions changed over time.
Introduction
The most common cause of severe diarrhea in young children around the world is rotavirus infection. According to statistics, rotavirus-related infections cause an average of 1600 child deaths annually, making them the second most common cause of mortality in children under five years old that can be prevented by vaccination after pneumococcal pneumonia [1][2][3][4][5][6].
RotaTeq (Merck and Co., PA, USA) and Rotarix (GSK Biologicals, Rixensart, Belgium) are two efficient rotavirus vaccines that have been licensed since 2006 and are advised for use in all countries by the WHO, particularly in those with high diarrhea-related mortality in children under the age of five [1].
The introduction of efficient and accessible rotavirus vaccines could significantly reduce the number of fatalities worldwide caused by diarrhea. The WHO broadened its recommendation for rotavirus vaccination use in 2009 to cover all nations, with a focus on those with high rates of death from diarrhea. To yet, however, the rotavirus vaccination has mostly only been distributed in nations with low rates of death due to diarrhea [5]. In high-and middle-income nations that have thus far used rotavirus vaccines, significant decreases in morbidity and death owing to rotavirus and diarrhea have been seen [7].
The effectiveness and reliability of rotavirus vaccination have been evaluated in a double-blinded, placebocontrolled, phase III study in 63,225 babies (31,675 in the vaccine group, 31,552 in the placebo group) from 11 South American countries [7]. It was found that the rotavirus vaccine was effective in preventing diarrhea and decreased the incidence of rotavirus gastroenteritis as well as hospital admissions [7]. In April 2009, the World Health Organization Strategic Advisory Group of Experts (SAGE) recommended that all national immunization schedules include rotavirus vaccination for infants [8].
Rotavirus infections occur in Turkey throughout the year; however, they are usually more common between September and May [9]. Although infections do vary with respect to regions, a recent study reported rotavirus frequency to be between 7.7% and 73.7% [9]. The death rate due to diarrhea in Turkey decreased, following the widespread application of oral fluid treatment. However, baby deaths may still occur due to diarrhea-related complications.
The Ministry of Health decides whether to add a vaccine to the national immunization program after consulting with an advisory board [10]. G1P(8) (54%), G2P(4) (12%), G3P(8) (3%), G4P(8) (9%), and, in recent years, G9P(8) and G9P(6) (both 4%), are the serotypes of the most common rotaviruses [11]. The most common serotypes seen in Turkey are G1-G4 and, increasingly in recent years, G9 [12]. However, in another study, it is reported that a large number of genotypes were observed, including common, uncommon, and mixed types, indicating a marked heterogeneity of rotavirus strains circulating in Turkey with major differences in the normally reported prevalences of the common genotypes, such that the prevalence of G3 and G1 was increased and that of G9 and G2 decreased from 2014 to 2016 [13][14].
Neither of the two types of commercially available rotavirus vaccines is government-funded in the Turkish routine vaccination program [10]. The rotavirus vaccine was only available and generally recommended, in private clinics for a fee. While some parents choose to vaccinate their children at their own expense, others receive partial reimbursement from private health insurance providers [10].
This study aimed to explore the changing attitudes of parents and parental characteristics over an approximately six-year period and what factors influenced their decision whether to have their children vaccinated against rotavirus based on the health belief model [15]. Fundamental components of the health belief model are perceived benefits, harm, susceptibility, severity, self-efficacy, and cues to action. All these components are usually evaluated simultaneously [16].
Materials And Methods
Two time periods were used to conduct this study. First, survey information was gathered from 262 parents in a semi-private pediatric outpatient clinic in Kocaeli, Turkey, between January 2009 and March 2010. A survey was once again used to collect the second set of data, this time from 302 parents, who responded to it while visiting the Kocaeli University Hospital's pediatric outpatient clinic during a routine visit between August 2015 and May 2016. They were healthy children between the ages of 0 and 18 years old. Before participating in the survey, all parents gave their informed consent. This study was approved by the Kocaeli University Ethical Committee (KOU KAEK 2015/242). Data were collected from parents who brought their children to the outpatient clinic for routine checks. They were approached at random, and once they gave their consent, the study was conducted face-to-face. Parents of the children with immune deficiencies, chronic diseases, or who were born prematurely or at a small for gestational age (SGA) were excluded from the study.
The questionnaire was created to investigate the rotavirus status of children and the socio-demographic characteristics of the families, including childbirth date and gender, family income, parents' level of education, parents' ages, and the number of other children they have. It also investigated the location of the residential address (urban/inner-city, suburban, or rural), the parents' line of work, and the parent's reasons for not vaccinating their children against rotavirus. The survey's content was based on the health belief statements for the rotavirus vaccine adapted from Taylor and Newman (2000) [15]. Using a five-point Likert scale, parents expressed their level of agreement with each statement, with options ranging from "strongly agree" to "strongly disagree." The responses were transformed into an ordinal scale with scores ranging from 1 to 5, with one indicating strong disagreement and five indicating strong agreement. In both studies, the parents were also asked about their reasons for accepting or refusing the vaccine. They were also questioned about their level of knowledge about rotavirus and its source.
Statistical analysis was conducted using SPSS, version 17 (IBM Inc., Armonk, NY, USA). The normal distribution test was evaluated with the Kolmogorov-Smirnov Test. Numerical variables with normal distribution were given as mean ± standard deviation, numerical variables not showing normal distribution as median (25th-75th percentiles), and categorical variables as frequency (%). Differences between groups were tested with the Mann-Whitney U test and Kruskal Wallis one way variance analysis and Dunn's multiple comparison test for numerical variables that do not have a normal distribution. Relationships between variables were determined by Spearman Correlation Analysis. For the testing of two-sided hypotheses, p < 0.05 was considered sufficient for statistical significance.
Results
For the study conducted in 2009-2010, 67.6% (n = 177) of the questionnaires were filled out by mothers only, 28.6% (n = 75) were completed by fathers and mothers together, and 3.1% (n = 8) were completed by fathers alone. The remaining (n=2) 0.8% were completed by other family members present. In 2015-2016, 80.1% (n = 242) of questionnaires were filled out by mothers only, and 19.9% (n = 60) were completed by fathers only. The socio-demographic characteristics of the families participating in 2009-2010 and 2015-2016 are compared, and only the mothers' jobs were comparable as shown in Table 1 In a separate study carried out in 2009 comparing parents' perceptions of the seriousness of various diseases, responses to "This vaccine is unneeded because this disease is a minor illness" for rotavirus in 2009-2010 were evaluated in order to gauge parents' perceptions of the seriousness of the disease or the significance of the rotavirus vaccine. It was discovered that the parents in 2009-2010 neither agreed or disagreed with the seriousness of the rotavirus (50.6%), and therefore had no strong opinion about rotavirus infection [17].
The "Risks of rotavirus vaccine outweigh benefits" statement looked into the worry about the side effects of the vaccine. Because the rotavirus vaccination can cause nausea, fever, and diarrhea, parents were reluctant to ask for it. While 118 parents, or 40%, disagreed with the statement regarding side effect hesitancy, 115 parents, or 38%, neither agreed nor disagreed, and 69 parents, or 22%, disagreed with the statement regarding the alleged side effects of the rotavirus vaccine in 2015-2016. Regarding the last statement, "I am uncomfortable with the number of shots my child receives," 30% (n = 90) of parents disagreed, 35% (n = 106 neither disagreed nor agreed, and 34% (n = 105) agreed with the statement in 2015-2016 ( Table 2).
The composite score is calculated as the ratio of the sum of the highest responses to the number of questions, as previously described in Ref. [15]. It basically represents the parents' composite health belief score and percentage. Using each parent's survey responses, the composite health belief score was calculated by dividing the sum of individual statement scores by the number of statements for which the parent indicated a level of agreement as shown in Figure 1. Each parent was also asked their reasoning if their response to the rotavirus vaccine was "Yes." Parents were more positively influenced, especially in 2015-2016, as shown in Table 3. Not having enough information and financial concerns, such as not being able to afford the vaccine, swayed parents' decisions. Parents' perceptions of the importance of rotavirus vaccination increased from 2010 to 2016, probably because of better and more specific information. At the end of the survey, each parent in the pediatric outpatient clinic in 2015-2016 was given detailed information on the rotavirus vaccine. After being fully and properly informed about rotavirus infection, the severity of the disease, and the availability of vaccines, parents' vaccine acceptance rates increased dramatically (p < 0.05) from 69.5% to 88.0% (n = 266). when parents also reflected that they were not willing to have rotavirus vaccination for their children even if it was funded and included in the national vaccination program [17]. A similar attitude is again reflected in 2016; however, this view changed dramatically after parents were given information on the effects and the burden of the disease after they filled out the survey.
Discussion
Additionally, when compared to parents with lesser levels of education, there was a statistically significant difference in vaccination intention, according to a finding similarly published by MacDougall et al. [18]. Given that parents with greater levels of education were much more inclined to have their children receive vaccinations, the mothers' and fathers' levels of education and their occupations are key factors. The amount of parental knowledge, parental wealth, access to vaccines, the accuracy of vaccine information, and the education level of the mothers all appear to have played a significant role in immunization acceptance [19][20]. One should also note that some of the socio-demographic factors are also different and better in the 2015-2016 study, such as income level, parents' education, and especially fathers' jobs, etc. Nevertheless, the education levels of the mothers and fathers and their job types appear to be important, as these parents were significantly willing to have their children immunized. The significance of social media should also be emphasized in a when looking at the characteristics of parental vaccine refusal.
Vaccine costs would mostly impact those in the low socioeconomic levels, as people in the middle and higher-income groups would be able to afford the vaccine [18]. The cost-effectiveness of a new health intervention is one of several crucial factors considered by decision-makers before an intervention is introduced [21][22]. Our study found that both the cost and a lack of high-quality information influenced parental decisions about rotavirus vaccines.
Objective and accurate information given to parents by healthcare workers, particularly by pediatricians, seemed to have an effect on parents' changing their decisions. Similar results were reported by Le Ngoc Tho et al. [23] where 93.7% of parents were positive for vaccination after being fully informed, and in another study, this rate was 90% [24].
It is clear that parents are positively influenced by the advice of doctors, especially that of pediatricians [10,17]. However, lack of awareness and knowledge of the potential health burden of rotavirus among parents is not taken seriously by health care providers or family physicians [20]. This was also determined in the present study. A number of parents replied that their doctors told them that not completing the vaccination course would be sufficient. A similar result was found by Bedford and Lansley (2006), who reported that, apart from providing the parents with information, the attitude and approach of doctors or health care providers were also important factors in accepting a vaccine [25]. This indicates that providing accurate and timely information on immunization issues to pediatricians, and probably other physicians and healthcare workers responsible for the health care for children is important.
In an internet-based study carried out in Germany with 6025 participants [26], 95% of the participants reported that the most important source of advice on vaccines is advice from pediatricians, the same view endorsed by the present study and others [10,17]. The majority of participants expressed a positive experience with immunizations in their children and relied on their pediatricians as the major source of information on this subject. Participants, who also mentioned books and the internet as information sources were less satisfied once they had been informed by their pediatricians. This indicated that providing accurate and timely information on immunization issues to pediatricians, and probably other physicians and health care workers responsible for the health care for children, as well as guidance to relevant internet sources would be important for providing objective information to parents [26]. In our view, providing information about vaccinations during pre-natal maternal education sessions may play a key role, a view also put forward by Hu et al. (2017) [27] where they also suggest that a strong partnership should be established between obstetricians and pediatricians or other vaccine related healthcare workers.
Furthermore, parents' views and perceptions towards all vaccines are not equal, as some conditions are considered more serious than others [10,14]. For this reason, when giving advice to parents, healthcare professionals should consider the parents' cognitive processes as well as the benefits of vaccination, and the seriousness and threat of the diseases. It would probably be advisable to re-educate healthcare providers and physicians and provide them with more support before starting a vaccine campaign, as they play a key role in the public acceptance of new vaccines [28]. In the absence of public endorsement by the government and the implementation of public health education programs not only for parents but also for healthcare workers, parents did not rate a disease such as a rotavirus infection as an important health concern. This was also the case with nurses, who would recommend the rotavirus vaccine if there was a national recommendation and the vaccine was publicly funded, as previously reported [16]. In this study, 20% (n = 60) of parents thought rotavirus illness was not a very serious disease and vaccination was therefore not necessary, 32% (n = 99) were not sure, and 49% (n = 148) thought rotavirus infection was serious enough to warrant rotavirus vaccination.
One of the other main parental barriers to vaccination was the confusion and difficulty in tracking vaccination schedules. In addition, parents cited a lack of awareness regarding the importance of vaccines, missing due dates, and fear of the possible complications and side effects of vaccines as reasons for not completing vaccination. As a result, it is critical to remind and reassure parents about vaccine efficacy and safety. Given the widespread use of mobile phones, the use of Android and iOS apps designed for vaccination reminders can be helpful. Existing apps have been reviewed, and a new app design was suggested by Abahussin and Albarrak [29].
According to McIntosh et al. (2016), tracking the degree and type of vaccine reluctance is necessary since these variables may change over time. Measuring vaccine hesitancy is also essential for the proper development of measures to increase vaccine coverage and for monitoring. This paper also documents the evolution of parental attitudes over time. Pediatricians may have a significant impact on parental vaccine decisions, and vaccine hesitation may be exclusive to certain vaccines but not all [30].
To lessen the pervasive impacts, vaccine hesitancy and refusal should be regularly observed, researched from medical, psychological, social, political, and ethical perspectives, and appropriately addressed [30]. In this study, the views and actions of parents concerning the rotavirus vaccine between 2010 and 2016 were compared. Parents in 2009 did not strongly agree or disagree with the seriousness of the rotavirus, hence there was no strong sentiment in favor of the vaccine. But in 2016, there was more understanding and acceptance. These could serve as a good guide for those making decisions on whether to add rotavirus vaccination to the national immunization program.
Conclusions
The acceptability of the rotavirus vaccine depends on a number of factors, although greater information, especially from a pediatrician, and government funding for the vaccine may sway parents' opinions. It would be crucial to include rotavirus vaccinations in the standard schedule for childhood immunization to prevent the childhood mortality brought on by rotavirus and deaths from diarrheal illnesses. The attainment of these significant and shared objectives might be greatly helped by increased efforts to make these vaccines available to all children.
Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Kocaeli University Ethics Committee issued approval KOÜ KAEK 2015/242. This study was approved by Kocaeli University Ethics Committee. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.
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2023-02-25T16:10:16.303Z
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2023-02-01T00:00:00.000
|
{
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255627479
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pes2o/s2orc
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v3-fos-license
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Investigation of the Effects of Glabridin on the Proliferation, Apoptosis, and Migration of the Human Colon Cancer Cell Lines SW480 and SW620 and Its Mechanism Based on Reverse Virtual Screening and Proteomics
Colon cancer is a relatively common malignant tumor of the digestive tract. Currently, most colon cancers originate from adenoma carcinogenesis. By screening various licorice flavonoids with anticancer effects, we found that glabridin (GBN) has a prominent anticolon cancer effect. First, we initially explored whether GBN can inhibit proliferation, migration, and invasion and induce apoptosis in SW480 and SW620 cells. Next, we exploited reverse virtual and proteomics technologies to screen out closely related target pathways on the basis of a drug and target database. At the same time, we constructed the structure of the GBN target pathway in colon cancer. We predicted that GBN can regulate the phosphatidylinositol 3-kinase (PI3K)–protein kinase B (AKT)–mammalian target of the rapamycin pathway (mTOR) pathway to fight colon cancer. Finally, through Western blot analysis and qRT-PCR, we verified that the expression levels of the PI3K, AKT, and mTOR proteins and genes in this pathway were significantly reduced after GBN administration. In short, the promising discovery of the anticolon cancer mechanism of GBN provides a reliable experimental basis for subsequent new drug development.
Introduction
Colorectal cancer (CRC) is one of the common malignant tumors of the digestive tract. It ranks third among all malignant tumors in terms of incidence [1]. This disease is mainly located in the colon or rectum [2]. Thus far, the molecular mechanism of its pathogenesis has not been completely elucidated. Current scientific research shows that the main mechanisms of this malignancy involve the instability of the genome [3], the loss of calcium receptors in colonic epithelial cells [4], the effect of cell adhesion [5], and the abnormal expression of epidermal growth factors [6]. Moreover, many risk factors, including age, high-fat and high-protein diets, genetic factors, polyps, and adenomas, can lead to colon cancer [7]. At present, CRC is mainly treated through surgery combined with radiotherapy, chemical, and targeted drug therapy. However, these therapies can cause serious adverse reactions. Traditional Chinese medicine for the treatment of colon cancer involves tumor prevention and treatment; this approach can reduce the side effects or adverse reactions, such as gastrointestinal reactions, caused by radiotherapy and chemotherapy [8,9].
Glabridin (GBN) is a flavonoid and one of the main active ingredients of Glycyrrhiza glabra L [10]. At present, GBN has a variety of pharmacological activities, including antiatherosclerosis [11], blood lipid reduction [12], nervous system protection [13], and anti-inflammatory [14] actions. In addition, it is commonly used to eliminate free radicals [15] and melanin [16]. GBN has been reported to have effects against liver cancer [17,18], cervical cancer [19], and breast cancer [20]. Our research team found for the first time that GBN has a prominent anticolon cancer effect. On this basis, we used a variety of modern technologies to elucidate comprehensively the mechanism of GBN against colon cancer.
Computer reverse virtual technology can predict potential targets and signal pathways by scoring the binding energy of compounds and proteins [21]. The differential proteins of drug action can be found through proteomics technology. The signaling pathways involving differential proteins are identified to verify the target pathways screened by using virtual technology. In recent years, a growing number of scholars have successfully used these two techniques to study the mechanism of action of biology and drugs [22], which has provided great enlightenment for our research.
In this study, we combine the above two technologies to outline a pathway that is closely related to GBN's anticolon cancer activity. The mechanism of action of GBN may be related to the inhibition of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of the rapamycin pathway (mTOR) signaling pathway. Finally, we experimentally verify the key indicators of proteomics and computer virtual technology results to clarify the mechanism of GBN in the treatment of colon cancer.
Materials and Methods
2.1. Reagents and Chemicals. GBN standard products were acquired from Shanghai Yuanye Biotechnology Co., Ltd. (batch number G006171216). Ammonia, iodoacetamide, and triethylammonium bicarbonate buffer were all procured from Sigma. Acetone and sodium lauryl sulfate were purchased from Shanghai Sinopharm. TMT 16Plex, Protein Ladder, Protease Inhibitor Cocktail, Pierce™ BCA Protein Assay Kit, NUPAGE 10% BT GEL 1.0MM 12 W, and Bond-Breaker™ TCEP Solution (TCEP) were produced by Thermo Fisher Scientific. Acetonitrile, methanol, formic acid, and other reagents required for the experiments were all of analytical grade. American Sigma Company provided dimethyl sulfoxide reagents. Fetal bovine serum (FBS) was sourced from Gibco, United States (10099-141). Beijing Soleibao Technology Co., Ltd., provided 0.25% trypsin-EDTA digestion solution and EDTA-free trypsin digestion solution.
2.2. Cell Culture. The human colon cancer cell lines SW480 and SW620 were purchased from Shanghai Luyu Biotechnology Co., Ltd. HT29 and HCT116 were provided by Procell Life Science & Technology Co. Ltd. All cells were cultured in L15 medium with 10% FBS. Double antibodies (100 μg/mL streptomycin and 100 units/mL penicillin) were added to the incubator at 37°C and 5% CO 2 . The medium was changed every 2 days, and the cells were passaged at 1 : 3. Cells in the logarithmic growth phase were used in the experiment. The cells were digested and pipetted with 0.25% trypsin digestion solution (Beijing Soleibao Technology Co., Ltd., China). Glycyrrhizin was dissolved in dimethyl sulfoxide solution (Sigma, USA). The medium was diluted to the concentrations of 40, 20, 10, and 5 μg/mL. The experiments were conducted in quadruplicate. In the blank experiment, dimethyl sulfoxide was used instead of glycyrrhizin.
Cell Proliferation Analysis.
First, different colon cancer cells (SW480, SW620, HT29, and HCT116) in the logarithmic growth phase were digested and resuspended. Second, GBN was added at different concentrations (100, 50, 25, and 12.5 μmol/L −1 ). After 24 h, 10 μL of CCK-8 solution was added to each well, and the cells were cultured for 3.5 h. Finally, the absorbance value at 450 nm was measured with a microplate reader for calculation. The calculation formula was as follows: cell survival rate = ½ðAs − AbÞ/ðAc − AbÞ × 100% (As: experimental well OD value; Ac: control well OD value; Ab: blank well OD value).
Cell Apoptosis
Analysis. The cell suspension (without trypsin-EDTA) was treated with different concentrations of GBN (20 and 40 μg/mL). At the same time, 5 μL of Annexin V-FITC was added. Subsequently, the cells were washed with PBS and suspended. Then, the cells were incubated with Annexin V-FITC/PI (Shanghai Beibo Biotechnology Co., Ltd.) at 37°C for 15 min in the dark. All data were analyzed by using a C6 flow cytometer (BD Biosciences, San Diego, CA, USA).
Nuclear Morphology Analysis.
A cover glass was placed in a six-well plate, and colon cancer cells were implanted. The cells were treated with 40 μg/mL GBN. After fixation and washing, 0.5 mL of Hoechst 33258 staining solution (Nanjing KGI Biotechnology Development Co., Ltd.) was added, and the cells were stained for 5 min. After sealing with a sheet, staining was observed with a fluorescence microscope (Olympus IX73), and images were collected.
2.6. Cell Migration Experiment. The cells were plated and incubated when their density reached 80%. Subsequently, the back of the plate was scratched, and the cells in treated medium containing 40 μg/mL GBN were added. After the cells were cultured at 37°C for 0 and 24 h, photographs were taken and recorded to determine the healing distance of the scratches. Each experiment was repeated three times. 2.10.2. Experimental Method. The target crystal structure, including 2119 active sites of 140 targets, corresponding to the disease genes was completely resolved by using the whole target library of the reverse virtual screening platform. The target structure corresponding to the disease gene was analyzed by using the part target library. These structures included 5487 active sites of 506 targets. The structural formula file of GBN was obtained by searching the DrugBank databank. Then, virtual molecular docking was carried out. The binding ability between the compound and protein was scored by applying the reverse virtual screening platform, and the top 200 results were returned. A high score indicated that the drug is bound to the target stably. Finally, target prediction and pathway analysis were carried out by comparing the results of the ProteinBank and KEGG databases.
2.11. Proteomics Analysis 2.11.1. Protein Preparation and TMT Labeling. Tumor cells were lysed in lysis buffer at 70 Hz for 90 s. The lysed tissue sample was centrifuged, and the supernatant was collected. A BCA protein quantification kit (Thermo Fisher Scientific) was used to detect the protein content of the total protein extract of each group of tumor cells. After quantification, the protein sample was reductively alkylated with 10 mmol/L TCEP reducing agent (Sigma) and 40 mmol/L iodoacetamide (Sigma). Subsequently, the TMT 16Plex kit (Thermo Fisher Scientific) was used for labeling. The peptides in each group were labeled with different TMT labels (Table 1). Eventually, all the labeled samples were mixed, vacuum dried, and stored at −20°C for further analysis.
High-pH Liquid Phase Separation and LC-MS/MS
Analysis. The TMT-labeled peptides were dissolved in UPLC loading buffer. A reversed-phase C 18 column ACQUITY UPLC BEH C18 Column (1.7 μm, 2:1 mm × 150 mm, Waters, USA) was used for high-pH liquid phase separation.
Easy-nLC 1200 combined with a Q Exactive HF-X mass spectrometer was used to identify proteins. Peptides were dissolved in mass spectrometry loading buffer and separated on a C 18 chromatographic column (75 μm × 25 cm, Thermo, USA) after 120 min with a volume flow rate of 300 μL/min. The EASY-nLC liquid phase was used as the gradient elution. Buffer phase A comprised 2% acetonitrile and 0.1% formic acid, and buffer phase B consisted of 80% acetonitrile and 0.1% formic acid. MS and MS/MS acquisitions were automatically switched. MS was performed with a full scan (m/z 350-1500). The acquisition mode was tDDA, and the cycle time was 2 s. Finally, TurboTMT was used to improve the resolution of the ion isotope.
2.11.4. Bioinformatic Analysis. Gene Ontology (GO: http:// geneontology.org/) was selected for the functional annotation of all differential proteins from the three aspects of biological process, cell composition, and molecular function. The Kyoto Encyclopedia of Genes and Genomes (KEGG: http://www.genome.jp/kegg//) pathway database was used to analyze the metabolic and signal pathways involving differentially expressed proteins. Table 2 shows all of the primers used for PCR and sequencing. First-strand cDNA was synthesized by using an RT kit (Thermo Fisher Scientific Technology Company). In accordance with the protocol provided in the instructions, 2 μg of total RNA was extracted for reverse transcription, and the final reaction volume was 20 μg. The PCR mixture contained 4 μL of 5× reaction buffer, 1 μL of Ribolock RNase Inhibitor (20 U/μL), 2 μL of 10 mm dNTP Mix, and 1 μL of RevertAid M-MuLV RT (200 U/μL) (Novazin Biotechnology Co., Ltd., China). All the PCR analyses were performed under the same conditions. The specificity of amplicons was verified through melting curve analysis (10 s at 95°C and 30 s at 60°C) after
4
Oxidative Medicine and Cellular Longevity 40 cycles. The expression of the target gene relative to that of the internal control was calculated by using the 2 −△△ Ct method. Finally, the relative quantitative detection of the gene to be determined was performed.
2.12.2. Western Blot Analysis. Proteins were extracted from human colon cancer cells and quantified as described in Section 2.11.3. An equal amount (20 μg) of total protein in each sample was separated by using 10% SDS-PAGE. Then, the bands were transferred to a PVDF membrane (Millipore, MA, USA). Next, the membranes were blocked with 5% with skimmed milk powder at 37°C for 2 h and incubated with anti-PI3K (diluted 1 : 100), anti-Akt (diluted 1 : 100), anti-mTOR (diluted 1 : 100), and anti-β-actin (dilution 1 : 2000) antibodies overnight at 4°C. Hereafter, the membranes were washed with 0.5% Tween-20/PBS for 4 × 10 min, then incubated with the horseradish peroxidase-labeled goat antirabbit secondary antibody (1 : 2000) for 1 h. After four washes with PBST, the strips were exposed in a fully automated chemiluminescence instrument and stored. Protein Oxidative Medicine and Cellular Longevity expression level was defined as the gray value quantified with ImageJ software (National Institutes of Health, USA) and normalized to β-actin expression.
2.13. Statistical Analysis. All the experimental data were expressed as the mean ± SEM. Statistical analyses were performed with SPSS software (version 26.0, SPSS Inc., Chicago, IL, USA). The t -test (if σ1 2 ≠ σ2 2 , then, the t-test was used) was performed to compare the means between the groups of samples. One-way ANOVA test (if σ1 2 ≠ σ2 2 , then, Dunnett's t -test was used) was conducted to compare the means between multiple groups of samples, and P < 0:05 indicated that the difference was significant.
GBN Inhibited the Proliferation of SW480 and SW620
Cells. Figure 1(a) shows the chemical structure of GBN.
The human colon cancer cell lines SW480, SW620, HT29, and HCT116 were selected to study the inhibitory effects of different concentrations of GBN (100, 50, 25, and 12.5 μmol/L) on tumor cell proliferation. The results showed that different concentrations of GBN had a significant inhibitory effect on colon cancer cells (P < 0:05; P < 0:01). The findings in Table 3 confirm that the human colon cancer cell lines SW480 and SW620 were more sensitive to GBN than other lines and can thus be used for follow-up experiments. The survival rates of the SW480 cells treated with 40 μg/mL GBN for 24 and 48 h were 43:75% ± 2:38% and 45:48% ± 0:23%, respectively (P < 0:001). The survival rates of each group of SW620 cells after 24 h of treatment with 20 and 40 μg/mL GBN were 50:29% ± 1:73% and 22:16% ± 2:08%, respectively (P < 0:001). Cell proliferation in each treatment group significantly inhibited cell proliferation by varying degrees (Figures 1(b) and 1(c)) after 24 h of treatment relative to that in the control group. The difference among groups was statistically significant (P < 0:001). On the basis of these results, the follow-up mechanisms of action and proteomics experiments were performed with 20 and 40 μg/mL GBN for apoptosis experiments and the action time of 24 h.
Effect of GBN on the Migration of SW480 and SW620
Cells. The cell scratch experiment revealed that the migration rates of SW480 and SW620 cells under 40 μg/mL GBN treatment were 34:10% ± 2:34% and 25:11% ± 0:73%, respectively. GBN significantly inhibited the migration of the human colon cancer cell lines SW480 and SW620 and decelerated the healing of scratches (P < 0:001) (Figure 3(a)).
3.4.
Effect of GBN on the Invasive Ability of SW480 and SW620 Cells. The number of invasive SW480 cells in the GBN group was 51:00 ± 6:48. The number of invasive SW620 cells in the GBN group was 51:00 ± 6:56. GBN can significantly inhibit the invasive ability of SW480 and SW620 cells and significantly reduced the number of invasive cells compared with the control treatment (P < 0:01) (Figure 3(b)).
Effects of GBN on the Expression Levels of Proteins and
Genes in SW480 and SW620 Cells. In SW48 and SW6200 cells, the protein expression levels of Bax, cleaved-caspase 7 Oxidative Medicine and Cellular Longevity 3, and cleaved-caspase 9 increased significantly after GBN drug intervention (P < 0:05, P < 0:001, and P < 0:001). Meanwhile, Bcl-2 and MMP9 protein expression decreased (P < 0:01) (Figure 4(a)). Figure 4(b) also illustrates that the MMP2 gene level in SW480 cells significantly decreased (P < 0:01). In SW620 cells, the levels of Bcl-2 and MMP2 genes obviously decreased (P < 0:001). These results confirmed that GBN can promote the migration and invasion of SW480 and SW620 cell apoptosis through the caspase apoptosis pathway.
3.6. Establishment of a Disease Target Database to Screen the Target Results. A total of 18 potential targets were found in accordance with the virtual screening scoring results and the KEGG database analysis of the main related pathways of GBN involved in colon cancer (Table 4). KEGG database analysis revealed that GBN had six possible targets, namely, ABL1, INS, SRC, IRAK4, CHEK1, and KDR. This result confirmed that the virtual binding proteins of GBN and colon cancer were relatively concentrated in the PI3K-AKT, mTOR, and MAPK signaling pathways.
Candidate
Target Determination for GBN. The screened GBN targets were compared with the CRC-related protein data obtained from a tumor genomics database (http:// www.cbioportal.org). The results showed that GBN could upregulate 64 related proteins in CRC cells (Table 5). By comparing these 64 proteins with those in Table 4, we found that 13 candidate targets upregulated proteins in CRC cells and acted on signal pathways (Table 6). In addition, by using the above database, we found five candidate genes for the anticolon cancer effect of GBN, namely, INS, KDR, ABL1, SRC, and CHEK1. These proteins were mainly involved in the PI3K-AKT, mTOR, MAPK, and Wnt pathways.
Comparison of Screened GBN Targets with Genes
Expressed in SW480 and SW620 Cells. First, we obtained the genetic information of SW480 and SW620 cell lines by using the Cancer Cell Line Encyclopedia. Then, through Table 5, we confirmed that the information of the five target genes in SW480 cells, including PTPN1, SEC14L2, PDE3B, PPARD, and TGFBR1, was consistent. Among these genes, PPARD and TGFBR1 were the same as the screened candidate target genes of GBN. Similarly, in the SW620 cell line, HSP90AB1 was the same as the screened candidate target gene of GBN. Finally, we confirmed that these target genes were involved in the Wnt, TGF-β, MAPK, and PI3K-AKT signaling pathways.
Proteomics Research and Analysis.
We conducted proteomics analysis to obtain a global overview of GBN regulatory proteins and to further understand the mechanism of GBN's anticolon cancer action. High-pH RPLC analysis revealed that 7-20 amino acids were distributed in most of the peptides. This distribution pattern conformed to the general rules based on enzymatic hydrolysis and HCD fragmentation. By searching the NCBInr database, we identified 57 965 peptides and 6750 proteins. A protein with a fold difference that had increased by 1.2 times or decreased by 0.83 Table 7: GBN_620 vs. c_SW620 differential protein GO classification statistics' table.
Bioinformatic Analysis of Differentially Expressed
Proteins. We first performed GO enrichment analysis to clarify the biological importance of the differentially expressed proteins in c_SW620 and c_SW480 under GBN treatment. The functions of these proteins were annotated in accordance with cell composition, molecular functions, and biological processes ( Table 7). The GO annotation analysis of differentially expressed proteins between GBN_620 and the control group c_SW620 showed that 19 proteins were involved in biological processes, Figure 6: (a) GBN vs. c_SW620 differential protein KEGG enrichment analysis diagram; (b) GBN vs. c_SW480 differential protein KEGG enrichment analysis diagram. Note: (1) In the column chart, the abscissa represents the pathway name, and the ordinate represented the enrichment rate (the greater the ratio, the greater the degree of enrichment). (2) The color gradient of the column indicated the significance of enrichment, and the darker the color indicated the higher the enrichment degree of the KEGG term ( * * * P < 0:001, * * P < 0:01, and * P < 0:05).
The enriched string diagram showed the corresponding relationship between the target protein and the KEGG pathway. The left side was the protein, and the order from top to bottom showed log 2FC. The greater the log 2FC, the difference in the expression of the upregulated protein was the greater. On the right was the KEGG pathway name and z score of the target protein. On the right was the KEGG pathway name and z score of the target protein. Among them, count represented the total number of target proteins involved in this pathway. Up represented the number of upregulated proteins involved in this pathway. Down represented the number of downregulated proteins involved in this pathway. If z score was greater than zero, it meant that there were more upregulated proteins than downregulated proteins involved in this pathway, and this pathway was more likely to be activated. Conversely, z score was less than zero, which meant that this pathway is more likely to be inhibited.
two were involved in cell components, and eight were involved in molecular functions. GO enrichment analysis revealed that the differentially expressed proteins had the highest degrees of enrichment in biological processes and molecular functions. When the enrichment of the differentially expressed proteins was corrected at P < 0:05, the enrichment rate was 100% ( Figure 5(b)). GO annotation analysis was performed on the differentially expressed proteins between the GBN_480 and c_SW480 group. These proteins were involved in 16 biological processes, two cell components, and seven molecular functions. GO enrichment analysis demonstrated that the differentially expressed proteins were enriched in the biological process function at the highest degree. The enrichment rate after concentration correction at P < 0:05 was approximately 40% (Figure 5(c)).
KEGG Functional Analysis of Differentially Expressed
Proteins. By using the KEGG database, we can classify differentially expressed proteins in accordance with the pathways they participate in or the functions they perform. The analysis between groups revealed that in SW620 cells under GBN action, the differentially expressed proteins participated in KEGG pathways (the first 20 are listed in Table 8). Table 9 illustrates that in secondary classification, differential protein pathways were mainly distributed in metabolic pathways, such as amino acids and nucleotides, translation, signal transduction, cell growth and death, and cancerrelated pathways. We discovered that the highly enriched pathways with P < 0:05 were the unsaturated fatty acid biosynthesis pathway, p53 signaling pathway, mTOR signaling pathway, and other pathways (Figure 6(a)).
The analysis of GBN_480 and c_SW480 KEGG annotations showed that the differentially expressed proteins participated in 83 KEGG pathways (the first 20 are listed in Table 10). Secondary classification revealed that the differential proteins mainly concentrated in pathways related to energy metabolism, lipid metabolism, translation, signal transduction, transportation and catabolism, cell growth and death, cancer-related pathways, gene folding and degradation, and infectious diseases (Table 11). KEGG enrichment analysis illustrated that the pathways with P < 0:05, such as apoptosis and platinum drug resistance, were highly enriched ( Figure 6(b)).
Differential Pathway and Protein Expression Analysis.
We mainly investigated the metabolic pathways involving the key proteins to analyze the functional effects of differential proteins as a whole. We found a total of 382 differential proteins in c_SW620 administered with GBN. These proteins were involved in 260 KEGG metabolic pathways that were distributed in six major categories and 44 subcategories. In addition, pathway classification showed that the 29 differential proteins with the largest proportion were distributed in the signal transduction pathway in the environmental information processing category (Figure 7(a)). Next, through KEGG pathway analysis, we finally confirmed that in c_SW620 administered with GBN, the largest number of proteins participated in the two signaling pathways of mTOR and PI3K-Akt. Through the cluster analysis of 29 differential proteins, we found that the administration of GBN had a good callback effect on most signaling pathways (Figure 7(b)). Protein levels changed significantly in c_SW480 cells administered with GBN. In the c_SW480 cell group, we found 348 differential proteins, which were involved in 225 KEGG metabolic pathways. The statistical results of pathway classification indicated that these pathways were distributed in six categories and 41 subcategories (Figure 7(c)). Among the differentially expressed proteins, 27 were distributed in the signal transduction pathway in the environmental information processing category. KEGG analysis indicated that that the largest number of proteins was involved in the two signaling pathways of cancer and PI3K-Akt. Cluster analysis indicated that the main downregulated proteins were serine protein kinase (P30154), guanine nucleotide-binding protein subunit-4 (Q9HAV0), fibronectin (P02751), 14-3-3β/α protein (P31946), Cyclin-dependent kinase CDK-4 (P11802), adhesion protein (P04004), 3-phosphoinositide-dependent protein kinase 1 (Q15530) telomere length regulatory protein TEL2 homolog (Q9Y4R8), V-type proton ATPase (O75348), and RhoA (P61586) (Figure 7(d)).
In short, the use of KEGG pathway enrichment analysis demonstrated that the largest number of proteins was involved in cancer, PI3K-Akt, mTOR, and other signaling pathways. After GBN acted together on the signal transduction pathway in SW620 and SW480 cells, the proteins that showed significant differences included NCSTN, V-ATP, CTNNA, CDK4, DUSP9, MKP4, ATP1B, and CD298.
Discussion
Computer reverse virtual screening technology is often used to predict the signaling pathways and main targets of smallmolecule compounds [23]. It has the advantages of low cost, fast speed, and high efficiency [24]. Our research group uses a system that is based on independent disease target databases (whole and partial) and molecular docking methods Figure 7: (a) Statistical histogram of pathway classification in SW620 cell administration group; (b) clustering heat map of 29 differential proteins in c_SW620 cell signaling pathway; (c) statistical histogram of pathway classification in SW480 cell administration group; (d) clustering heat map of 29 differential proteins in c_SW480 cell signaling pathway. Note: 7a and 7c represented 6 major categories and 44 subcategories of KEGG metabolic pathways of differential proteins common to the drug-administered group and the control group. 7b and 7d showed that each column represented a sample, and each row represented a protein. On the left was a dendrogram of protein clusters, and on the right was the name of the protein. The closer the two protein branches were, the closer their expression levels were.
(AutoDock Vina). We also established a reverse virtual screening system with the help of the SDF format in the DrugBank database [25]. This system has been used by numerous researchers to verify the accuracy and practicality of anticancer targets [26][27][28]. Proteomics can detect the full set of proteins expressed by the complete genome in a cell [29,30]. This technology can visually identify the regulation of drugs at the cellular protein level [31], then find targets and related pathways [32]. Therefore, on the basis of our experimental results, we confirmed for the first time that GBN can inhibit the apoptosis, migration, and invasion of SW620 and SW480 cells. At the same time, through computer reverse virtual screening technology and proteomics research, we predicted that GBN may inhibit the occurrence of colon cancer through the PI3K-AKT-mTOR pathway.
The PI3K-AKT-mTOR is closely related to the occurrence and development of CRC [33,34]. PI3K is a key factor in this pathway [35]. Activated PI3K promotes the phosphorylation of PIP2 on the cell membrane into PIP3. The THr308 and Ser473 in the AKT protein are phosphorylated to bind with PIP3 [36]. PI3K activation can affect a variety of downstream effector molecules that can lead to reduced apoptosis [37], stimulate cell growth [38], and promote proliferation [39]. Activated Akt can also mediate apoptosis by regulating the expression of genes, such as Bcl-2 and caspase 3 [40]. mTOR, an important target gene downstream of Akt, is considered to be a member of the PI3K-related protein kinase family [41]. Activated mTOR can phosphorylate the translation repressor molecule eIF4E binding protein 1 and ribosomal protein p70S6K [33]. This process can inhibit tumor cell apoptosis [42]. Furthermore, the activated mTOR signaling pathway can increase the expression of matrix metalloproteinases to enhance extracellular matrix degradation. Therefore, this process can enhance tumor cell invasion and promote tumor cell migration [43]. Similarly, a large number of studies have reported that the PI3K-AKT-mTOR pathway can inhibit the growth, proliferation, and invasion of colon cancer cells [44]. Therefore, through
Oxidative Medicine and Cellular Longevity
Western blot analysis and qRT-PCR experiments, our team verified that GBN can exert its mechanism of drug action through the PI3K-AKT-mTOR pathway.
Conclusions
GBN has a certain application value because of its various anti-inflammatory, antibacterial, antitumor, anticardiovascular disease, and nervous system protective effects. Although it is currently not used as a clinical drug, it has an important curative effect without obvious side effects. Therefore, it is a highly promising potential drug. In summary, we used the combination of computer reverse virtual and proteomics technologies to study the mechanism underlying GBN's anticolon cancer effect. GBN can inhibit the proliferation of the human colon cancer lines SW480 and SW620 in vitro and induce tumor cell apoptosis. Its mechanism of action may be related to the inhibition of the PI3K-AKT-mTOR signaling pathway. Our research results demonstrated that the combination of computer reverse virtual and proteomics technologies is an effective tool for evaluating and exploring the efficacy and mechanism of action of Chinese medicine. At the same time, the results of this study provide a new understanding of how GBN inhibits proliferation and migration and promotes apoptosis in the human colon cancer cell lines SW480 and SW620 in vitro and evidence for GBN as a potential candidate drug for colon cancer.
Data Availability
The data that support the findings of this study are available from the corresponding authors upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.
Conflicts of Interest
The authors declare no conflicts of interest.
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2023-01-12T17:36:16.909Z
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2023-01-05T00:00:00.000
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"year": 2023,
"sha1": "fc5f2198eb41c78eb7ef48e6e57227d5f56e8def",
"oa_license": "CCBY",
"oa_url": "https://downloads.hindawi.com/journals/omcl/2023/1117431.pdf",
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"Medicine"
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216125237
|
pes2o/s2orc
|
v3-fos-license
|
Algorithms of Cause-and-effect Approach to Increase Service Net Efficiency
Service nets distribute goods and services that is why their improvement is one of the important tasks of any production chain. There is many models related to the sphere however, in many of them it is possible to see some weaknesses. At the same time since the task mentioned is complicated and large scaled some systematical approach should be applied that needs to be modified taking into consideration presence in the systems objects, processes, events and phenomena of various nature and origin. As possible approach one consideres so called cause-and-effect one that provides a universal description of complex systems and possibility of descision making in undefined or under-defined situations. In the artcile below this approach is considered and informational logic diagrams and algorithms to increase service net efficiency are presented. Gas stations were taken as examples and sphere of practical application, that results are discussed.
Introduction
Service nets are important parts of present economy [1]. The statement is true for petroleum supply also [2,3] that may be considered as a good example. To develop it there should be improvements of the existing models and optimization methods [4][5][6] to make management and control more efficient considering last as one of the main tasks of the present century [7].
A Service net (company, structure, etc.) as any complex system works under requirements of the upper, goal-orienting and parents systems, follows demands of consumers, takes into consideration possibilities of competitors and suppliers, fulfills laws, makes innovations and so on. These demands, influences and restrictions (further-factors) on/to/from a system and its surroundings may be formalized as elements of mathematical sets that permit to create in a parameter/factor space some feasible regions, goal areas and so on.
Under the process approach if one considers gas stations as an example of service nets it is usually determined the station themselves (that serve consumers), station complexes or gas station nets (that provide work of the stations in a region) and companies as legal entities. The investigation object is a large scale complex territorially distributed hierarchical human-machine system [8] tasks to increase efficiency of which are the ones of many criteria optimization [9]. Existing models and methods to resolve modern practical tasks are frequently not enough since they are not systemized. In addition, there are mainly considered state-level or object-level structures without due attention to regional service nets (station complexes), transportation (clients) flows are modeled as simplistic that is non-adequate, modern petroleum equipment like OPT (Outdoor Payment Terminal) and so on is not considered, etc.
The task is to bring efficiency or key performance indicator (KPI) K to its maximum at given and perspective factors of the system and surroundings G during ∆t by developing structures S = X, U, GR and choosing control actions, C, A, X, U, R that is where X -set of control means, U -relation between them, GR -structure graphs, С -control functions, A -control algorithms, R -variant of a control structure. In given formulation the task is not resolved in general because of large scale, diversity of components and non-linearity of their interactions that demands a new approach.
Method: Causal-and-effect Approach to Increase Efficiency of Complex Systems
The most important regularity of complex system behavior is historicity or development in time [10,11]. These ideas are formulated in all fields of knowledge and practical activity [12]. At the same time relative simplicity and observability of cause-and-effect (causal) interactions is the reason for their particular «refusal», since for example from «the earlier» is not exactly derived «in accordance with/because of». This situation caused necessity of the following research that is done in some spheres [13].
In general, it is possible to consider that modern scientific knowledge is based on the determination of causal interactions between objects, processes, events and phenomena (further -objects). They unify visions from intuitive through scientific to philosophical permitting formal logical description at deep investigation of the functional spheres, taking into consideration origin of the events, their vicinity and development in time and provide matching of knowledge that comes from various spheres of theory and practical activity.
Every object of a system, a process realized, event as changing of state or phenomena of surroundings has its reason of origin and development that connects them with other objects. Goals as future states of a system are achieved at conditions determined by mentioned factors (condition 1). Results (effects) of causal interaction changes a system and surroundings that performs some new conditions (or condition 2). For analytical description the cause-and-effect cell operation algebra is used similar to finite-state machine one [6]. The model of the cell (general at the first level of decomposition) is as follows: Complements (extra-and interpolation of parameters between parts of the system with the most trustable data); Composition (developing of the whole system model by following flowcharts of processes of known systems and models, so-called base or etalon models); And substitution (synthesis of structures optimal on criteria).
To increase efficiency the model of an elementary causal cell structure mentioned was changed. Achieving of a simple goal by means of elementary control task solution is modeled by the elementary cause-and-effect cell. Part of the system at state SA with KPI K to achieve goals at the factors of surroundings GA under control СА by converting of resources WA is brought by means of functions and algorithms, contained in the kernel of a causal cell, to the state SB with conditions GB and control CB corrected accordingly to the goal achieving degree ( GA GB − ), new K* («*» -after interaction) and output resources flow WB. It is possible to express K, G, S through each others. For nonelementary cases some cause-and-effect (or reasone-andconsequence, RC) complexes are created for the part or whole of the system. Casual components interact accordingly to the flowcharts of base models using operations of OC set. There fore new model of RC-cell on the first level of decomposition at matrix view is presented, where А -before and B -after interaction: The solution of tasks in known situation is achieved by putting general, known or theoretically and eхperimentally proved RC-cells, its components decomposition till the level understandable by decision-makers using base models, practice performance checking, necessary feedback and correction.
If there are unknown situations or in case when there is no enough information about the system, surroundings and their interaction some parts with the most trustable data are determined. For them procedures mentioned to known situations are realized, general RC-models of the whole system are developed and optimization tasks are resolved considering known models with accuracy of data available. Results are step-by-step improved while the system is developed and/or one gets new data. Information about results put in some Petrol Data-Base (PDB). Since there are no restrictions on types of functions and algorithms of kernels, it is possible to describe interactions of objects of different nature.
Results (Algorithms and Diagrams) to Improve Service Station Net
Optimal control parameters task causal formulation is shown as folows: where n inp -input transport flow, n out -outpuit flow, G uv ∈ G -factors of the system and surroundings (u=1.. U -type, v=1.. V -kind), int -quasi-stationary time intervals, where linear dependence or constancy are adequate and possible, α -statistical significance, K station -efficiency indicator to be increased, К station ↑. Generalized algorithm to determine efficient parameters of gas stations at given factors of surroundings is presented on Figure 1, where R -factors/parameter space, R*-feasible region, X and Y -data of comparable objects, av -average, ∆K -error. «End» operator is dotted since the algorithm is cycled due to necessity of step-by-step improvement of the system. The task to synthesize the structure of complex multicircuit systems, optimal on K petrol at given G, where K petrol is brought to MAX by development of structures and selecting of control actions at causal formulation is as follows: ( ) where w 1..6 -resources (1 -staff, 2 -technology, 3 -energy, 4 -knowledge, 5 -finances, 6 -materials), P -set of processes, GR, GR 1 -GR 4 -structure graphs of, correspondingly, infra-system (non-active and needed control), control (1), decision making (2), organizationtechnical (3) and information (4) systems. It is supposed [14] that the models are enough to describe a whole system. A system structure is synthesized accordingly to the following informational logic diagram presented in written form.
On the I-st stage there is an analysis of system at surrounding conditions. a) Designation and specification the goals as components of the X vector given by decision-makers. It depends on factors of surroundings G, flow chart of processes S, control means characteristics X and relations between them U, i.e. X (G, S, X, U). Quantitively they are determined by data of real working objects. b) Specifying the system in surrondings adding to it some controllable components data of which descisionmakers can evaluate goals achievability. c) Determination of boundaries between controllable and control systems accordingly to activity (deliberate changing of information) of components. Non-active or infra-system does not have the property and needed control. areas Е ef (see Figure 1).
Figure 2. Typical relations between goals, processes and objects in gas station nets.
On the II-nd stage the structure of control system is formed. a) Formation of the process flow charts and object structures accordingly to the known models that are acumulated in PDB. b) Determination of permitted dominative and sequence relations between objects N, processes P and goals G using results of the sphere analisis [15] done ( Figure 2). On Figure 2 P 1 -petroleum product supply, P 2operational activity, P 3-sales and consumer service, P 4accounting and reporting, P 5 -maintenance and repairing, P 6 -staff training, P 7 -security, P 8 -energy provision, P 9 -transport, P 10 -information service, P 11 -purpose-oriented direction, P 12 -procurement, P 13analysis, P 14 -decision making, P 15 -control; X pqcontrol means (p=1.. P -type, q=1.. Q -level); N 1.. rinfra-system objects non-active at the view. c) Formation of controllable system structure as interconnected recource-converting objects accordingly to the processes structure of Figure 2. On III-rd stage control system structure is formed. a) Specifing the control time periods H k (k=1.. K), control functions C i (i=1.. I) and control means X pq accordingly to the models of PDB mentioned. b) Forming sets of elementary control tasks : and circuits Cartesian products. c) Control system structure model creation or Ω-synthesis and refusal circuits with the low efficiency, w/o necessary automation level or meaningless. On the IV-th stage there is a synthesis of control system structure by circuits convolutions. Some better X pq may be added and convolutions are done until limits of their properties, considering efficiency and level of automation. a) C-convolution (synthesis) as integration of control functions alongside control circuits and designation more C i to the smaller number of X pq . b) P-convolution as an integration of control functions belonging to various control circuits of processes P j by designating of more C i to be performed by the same X pq . c) Н-convolution as an integration of control functions C i on various time periods by lower number of X pq . On the V-th stage one looks optimal variants of the system structure.
d) Forming of control circuit set (Ω'synthesized) and determination of КPetrol. e) Designation as optimal those structures, KPI of which are closest to Eef. If it is not achieved, one goes to Stage I. f) Forming of organization-and-technical system structures by bringing new and/or improving existing control means accordingly to the control system structure (see p. 5.2) and requirements to control means Xpq. g) Forming the information system structure by putting data arrays and transmission channels to control and organizational-technical system structures already done (see pp. 5.2 and 5.3). h) Forming the decision-making structure by designation to the components of organization-and-technical system structures and the same for information system types of decision-making acts using PDB mentioned [15]. i) Proposal to improve the model and algorithm, transition to Stage II.
The task to form structures and choose optimal control actions for service nets using system cause-and-effect approach is resolved by the following informational logic diagram ( Figure 3). Figure 3. Information-logic diagram to form structure and choose control actions for service (gas) nets, optimal on the given criteria with using system reason-and effect approach.
Discussion
As a result of the causal approach application proposed it was developed the complex of inter-related informational logic diagrams and algorithms to impove service nets as a part of the methodology of rational development and continious improvement of service station nets and effecient automatical control of processes and objects in the systems (Methodology). Basic components of the methodology are presented on Figure 4.
On the base of the Methodology there were solved some practically important tasks [15].
In particular, there were found optimal gas station parameters at various types of street-and-road nets. It was shown that as optimal on the criterion of minimum outage of clients and service channels may be considered (if one minds fuel sales only) the structure of two dispensers with all of the fuels being sold on the station. Outdoor payment terminals built in dispencers provide at least 10% higher productivity. During 2000-2014 the model was applied on more than 150 objects.
For gas station nets it was proved that up to 80% of the modern and perspective flows of clients may be served by smaller quantity of stations. For various types of street-androad nets there were found some characteristics (quantity of cross-roads, distances between neighboring stations and their quantity), providing minimal redistribution of clients bertween objects of the same net for small (up to 500 thousand residents) and medium (up to 1,5 million residents) cities and towns for non-dominating petroleum supply company, operating smaller that 25% of a region stations, and the same on highways. Also in some regions of middle Russia there were developed optimal structures to serve card clients that increased sales in volume in 6 times. In these regions and some CIS countries there were changed technical maintanance systems that provided cost reduction in 3-15% at better service. Moreover there were prepared efficient system structures to serve clients near pumps, security, automation, procrurement counter-actions, capital construction, etc. Finally, it was done modelling of processes on stations that permitted to increase staff training skills in newly built trainng centers in Saratov and Volgograd-cities.
Conclusion
Service nets are important for an economy and require their continuous improvement.
A causal-and-effect approach was suggested and new informational logic diagrams and algorithms were developed. They are characterized by co-synthesis of controllable and control systems, descision-making in case of not enough trustable data from systems and surroundings, possibility to match objects, processes, events and phenomena of various nature and so on.
Adequatnes of the methodology is cofirmed by the proximity of the known and developed models on the similar feasible regions, reliability of results by statistical data for more than 15 years of observation, validity of conclusions by results of approbation and successful multiple applications. Said above permits to use it for other service nets and complex system at all.
Conflict of Interest Statement
All the authors do not have any possible conflicts of interest.
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2020-04-24T23:54:15.987Z
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2020-03-24T00:00:00.000
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251698131
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Insights into Circulating Tumor Cell Clusters: A Barometer for Treatment Effects and Prognosis for Prostate Cancer Patients
Simple Summary Circulating tumor cells (CTCs) are a promising biomarker for the risk of prostate cancer aggressiveness and metastasis and play a role in the processes of tumor migration and metastasis. CTC clusters, which have different physical and biological properties from individual CTCs, are collections of tumor cells and non-malignant cells, resulting in greater metastatic potential. Therefore, this review aims to summarize the current knowledge of CTC clusters in metastasis as well as related biological properties and to suggest possibilities for their usage in diagnostic and therapeutic practice. Abstract Prostate cancer (PCa) exhibits high cellular heterogeneity across patients. Therefore, there is an urgent need for more real-time and accurate detection methods, in both prognosis and treatment in clinical settings. Circulating tumor cell (CTC) clusters, a population of tumor cells and non-malignant cells in the blood of patients with tumors, are a promising non-invasive tool for screening PCa progression and identifying potential benefit groups. CTC clusters are associated with tumor metastasis and possess stem-like characteristics, which are likely attributable to epithelial–mesenchymal transition (EMT). Additionally, these biological properties of CTC clusters, particularly androgen receptor V7, have indicated the potential to reflect curative effects, guide treatment modalities, and predict prognosis in PCa patients. Here, we discuss the role of CTC clusters in the mechanisms underlying PCa metastasis and clinical applications, with the aim of informing more appropriate clinical decisions, and ultimately, improving the overall survival of PCa patients.
Introduction
In the United States, prostate cancer (PCa) is the most common cancer diagnosed in males [1][2][3]. Since prostate-specific antigen (PSA) was widely applied to the detection of asymptomatic PCa during the early 1990s [4], overall PCa incidence in males has generally decreased. This has revealed the immense benefit of inspection tools. Recently, however, it has been found that early detection of PCa by PSA testing has led to overdiagnosis and overtreatment. Thus, there is an urgent need for alternative tools [5]. Liquid biopsy, defined as the analysis of tumor cells and tumor-derived products in the blood and other body fluids [6], is an alternative to tissue biopsies. It can be used to diagnose and screen for tumors in real-time. Additionally, it is a noninvasive and replicable way of monitoring circulating tumor cells (CTCs), cell-free DNA (cfDNA), cell-free RNA (cfRNA), and extracellular vesicles and particles (EVPs). Studies have demonstrated that CTCs' diagnostic Many questions still remain about CTC clusters. For instance, is the direct derivation of CTC clusters from primary tumors or single CTCs in the peripheral blood? Likewise, the relationship and interaction between CTC clusters and single CTCs remain unclear. How do CTC clusters metastasize? How are CTC clusters related to stem cells? In this context, these topics have been reviewed with an emphasis on the relationship between CTC clusters and PCa metastasis, as well as the prospective application of CTC clusters in clinical settings.
CTC Clusters and Single CTCs
To date, despite there being no solid evidence that CTC clusters and single CTCs are two totally different and independent cells, the contrast between CTC clusters and single CTCs based on physical properties and biological features is reported in many tumors. In patients with tumors, single CTCs are more prevalent than CTC clusters, and CTC clusters consisting of several tumor cells are larger than single CTCs. This lessens the potential for extravasation. However, CTC clusters have been observed reversibly unfolding into single chains when they go through vessels [34]. Non-malignant cells in CTC clusters are involved in extravasation and stabilize CTC clusters in peripheral blood [35]. In addition, single CTCs are unable to form polyclonal metastatic foci in distant organs, but CTC clusters have a greater ability to metastasize and also have the potential to form polyclonal metastatic foci [36]. In conclusion, the composition, survival advantage, and metastatic potential are the main differences between CTC clusters and single CTCs.
Locations of Cells with Different E/M States in CTC Clusters and Invasion
EMT is a complex cellular pathway in which epithelial cells lose epithelial characteristics (e.g., cell-to-cell adhesion) and gain mesenchymal characteristics (e.g., increased migratory capabilities) [37]. Experimental evidence that has accumulated over decades has indicated that tumor cells in CTC clusters undergo EMT, as demonstrated by EMT biomarker detection [30,38,39]. The evidence also reveals that EMT has potential relevance to mechanisms underlying tumor metastasis, cancer stem cell (CSC) generation and maintenance, as well as drug resistance [40]. At the molecular level, the loss of adherens junction protein E-cadherin is considered a hallmark of EMT [41]. It results in the gain of mesenchymal markers such as vimentin, N-cadherin, α-smooth muscle actin (α-SMA), and fibronectin [42]. Satelli et al. reported that FOXC2, an EMT-specific marker, was detectable in CTCs from 10 metastatic PCa patients. However, the epithelial markers EpCAM and E-cadherin were absent in these cells, indicating a mesenchymal phenotype [32]. Surprisingly, Yu et al. found an association between the expression of mesenchymal markers and CTC clusters in human breast cancer specimens, rather than single migratory cells [43]. This has focused following researches on CTC clusters and EMT. In PCa-based PDX models, CTC clusters have been observed to contain a mix of cell phenotypes, and the location patterns of different cell phenotypes are worthy of inquiry. For instance, epithelial-like cells have been located on the periphery of the cluster, surrounding hybrid or mesenchymal-like cells in PCa-based PDX models [30]. The epithelial phenotype has been implicated in metastatic colonization [40]. Lori E. et al. demonstrated that PCa with an increasingly mesenchymal phenotype shed greater numbers of CTCs more quickly and with greater metastatic capacity than PCa with an epithelial phenotype in 4 PCa models with progressive epithelial (LNCaP, LNCaP-C42B) to mesenchymal (PC-3, PC-3M) phenotypes [44]. Thus, epithelial-like cells on the periphery of CTC clusters might be essential to understanding mechanisms underlying distant metastasis. However, tumor cells with an EMT phenotype have exhibited a reverse location pattern in solid tumors. In primary and secondary PCa, the expression of the EMT phenotype at the invasive tumor front is higher than that at the center [45]. A similar phenomenon has also been observed in other solid tumors such as breast cancer [46]. Thus, it is the different environments that might result in some variation in cell phenotypes in CTC clusters or solid tumors. Additionally, it is unclear whether the distribution of CSC-like cells might have differential effects on cell phenotype dedifferentiation or conversion within CTC clusters or solid tumors. Collective cell migration has been observed not only in tumors but also in wound healing and tissue renewal. Cells in collective migration can perceive the microenvironmental chemotaxis and initiate the cellular migration by dividing into "leader" cells and "follower" cells [47].
Single-cell RNA-sequencing analysis of leader-like and follower-like cells has revealed differentially expressed gene profiling pertaining to cellular locations within the migrating collective [48]. For example, genes associated with Wnt/planar cell polarity (PCP) signaling were overexpressed in cells at the invasive front. This indicates that Wnt/PCP signaling is involved in tumor invasion [48,49]. Luo et al. demonstrated that the crosstalk between androgen receptor (AR) and Wnt signaling promotes the androgen-independent growth of PCa by maintaining LNCaP cells under androgen-depleted conditions. WNT5A and LEF1 are reported to be downregulated in low-grade PCa while upregulated in metastatic PCa [50]. In addition, a retrospective analysis suggested that non-canonical Wnt signaling was activated in CTCs of 13 PCa patients with AR inhibitor, resulting in antiandrogen resistance [51]. In PCa, ectopic expression of Wnt5a suppresses the anti-proliferative effect of the inhibition of AR, whereas this suppression restores partial sensitivity in drug-resistant cells. Wnt5a, a vertebrate Wnt ligand, triggers the Wnt/PCP signaling pathway. Ultimately, it regulates cytoskeletal remodeling, such as cell polarity, migration, and subsequently, tissue re-arrangement and organ formation [52]. The assessment of noncanonical Wnt signaling pathway components seems to be a promising way to identify patients with metastatic castration-resistant prostate cancer (mCRPC). These patients are likely to have poor prognoses after treatment with androgen deprivation therapy (ADT). Therefore, the interaction between the Wnt signaling pathway and the AR signaling pathway might be relevant to the ADT resistance. In patients' CTC clusters, the distribution of tumor cells in different E/M states might imply that tumor cells perform various roles in the migration process.
Stem-Like State of Hybrid E/M Phenotype Cells and Metastasis
It has been assumed that many intermediates between epithelial and mesenchymal phenotypes co-exist in the migrating CTC cluster. The hybrid E/M phenotype cells form heterogeneous CTC clusters with cells in various EMT states and maintain cell-cell junctions on the basis of E-cadherin [53,54]. However, the function of each phenotype remains controversial. Several recent studies have suggested that it is hybrid E/M cells that act as pluripotent CSCs and promote invasiveness [42]. To date, there have been consistent observations in hybrid E/M cells isolated from mouse PDX models of PCa [55], as well as in the co-culturing of epithelial PCa cells with post-EMT PCa cells in vitro [56]. Further, this hypothesis has received the support of clinical evidence [57], which suggests that hybrid phenotypes "gain" features of both epithelial and mesenchymal phenotypes in carcinoma specimens [58,59]. Ruscetti et al. demonstrated that mesenchymal and epithelial states in PCa cells of Cre +/− ; Pten L/L ; Kras G/+ ; Vim-GFP mouse models contribute differentially to their capacities for tumor initiation and metastatic seeding, respectively [55]. Mesenchymal tumor cells display an enriched tumor-initiating capacity, and epithelial tumor cells can exist with the capacity to form macro-metastases. A hybrid phenotype that possesses both properties of mesenchymal and epithelial tumor cells has an enhanced ability to migrate and form distant foci in a complex environment ( Figure 1). However, some researchers have held opposing views. Tsuji et al. demonstrated that only appropriate cooperation between EMT cells and non-EMT cells of HCPC-1 cells can promote successful metastasis; each kind of cell alone is unable to metastasize [60]. In this study, the researchers did not consider the hybrid E/M state. Furthermore, several findings have indicated that EMT is dispensable for cancer cell-mediated migration. For instance, Fischer et al. demonstrated that inhibiting EMT by targeting ZEB1 and ZEB2 overexpression via miR-200 does not impair breast tumor cells' ability to form distant lung metastases [61]. Likewise, Zheng et al. suggested that EMT inhibition by deleting Snail or Twist is critical to neither robust invasion nor the metastasis of pancreatic cancer [62]. Together, CTC clusters undergoing EMT are pivotal for migration, but hybrid E/M phenotype cells' exact role in metastasis is worth pursuing further. , cancerassociated fibroblasts (CAFs), and platelets. Tumor cells undergo the epithelial-mesenchymal transition (EMT), and they exhibit three phenotypes: epithelial phenotype, mesenchymal phenotype, and hybrid E/M phenotype. The epithelial phenotype has the capacity for tumor initiation, and the mesenchymal phenotype has the capacity to form metastasis. The hybrid E/M phenotype is equipped with both capacities. TAMs, CAFs, and platelets each play roles in metastasis and prostate cancer invasion.
The hybrid EMT cell state, with both epithelial and mesenchymal features, is reconcilable with the state of highly plastic stem-like cells. Similarly, in the process of blood dissemination, CTCs undergoing EMT possess features of CSC-like tumor cells that are responsible for generating most of the metastatic foci, as well as capabilities of resistance to radio-and chemotherapy-based treatment [11,63]. Likewise, CTC clusters have been observed expressing more mesenchymal transcripts in patients receiving cancer treatment [64]. A study suggested that 35/46 (76%) CTCs were CD133-positive, a putative prostate cancer stem cell marker, in 35 patients with high-risk, localized prostate cancer; the researchers demonstrated that the CD133 and E-cadherin-positive CTC fragments were associated with biochemical recurrence at 1 year [65]. Compared to single CTCs in the DNA methylation landscape of 43 breast cancer patients and 3 mouse models, CTC clusters resulted in hypomethylation in the binding sites of OCT4, NANOG, SOX2, and SIN3A. However, single CTCs featured hypomethylation of other TFBSs, including those that are occupied by MEF2C, JUN, MIXL1, and SHOX2. [19]. Additionally, most of these binding sites were occupied by master stemness and associated with proliferation regulators. The patterns were similar to those of embryonic stem cells. Of note, NANOG is essential to the Figure 1. Cells that make up circulating tumor cell (CTC) clusters. CTC clusters include tumor cells and non-malignant cells that consist primarily of tumor-associated macrophages (TAMs), cancerassociated fibroblasts (CAFs), and platelets. Tumor cells undergo the epithelial-mesenchymal transition (EMT), and they exhibit three phenotypes: epithelial phenotype, mesenchymal phenotype, and hybrid E/M phenotype. The epithelial phenotype has the capacity for tumor initiation, and the mesenchymal phenotype has the capacity to form metastasis. The hybrid E/M phenotype is equipped with both capacities. TAMs, CAFs, and platelets each play roles in metastasis and prostate cancer invasion.
The hybrid EMT cell state, with both epithelial and mesenchymal features, is reconcilable with the state of highly plastic stem-like cells. Similarly, in the process of blood dissemination, CTCs undergoing EMT possess features of CSC-like tumor cells that are responsible for generating most of the metastatic foci, as well as capabilities of resistance to radio-and chemotherapy-based treatment [11,63]. Likewise, CTC clusters have been observed expressing more mesenchymal transcripts in patients receiving cancer treatment [64]. A study suggested that 35/46 (76%) CTCs were CD133-positive, a putative prostate cancer stem cell marker, in 35 patients with high-risk, localized prostate cancer; the researchers demonstrated that the CD133 and E-cadherin-positive CTC fragments were associated with biochemical recurrence at 1 year [65]. Compared to single CTCs in the DNA methylation landscape of 43 breast cancer patients and 3 mouse models, CTC clusters resulted in hypomethylation in the binding sites of OCT4, NANOG, SOX2, and SIN3A. However, single CTCs featured hypomethylation of other TFBSs, including those that are occupied by MEF2C, JUN, MIXL1, and SHOX2 [19]. Additionally, most of these binding sites were occupied by master stemness and associated with proliferation regulators. The patterns were similar to those of embryonic stem cells. Of note, NANOG is essential to the establishment of pluripotency, self-renewal, and reprogramming [66], which regulate the gene expressions involved in the mitochondrial metabolic pathways required to maintain tumor-initiating stem-like cells in PCa and breast cancer [67]. Tumor heterogeneity, which is generated evolutionarily not only as a result of genetic alterations but also by the presence of cancer stem cells, is a driving factor behind the failure of cancer treatment modalities. Therefore, elucidating the molecular underpinnings of CSCs' biological features is crucial to the development of novel cancer therapies for PCa.
Sources of CTC Cluster
It has been demonstrated that CTC clusters can reduce CTC apoptosis, elevate cell viability, and promote the ability to re-form clusters [12,68]. Furthermore, CTC clusters have been found to exhibit stronger resistance to anti-tumor drugs than single CTCs [69,70]. However, how CTC clusters form remains controversial. To date, there are two main hypotheses explaining how CTC clusters form ( Figure 2). The first is that as tumor pieces, CTC clusters fall off the primary tumors and then travel through the vessels. The other is that CTC clusters are aggregated from single CTCs in the blood. Alone, CTC clusters are considered to be a part of the primary tumor, shedding into the blood spontaneously or passively. However, recent findings have challenged this hypothesis. Liu et al. have observed that individual CTCs derived from patient-derived breast cancer models aggregate into CTC clusters in the blood vessels, suggesting the potential resource of CTC clusters. Most of the clusters contain two types of tumor cells as labeled by fluorescent indicators, resulting in a high ratio of polyclonal CTC aggregation within 2 h in the lungs. However, the ratios of dual-color aggregates in the lungs gradually decrease over time [71]. This study provides evidence that CTC clusters are directly formed by individual CTCs in the peripheral blood. Then, what is the relationship between metastatic foci and CTC clusters? Maddipati et al. reported that most metastatic foci in the distant organs were polyclonal populations in a mouse model of pancreatic cancer; whereas metastatic foci became increasingly dominated by a single clonal population as they increased in size ( Figure 2) [36]. In addition, genes that are highly expressed in primary tumors compared to CTCs are consistent with genes expressed in primary tumors compared to metastases. Furthermore, it has been reported that single CTC genomic profiling displays high concordance with metastatic biopsies from the same patients [72]. These studies imply that CTCs might be an origin of metastatic foci, and there is a differentiation between primary tumors and CTCs [51]. Cheung et al. determined that polyclonal lung metastases arise via colonization by a multicellular cluster of tumor cells instead of the serial seeding of single tumor cells. Additionally, CTC clusters were reported to exist in five different stages of metastasis: collective invasion, locally disseminated clusters in the adjacent stroma, intravasation tumor emboli, CTC clusters, and distant metastases [68]. Taken together, these observations suggest CTC clusters might form when there are a variety of heterogeneous tumor cells in the vasculature, and then gradually convert from polyclonal to oligoclonal foci after colonizing distant tissues. If
Role of CTC Cluster Components
CTC clusters consist of not only tumor cells, but also non-malignant cells, including TAMs, CAFs, immune cells, epithelial cells, and platelets. In CTC clusters, non-malignant cells act as protectors and supporters for cancer cells in the blood and increase cancer cells' ability to migrate and invade ( Figure 1). In solid tumors, CAFs promote tumor invasion by multiple tumor types with multiple mechanisms, ranging from typical cell-cell signaling to dynamic alteration of ECM, which is the result of fibroblast remodeling activity. Likewise, PCa patients' CTC clusters go through a high magnitude of fluid shear stress
Role of CTC Cluster Components
CTC clusters consist of not only tumor cells, but also non-malignant cells, including TAMs, CAFs, immune cells, epithelial cells, and platelets. In CTC clusters, non-malignant cells act as protectors and supporters for cancer cells in the blood and increase cancer cells' ability to migrate and invade ( Figure 1). In solid tumors, CAFs promote tumor invasion by multiple tumor types with multiple mechanisms, ranging from typical cellcell signaling to dynamic alteration of ECM, which is the result of fibroblast remodeling activity. Likewise, PCa patients' CTC clusters go through a high magnitude of fluid shear stress (FSS) in the peripheral blood, during which process reactive CAFs can conserve tumor cells' proliferative capability by cell-cell contact and secreting paracrine factors [24]. The reactive CAF phenotype emerges from normal fibroblasts (NFs), which are activated by cytokines secreted by tumors. Additionally, reactive CAFs can be identified by the overexpression of α-SMA, fibroblast specific protein 1, and fibroblast activation protein [73]. Further, Duda et al. demonstrated that CAFs spontaneously spread to the lung tissue along with metastatic cancer cells and accelerate the growth of secondary tumors [74]. This evidence indicates that CAFs are particularly relevant in CTC clusters with metastasis as well as invasion. CAFs are one of the abundant stromal cell populations in the tumor microenvironments. In primary tumors, CAFs facilitate a breach of the basement membrane (BM) by altering the cellular organization and the physical properties of BM, making it less compact for tumor cell invasion [75]. Tumor protrusion and processes can be inhibited when tumor cells are exposed to healthy extracellular matrixes, indicating CAFs' critical role in promoting metastasis [76]. Recently, it has been reported that a heterophilic adhesion among CAFs and tumor cells via N-cadherin or E-cadherin guides tumor cell migration within connective tissue [77]. That is in line with a previous report demonstrating N-cadherin-and E-cadherin-mediated cancer cell migration by cell-cell junction [78]. In sum, CAFs appear to be involved in cancer cell migration and invasion, especially in intercellular adhesion. In PCa, TAMs uniquely co-isolated with CTCs have been shown to promote conversion from epithelial-mesenchymal plasticity, resulting in the adaptation of cancer cells to mechanical stress. Thus, CTC clusters are conferred with adaptive resistance to shear stress and maintain integrity [20]. Additionally, platelets can act as a coating of CTCs to protect them from violent FSS, and their adhesive proteins (fibronectin and von Willebrand factor) have been found to interact with CTCs through integrins, supporting CTC cluster formation [78]. In addition, platelets have been shown to protect CTCs from apoptosis, promote EMT and extravasation, and facilitate escape from immune system surveillance such as by inhibiting natural killer (NK)-cell-induced lysis in PCa [79,80]. For instance, Labelle et al. demonstrated that platelet-derived TGFβ and the contact between platelets and tumor cells synergistically activate the TGFβ/Smad and NF-kB pathways in colon carcinoma and breast carcinoma cells. This results in the transition to an invasive mesenchymal-like phenotype [81]. Moreover, various immune cells also protect CTC clusters from anti-tumor immune attacks, promoting tumor cell migration. Szczerba et al. observed CTC-white blood cell clusters in breast cancer by staining EpCAM, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), and CD45 [82]. Furthermore, neutrophils have also exhibited promotive effects on CTC migration. In conclusion, non-malignant cells within CTC clusters play a critical role in promoting extravasation by altering cell-cell junctions and offering protection from anti-cancer immune attacks and FSS in the process of cancer cell migration and colonization.
Distant Metastatic Foci and CTC Clusters
Considering that CTCs can grow into emboli, researchers previously assumed that CTC clusters were unable to transit through capillaries 5-10 µm in diameter in view of cluster size. However, Au et al. recently presented evidence that over 90% of CTC clusters containing up to 20 cells successfully traverse 5-to 10-µm porous constrictions during detection by means of microfluidic devices. It has been noted that CTC clusters can rapidly and reversibly unfold into single chains by selective cleavage of intercellular adhesions when CTC clusters transit through narrow blood vessels (Figure 2) [34]. Similarly, Green et al. utilized a microfluidic device (a Pillar device and an X-magnetic device) to isolate single CTCs and clusters from whole blood in mouse models and patients with metastatic breast cancer. Surprisingly, they observed that some of the clusters could maintain weak intercellular adhesion. These clusters re-arranged their shape to travel through the pillars, and then clusters re-formed in the X-magnetic device [83]. This process provided direct evidence that CTC cluster cohesion can be regulated by the dynamic expression of E-cadherin, as reflected by the adaptive alteration of cluster shapes. Additional evidence revealing the metastatic potential of CTC clusters has been based on Na + /K + ATPase inhibitors, which lead to DNA methylation remodeling at critical sites and metastasis suppression by dissociating CTC clusters into single cells [19]. These studies provide novel ideas on how CTC clusters transit to distant metastatic foci. CTC clusters exhibit much higher intercellular adhesion levels than single CTCs, as evidenced by a report that CTC clusters in breast cancer overexpress plakoglobin (by 219-fold), an important component of desmosomes and adherence junctions, in comparison to single CTCs [12]. In a CHD1-normal cohort of PCa patients, high junction plakoglobin expression, which was linked to strong androgen receptor expression, high cell proliferation, and PTEN and FOXP1 deletion, was an independent predictor of poor prognosis and early biochemical recurrence [84]. Now, the precise role of intercellular adhesion in CTC cluster migration is still not well understood. Thus, identifying intercellular adhesion is crucial for elucidating the mechanism underlying PCa metastasis.
Separation Techniques and Devices
Despite the growing number of methods associating biological attributes and physical properties, particularly EpCAM and size, with the isolation and detection of CTC clusters, it is still a major challenge to isolate CTC clusters. The current CTC cluster enrichment technologies mainly use biological or physical properties of the CTC clusters for isolation. The CellSearch ® system was the first and only US Food and Drug Administration (FDA)authorized system for the enumeration of CTCs in 7.5 mL of blood to rely on the expression of EpCAM only [34], resulting in overlooking the more invasive EpCAM-negative CTCs that are in the process of epithelial-mesenchymal transition (EMT) [20]. Of note, some studies have suggested that non-blood-derived aneuploid circulating tumor-derived endothelial cells (CTECs) have properties in common with CTCs, such as the expression of EpCAM. Thus, CTECs' influence on CTC detection is deserving of much attention [15]. Recently, to purify CTC clusters out of individual CTCs, researchers have utilized the physical and biological properties in combination, including the size, deformability, and expression of cellular markers [35,36]. This is a great opportunity to understand CTC clusters.
Microfluidic devices, which reduce cluster separation, accelerate processing time, and collect live CTC clusters for downstream analysis, display the potential to be the most favorable platform for isolating CTC clusters [85]. A number of intrinsic biomarkers have been used to identify and isolate CTC clusters: for example, size, electrical polarizability, and hydrodynamics in microfluidic systems [86]. For instance, due to the strong sizedependence of inertial migration, CTC clusters and single CTCs can be selected by precisely controlling the channel length in untreated whole blood [87]. Additionally, deterministic lateral displacement has been used to continuously separate CTC clusters by size. CTC clusters, having greater sizes than individual CTCs, are removed from whole blood by deterministic lateral displacement of the microfluidic device. CTC clusters are collected through successful deflection, while the remaining parts are sorted by discriminating asymmetric clusters from symmetric single cells. Au et al. [88] demonstrated that the recovery of cultured breast cancer CTC clusters from whole blood using this integrated two-stage device results in minimal cluster separation, 99% recovery of large clusters, and cell survival rates exceeding 87%. Moreover, the ability of electric fields to exert forces on particles has been used as a means of manipulation in stand-alone separation devices. Chiu et al. [89] reported that optically induced dielectrophoresis-based cell manipulation in a microfluidic system was able to isolate H209 small cell lung cancer cell clusters with a cell purity and recovery rate of 91.5 ± 5.6%, and 70.5 ± 5.2%, respectively, without compromising integrity. However, electrochemical reactions can result in free radicals, leading to significant cellular damage. Microstructural protrusions have been shown to be useful for entrapping cells and performing cell separation based on size, deformability, and density. Herringbone grooves form a flow pattern suitable for separating particles of similar size based on density. The Herringbone-Chip with low shear flow properties revealed the presence of CTC clusters in patients with metastatic prostate cancer [90]. In sum, microfluidic chips exhibit the ability of efficient cell sorting without purification, high system throughputs, and low sample volume.
CTCs in Clinical Application
Liquid biopsy is the process of collecting information relevant to disease in body fluid (e.g., blood), including CTCs, cfDNA, cfRNA, and EVPs. In PCa, liquid biopsy has the advantages of easy acquisition, high replicability, and superior reflection of cellular properties, treatment efficacy, and cancer prognosis. Regarding PCa patients with bone metastasis, liquid biopsy is superior to tissue biopsy, since the latter is technically challenging. In addition, in bone metastasis, disseminated tumor cells (DTCs) are a special kind of CTC that can home to the bone marrow. The amount of DTCs isolated from bone marrow is larger than that of CTCs isolated from blood [91,92]. During this process, DTCs are also a candidate for tumor sampling. From another perspective, cfDNA, also called circulating tumor DNA, is a DNA fragment released into the blood circulation from apoptotic, necrotic or secreted tumor cells. The genetic variation and epigenetic features of tumor cells can be well preserved in cfDNA, making it a potential biomarker [93][94][95]. However, the amount of cfDNA is hard to quantify due to the high degree of fragmentation and low circulating concentration. Moreover, cfDNA measurement can be complicated by heart dysfunction associated with heavy smoking or physical exercise. Alternatively, EVPs can also serve as an indicator of cancer treatment efficacy, and even for early diagnosis and prognosis estimation [96][97][98]. Hoshino et al. investigated EVP proteomic profiles in 120 human samples and demonstrated that plasma-derived EVPs can be utilized to detect early oncogenesis with 95% sensitivity and 90% specificity [99]. In conclusion, liquid biopsy (especially CTCs), as an emerging non-invasive testing method, plays a vital role in aiding diagnosis, assessing efficacy and prognosis, and monitoring early recurrence and drug resistance. As such, it may become an important component of future cancer management.
CTC Enumeration
For PCa, extensive findings have suggested that CTCs play a pivotal role in guiding chemotherapeutics [100,101], predicting prognosis [102][103][104][105][106][107], and reflecting treatment effects [108][109][110] (Table 2). With regard to the limited technological methods for purifying single CTCs and CTC clusters, most studies involving CTCs do not distinguish CTC clusters from single CTCs when they explore the role of CTC counting. For instance, several studies have established the prognostic role of CTC enumeration in both localized and metastatic PCa patients, demonstrating a higher prognostic performance. Choi et al. demonstrated that localized PCa patients who were CTC-positive were at a higher risk of up-staging and up-grading [111]. However, Meyer et al. suggested that there is no significant correlation between CTC detection and PSA, disease characteristics, or the development of biochemical recurrence in patients with localized PCa [112]. Zapatero et al. also did not observe any association between CTC count and OS in 65 patients with advanced high-risk localized PCa [113]. In sum, the effect of CTC counts on localized PCa has been controversial. Therefore, larger-scale trials with more sensitive techniques are needed to confirm it, and in metastatic PCa patients, the prognostic role of CTC enumeration has more practical applicability. Lozano et al. demonstrated that baseline CTC enumeration of 80 mCRPC patients treated with docetaxel had greater significance than PSA quantitation in predicting overall survival [114]. Kruijff et al. summarized 114 mCRPC patients treated with cabazitaxel in the second round of chemotherapy. They determined that the CTC count has a strong prognostic value, for both progression-free survival (PFS) and overall survival (OS) [115]. In addition, Goldkorn et al. conducted a phase 3 prospective randomized trial in metastatic castrate-sensitive prostate cancer (mCSPC) patients treated with ADT combined with orteronel or bicalutamide. After adjusting for disease extent and distinguishing between patients likely to experience poorer survival outcomes, they found that baseline CTC counts in mCSPC had a high predictive value for 7-month PSA and 2-year PFS (N = 264 and N = 336, respectively) [116]. In sum, the CTC count is a promising predictor of outcomes in patients with mCRPC. Researchers have further differentiated PCa patients by CTC cutpoints of 0, 1-4, and ≥5, hoping for a more reasonable prognostic prediction. A pilot study suggested that CTC-positive (≥1 CTC/7.5 mL blood) patients with recurrent PCa have worse pathological and short-term oncological outcomes [117]. In addition, Yang et al. demonstrated that total CTC count ≥5/5 mL blood was an independent predictor of early progression to mCRPC and shorter cancer-specific survival after cytoreductive radical prostatectomy for 54 oligometastatic hormone-sensitive PCa patients [118]. These findings indicate that CTC enumeration, as an early treatment response biomarker, is helpful in supporting the utilization of CTC quantification to identify patient cases with early progression. It is also useful in selecting intensive treatments to prevent unnecessary exposure to ineffective therapy with undesirable toxicities [114]. Taken together, there is extensive evidence of an association between CTC count, prognostic prediction, and therapeutic efficacy in PCa patients, especially those with metastatic PCa.
To date, increasing CTC-involved experiments have provided insight regarding both single CTCs and clusters. Recently, Zhu et al. observed that CTCs exhibited random bursts during cancer progression in an orthotopic mouse model of human PCa. The bursting activity was more intense in early stages and declined in late stages in PCa. CTC counts have been found to peak during night time, suggesting that the release of single CTCs might be regulated by circadian rhythms [119]. Thus, for the purposes of CTC detection, blood sampling from patients at appropriate timepoints should be considered. Table 2. CTC enumeration.
Cancer Type
Results (from Published Trials) Localized prostate cancer CTC in patients with localized prostate cancer had a larger number than in healthy volunteers. In patients with stage T2 tumors, the presence of Gleason pattern 5 was positively correlated with CTC positivity (rho = 0.59, p < 0.001) [111].
Localized prostate cancer
CTCs were detected in 17 patients (range: 1-clusters with >100 epithelial cells) without significant correlations to PSA levels or Gleason scores [112].
Oligometastatic hormone-sensitive prostate cancer
CTCs were detected in 51/54 patients, and M-CTCs detection rates were 67%. A positive correlation was found between the M-CTC count and number of bone metastases [118].
Metastatic castration-resistant prostate cancer
Higher baseline CTC count was significantly associated with worse OS, PFS and time to PSA progression [114].
Metastatic castration-resistant prostate cancer
In 114 metastatic castration-resistant prostate cancer patients treated with cabazitaxel, CTC counts were independently associated with PFS and OS [115].
Androgen Receptor V7
Androgen receptor V7 (AR-V7) in CTCs has been studied widely for PCa as well (Table 3), and it has been found that the expression of AR-V7 is associated with cancer prognosis and drug resistance (Figure 3). AR-V7 is a truncated isoform of the canonical AR-FL protein, and it lacks the ligand-binding domain, which is the target of enzalutamide and retains both the DNA-binding domain and the amino-terminal domain [120]. Areti et al. suggested that AR-V7 detected in 34/69 (49.3%) CTCs is superior to that detected in 4/52 (7.7%) plasma-derived EVP as an indicator of cancer treatment efficacy in metastatic PCa patients [121]. Moreover, Sepe et al. confirmed that AR-V7 expression had a significant negative effect on radiological OS in 37 mCRPC patients treated with enzalutamide or abiraterone [122]. The detection of AR-V7 in CTCs is associated with shorter PFS and OS in mCRPC patients treated with abiraterone or enzalutamide, but such AR-V7-positive patients still derive similar benefits from subsequent taxane chemotherapy [101]. In a cross-sectional cohort study, Scher et al. found that 161 mCRPC patients who had pretherapy AR-V7-positive CTCs treated with taxanes had a more favorable survival time with taxanes relative to AR signaling (ARS) inhibitor. Additionally, it was demonstrated that cabazitaxel improves overall OS in mCRPC patients after docetaxel [123]. Gurioli et al. suggested that the initial reduced dose of cabazitaxel (20 mg/sqm) had a correlation between AR-V7 expressions and worse outcomes in eight mCRPC patients [102]. In sum, the detection of AR-V7 in CTCs is associated with poor outcomes in abiraterone-treated patients and enzalutamide-treated patients, but the presence of AR-V7 can be a treatment-specific biomarker that is associated with superior survival with taxane therapy over ARS-directed therapy in the clinical setting. As for 193 mCRPC patients whose CTCs were determined to be AR-V7-negative, Graf et al. observed that AR-V7-negative patients had superior survival on ARS inhibitors over taxanes [106]. In addition, Gupta et al. suggested that PTEN loss and BRCA2 gain were associated with significantly worse outcomes in 40 AR-V7 negative mCRPC patients treated with abiraterone/enzalutamide [124]. Thus, AR-V7-negative patients are more likely to have better survival time among metastatic PCa patients treated with enzalutamide or abiraterone. Intriguingly, Hench et al. observed that the expression of AR-V7 mRNA underwent a dynamic change in the progression of mCRPC, including recovery of AR-V7 expression in six patients' CTCs and the reversion from AR-V7positive CTCs to either AR-V7-negtiveCTCs or "no CTCs" [125]. This conversion may be due to the inactivation of the AR signaling axis, resulting in reduced selective pressure for AR-V7 expression. Nevertheless, Belderbos et al. suggested that AR-V7 expression in CTCs had no additional prognostic value in 127 mCRPC patients who progressed after administration of docetaxel and/or enzalutamide or abiraterone [126]. Further prospective validation is needed to explore the exact role of AR-V7 as a useful predictive biomarker in PCa patients treated with different chemotherapeutic drugs. In view of the different treatment modalities for PCa patients in the aforementioned studies, AR-V7 remains a prospective predictor of PCa. Table 3. AR-V7 in metastatic castration-resistant prostate cancer.
Ref.
Results (from Published Trials) [121] AR-V7 was detected in CTCs of 34/69 metastatic castration-resistant prostate cancer patients. AR splice variants were expressed in higher levels in CTCs than in paired extracellular vesicles.
[122] 21/37 patients CTC-positive before starting treatment with enzalutamide or abiraterone: 24% of CTC-positive patients were defined as AR-V7-positive. Positivity for each variable was significantly associated with poorer rPFS and OS. [123] Patients with AR-V7-positive CTCs before ARS inhibition had resistant posttherapy PSA changes, shorter rPFS, and shorter OS than those without AR-V7-positive CTCs.
[124] Consistent PTEN loss and inconsistent BRCA2 gain were associated with significantly worse outcomes in AR-V7-negative CTC patients treated with abiraterone/enzalutamide. [126] AR-V7 expression in CTCs was not associated with OS. [102] CTC expression of AR-V7 was significantly associated with OS. In patients treated with cabazitaxel 20 mg/sqm, median OS was shorter in AR-V7-positive than -negative patients (6.6 vs. 14 months).
Abbreviations: AR-V7-androgen-receptor splice variant 7; CTC-circulating tumor cell; AR-androgen receptor; PFS-progression-free survival; PSA-prostate-specific antigen; OS-overall survival; ARS-; rPFSradiographical progression-free survival. . mCRPC patients who have pretherapy AR-V7positive circulating tumor cells (CTCs) are associated with poorer outcomes for enzalutamide/abiraterone over taxanes. However, mCRPC patients who have pretherapy AR-V7-negative CTCs are associated with better outcomes on enzalutamide/abiraterone; subsequently, the alterations of PTEN loss and BRCA2 gain in CTCs indicate worse outcomes. In addition, cabazitaxel at a lower dose is associated with poorer outcomes after docetaxel in AR-V7-positive patients, compared to AR-V7-negative patients. Note that this figure should not be used for clinical decision-making.
CTC Clusters
To date, only a few studies have reported the experimental acquisition and application of CTC clusters in clinical settings. These studies have shown that these clusters reflect the innate properties of primary tumors. Ortiz-Otero et al. demonstrated that CTC clusters can serve as predictive biomarkers for cancer recurrence in primary PCa. They observed that 2/15 patients experienced cancer recurrence within 2 months after primary tumor resection, and levels of individual CTCs and CTC clusters were increased in these patients at the time of surgery or after surgery. Moreover, the levels did not normalize after 2 weeks, suggesting levels of individual CTCs and CTC clusters vary considerably in PCa progression [22]. Additionally, combining CTC clusters with routine single CTC detection exhibited independent predictive value in improving prognostic stratification in mCRPC patients [127]. Furthermore, the detected CTC clusters, especially AR-V7-positive CTC clusters, are essential for the response to abiraterone and enzalutamide therapy and for predicting disease outcomes. Okegawa et al. found that 26/98 CTC cluster-positive/AR-V7-positive patients treated with abiraterone or enzalutamide had more severe bone metastasis at diagnosis, pain, higher alkaline phosphatase levels, and visceral metastases [27]. Consistently in breast cancer, larger CTC clusters have been indicative of a higher risk of patient death [14]. Therefore, CTC clusters display the ability to further stratify death risk for patients with a high level of single CTCs.
With the development of technologies such as single-cell sequencing, the understanding of CTC has become more comprehensive on a micro-level scale. However, these advanced technologies face mounting issues when they are utilized in clinical practice. For instance, single-cell sequencing, a promising method for studying PCa properties and molecular mechanisms, has revealed CTC heterogeneity on a single-cell scale. In PCa Figure 3. AR-V7 (androgen receptor V7) as an indicator of chemotherapy in patients with metastatic castration-resistant prostate cancer (mCRPC). mCRPC patients who have pretherapy AR-V7-positive circulating tumor cells (CTCs) are associated with poorer outcomes for enzalutamide/abiraterone over taxanes. However, mCRPC patients who have pretherapy AR-V7-negative CTCs are associated with better outcomes on enzalutamide/abiraterone; subsequently, the alterations of PTEN loss and BRCA2 gain in CTCs indicate worse outcomes. In addition, cabazitaxel at a lower dose is associated with poorer outcomes after docetaxel in AR-V7-positive patients, compared to AR-V7-negative patients. Note that this figure should not be used for clinical decision-making.
CTC Clusters
To date, only a few studies have reported the experimental acquisition and application of CTC clusters in clinical settings. These studies have shown that these clusters reflect the innate properties of primary tumors. Ortiz-Otero et al. demonstrated that CTC clusters can serve as predictive biomarkers for cancer recurrence in primary PCa. They observed that 2/15 patients experienced cancer recurrence within 2 months after primary tumor resection, and levels of individual CTCs and CTC clusters were increased in these patients at the time of surgery or after surgery. Moreover, the levels did not normalize after 2 weeks, suggesting levels of individual CTCs and CTC clusters vary considerably in PCa progression [22]. Additionally, combining CTC clusters with routine single CTC detection exhibited independent predictive value in improving prognostic stratification in mCRPC patients [127]. Furthermore, the detected CTC clusters, especially AR-V7positive CTC clusters, are essential for the response to abiraterone and enzalutamide therapy and for predicting disease outcomes. Okegawa et al. found that 26/98 CTC cluster-positive/AR-V7-positive patients treated with abiraterone or enzalutamide had more severe bone metastasis at diagnosis, pain, higher alkaline phosphatase levels, and visceral metastases [27]. Consistently in breast cancer, larger CTC clusters have been indicative of a higher risk of patient death [14]. Therefore, CTC clusters display the ability to further stratify death risk for patients with a high level of single CTCs.
With the development of technologies such as single-cell sequencing, the understanding of CTC has become more comprehensive on a micro-level scale. However, these advanced technologies face mounting issues when they are utilized in clinical practice. For instance, single-cell sequencing, a promising method for studying PCa properties and molecular mechanisms, has revealed CTC heterogeneity on a single-cell scale. In PCa patients, the biomarkers and RNA profiling of single CTCs from each display considerable heterogeneity, including expressions of AR gene mutations and AR-V7. Moreover, tumor cells in CTC clusters also exhibit various expressions of biomarkers and genes. Therefore, research on more relevant CTCs is crucial to acquiring specific and sensitive outcomes for PCa patients. Using epithelial biomarkers (e.g., EpCAM) to identify CTCs, researchers established single-cell sequencing profiling of single CTCs that exhibit immense heterogeneity [51]. This indicates that more specific and effective selection criteria for CTCs are needed to obtain more accurate results. Thus, the CTC cluster, possessing greater metastatic potential and CSC-like characteristics, is a competitive candidate for analysis by single-cell sequencing. Research on PCa on a micro-level scale means any tiny mistake can contribute to drastically different conclusions. Researchers must stay alert for this.
Conclusions
Technical difficulty remains a challenge for the purification of CTC clusters. Recently, a variety of "label-free" enrichment technologies for CTC clusters have emerged with improved harvest efficacy [128]. However, they remain marginally satisfactory in view of weak specificity and inadequate harvesting. Therefore, the development of enrichment and isolation techniques with high acquisition efficiency and convenient downstream cellular analysis is in urgent demand.
The metastatic and pluripotent characteristics of CTC clusters are of great significance to cancer research. The formation of CTC clusters is pivotal to understanding how and why CTC clusters are heterogeneous. CTC clusters undergo EMT and contain a mix of cell phenotypes, especially the hybrid E/M phenotype, which plays various roles in metastasis. This might provide clues to a better understanding of tumor cell migration and colonization.
The CTC cluster is a promising biomarker of tumor management, yet research on its application has been insufficient. In the future, researchers will be able to study CTC clusters from the perspectives of enumeration, genome profiling, protein, and telomere. Future work on the origin, formation, and migration of CTC clusters will provide researchers with further insight into cancer biology and liquid biopsy.
Conflicts of Interest:
The authors declare no conflict of interest.
|
2022-08-21T15:12:11.613Z
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2022-08-01T00:00:00.000
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214048522
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pes2o/s2orc
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v3-fos-license
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Sketch-Based Subspace Clustering of Hyperspectral Images
Sparse subspace clustering (SSC) techniques provide the state-of-the-art in clustering of hyperspectral images (HSIs). However, their computational complexity hinders their applicability to large-scale HSIs. In this paper, we propose a large-scale SSC-based method, which can effectively process large HSIs while also achieving improved clustering accuracy compared to the current SSC methods. We build our approach based on an emerging concept of sketched subspace clustering, which was to our knowledge not explored at all in hyperspectral imaging yet. Moreover, there are only scarce results on any large-scale SSC approaches for HSI. We show that a direct application of sketched SSC does not provide a satisfactory performance on HSIs but it does provide an excellent basis for an effective and elegant method that we build by extending this approach with a spatial prior and deriving the corresponding solver. In particular, a random matrix constructed by the Johnson-Lindenstrauss transform is first used to sketch the self-representation dictionary as a compact dictionary, which significantly reduces the number of sparse coefficients to be solved, thereby reducing the overall complexity. In order to alleviate the effect of noise and within-class spectral variations of HSIs, we employ a total variation constraint on the coefficient matrix, which accounts for the spatial dependencies among the neighbouring pixels. We derive an efficient solver for the resulting optimization problem, and we theoretically prove its convergence property under mild conditions. The experimental results on real HSIs show a notable improvement in comparison with the traditional SSC-based methods and the state-of-the-art methods for clustering of large-scale images.
Introduction
Hyperspectral images (HSIs), acquired by the hyperspectral cameras, record the spectrum of materials covering a wide range of wavelengths. The rich spectral information of HSIs enables discriminating the materials that are often visually indistinguishable, which led to a number of applications in remote sensing, such as target detection [1,2], environmental monitoring [3], geosciences, defense and security [4]. It is often desired to categorize pixels in the imaged scene into different classes, corresponding to different materials or different types of objects. When no training data is available, this task is called clustering. Hence, clustering is also referred to as unsupervised classification.
Two most popular clustering methods are fuzzy c-means (FCM) [5] and k-means [6,7] due to their simplicity and superior computational efficiency. They group data points by finding the minimum distance between test data and each cluster centroid which is updated iteratively. However, their performance is sensitive to initial conditions and noise.
Recently, spectral clustering-based methods [8][9][10][11][12][13] have achieved a great success and have been widely applied in various fields due to excellent performance and robustness to noise [14]. In general, these methods first define a similarity matrix to construct a graph of data points, which is learned from the input data under different critera. Then, the resulting similarity matrix is used within the spectral clustering framework. The performance of spectral clustering heavily depends on the similarity matrix [14], hence, its construction is a crucial step. Many of these methods, like local subspace affinity (LSA) [15], spectral local best-fit flats (SLBF) [16] and locally linear manifold clustering (LLMC) [17] build the similarity matrix with k nearest neighbours (KNN) using angle or Euclidean distance between two data points. This approach tends to treat erroneously the data points near the intersection of two subspaces because their closest points often lie in another subspace.
The recent sparse subspace clustering (SSC) method [11] constructs the similarity matrix based on the self-expressiveness model where the input data is employed as the representation dictionary. SSC models a high-dimensional data space as a union of low-dimensional subspaces. The key insight is that, for each data point in the subspace S i , the global solution of the sparse coding problem with the self-representation dictionary automatically selects the data points in the same subspace S i . Thus, each data point gets automatically represented as a sparse linear or affine combination of other points in the same subspace. This is called subspace preserving property and is explicitly expressed by non-zero entries of the coefficient matrix C: i-th and j-th data points are in the same subspace if C i,j = 0. The coefficient matrix leads directly to the similarity matrix for spectral clustering.
As the SSC model calculates sparse coefficients individually and independently for each input data point, the clustering performance is sensitive to noise. In order to solve this problem, various extensions have been proposed with the aim to encode the spatial dependencies among the neighbouring data points in hyperspectral data, and obtain thereby more accurate similarity matrices and improved clustering results [18][19][20][21][22][23][24][25]. Guo et al. [18,19] focus on the clustering of 1-D drill hole hyperspectral data and regularize the coefficients of neighbouring data points in depth to be similar by a 1 norm based smoothing regularization. For the 2-D spatial-wise hyperspectral images, a smoothing strategy was introduced in Reference [20] by minimizing the difference between coefficients corresponding to the central pixel and to the mean of pixels in a local square window. A kernel version of SSC incorporating max pooling of the sparse coefficient matrix was presented in Reference [21]. The spectral-spatial SSC method of Reference [22] integrates an 2 spatial regularizer with the SSC model (L2-SSC), to penalize abrupt differences between the coefficients of nearby pixels. In Reference [23,25], an 1,2 norm constraint on the coefficients of pixels in each local region was incorporated in the SSC model. Based on the collaborative representation with an 2 norm constraint on the coefficients, a novel model with a locally adaptive dictionary was proposed in Reference [24].
While showing excellent performances, the above mentioned methods are also of considerable computational complexity, resulting from iterative optimization. The time complexity in each iteration is typically in the range of O((MN) 3 ), where M and N are the number of rows and columns in each band. For large scale HSIs with millions of pixels in each band, this bound can thus exceed 10 18 elementary operations per iteration, and such processing becomes often infeasible on the common computing platforms. The approaches reported in References [26,27] addressed this problem by constructing a graph based on a set of selected representative samples. In combination with modified spectral clustering methods, a lower complexity has been reached, but the clustering results are sensitive to the initially selected samples. Recently, some generalized large-scale methods [28][29][30] based on SSC have been proposed for clustering tasks in computer vision. In Reference [28], a scalable SSC method was designed for large-scale data sets, where a small part of samples are first randomly selected and clustered with the SSC model, and then clustering of remaining samples is executed by sparse coding with respect to the dictionary constructed from previously selected samples. The work in Reference [29] studied an efficient SSC model based on orthogonal matching pursuit (OMP) and discussed theoretical conditions for subspace preserving representation. A recent sketched SSC model of Reference [30] lowers the computational burden of SSC by using a clever random projection technique to sketch and compress the input data to a computationally affordable level. While these large-scale SSC-based methods demonstrated success in real applications with facial images, handwritten text and news corpus data, to the best of our knowledge none of them was applied before in the clustering of HSIs. Our experiments show that despite the scalability of these methods, their clustering performance in HSIs turns out to be poor. This can be attributed to the complex spatial structure of HSIs, spectral noise and spectral variability.
In view of this, we propose a sketched sparse subspace clustering method with total variation (TV) spatial regularization, termed Sketch-SSC-TV, which can handle large-scale HSIs while achieving a high level of clustering performance. A sketching matrix constructed by a random matrix is firstly employed to build a sketched dictionary, which is much smaller than the self-representation dictionary, resulting in a significant reduction of the number of coefficients to be solved. By incorporating the spatial constraint as the TV norm on the coefficient matrix, the proposed model greatly promotes the connectivity of neighbouring pixels and improves the piecewise smoothness of clustering maps. Furthermore, we propose an algorithm with theoretically guaranteed global convergence to solve the resulting optimization problem. By adopting the sketching matrix, the optimization complexity of the TV-related sub-problem reduces from O((MN) 2 log(MN)) to O(MNnlog(MN)) (n << MN), facilitating thus greatly the processing of large-scale data. The similarity matrix is constructed by applying KNN on the obtained coefficient matrix, and further employed within the spectral clustering method. Experiments conducted on four HSIs show superior clustering performance compared to both traditional SSC-based methods and the related large-scale clustering methods. The major contributions of the paper can be summarized as follows.
1.
The most important contribution of this paper is a new SSC-based framework, which can be applied on large-scale HSIs while achieving excellent clustering accuracy. To the best of our knowledge, this is the first time to address the large-scale clustering problem of HSIs based on the SSC model.
2.
Different from the traditional SSC-based methods which use all the input data as a dictionary, we adopt a compressed dictionary by using random projection technique to reduce the dictionary size, which effectively enables a scalable subspace clustering approach.
3.
To account for the spatial dependencies among the neighbouring pixels, we incorporate a powerful TV regularization in our model, leading to a more discriminative coefficient matrix. The resulting model proves to be more robust to spectral noise and spectral variability.
4.
We develop an efficient algorithm to solve the resulting optimization problem and prove its convergence property theoretically.
The rest of this paper is organized as follows. Section 2 briefly introduces the clustering of HSIs with the SSC model. Section 3 describes the proposed Sketch-SSC-TV model and the resulting optimization problem. Experimental results on real HSIs are presented in Section 4. Section 5 concludes the paper.
HSI Clustering with the SSC Model
Let a B-band HSI be denoted as Y ∈ R B×MN , where the i-th vector y i ∈ R B represents the spectral signature of the i-th pixel in HSI and MN is the total number of pixels. Sparse subspace clustering (SSC) partitions the high-dimensional data space into a union of lower dimensional subspaces. Concretely, it assumes that all high-dimensional data points y i 's, that is, spectral signatures of all the pixels from a given HSI Y, are drawn from a union of subspaces, each of which corresponds to a particular class.
The key idea is that among infinitely many possibilities to represent a data point y i in terms of other points, a sparse representation will select a few points that belong to the same subspspace as y i . This is known as the subspace preserving property. Thus, SSC starts from a self-representation model where the input data matrix Y is employed as a dictionary: Y = YC and infers the coefficient matrix C ∈ R MN×MN by solving the sparse coding problem (requiring that C is sparse) and ensuring that the trivial solution where each sample would be simply represented by itself is excluded. The non-zero entries in C will then indicate directly which data points lie within the common subspaces. Formally, SSC solves the following optimization problem: where is an all-one vector; diag(C) is a diagonal matrix whose entries outside the main diagonal are zero and λ is a parameter, which controls the balance between the data fidelity and the sparsity of the coefficient matrix. The constraint diag(C) = 0 is introduced to avoid the trivial solution of representing a sample by itself and the second constraint 1 T C = 1 T ensures that each data point is an affine combination of other data points. The problem in (1) can be solved by the ADMM algorithm [31], with the time complexity of O((MN) 2 B + (MN) 3 (I + 1)) where I is the number of iterations. The coefficient matrix C yields directly the dependence structure among the data points: a non-zero entry C ij indicates that the samples y i and y j are in the same class. Thus, it is reasonable to construct the similarity matrix W ∈ R MN×MN as where |C| takes the absolute values of C. The symmetric structure of W ensures that each pair of samples are connected to each other if either side is selected to represent another, which results in a strengthened connection of the graph. The similarity matrix W is then used as an input to spectral clustering [32] to produce the clustering result. Specifically, the Laplacian matrix L ∈ R MN×MN is first formed by where D ∈ R MN×MN is a diagonal matrix with D ii = ∑ j W ij [33]. Then the c eigenvectors {v k } c k=1 of L corresponding to the c smallest non-zero eigenvalues of L are calculated via singular-value decomposition (SVD). Finally, the clustering result is obtained by running k-means clustering on the MN × c matrix
Sketch-SSC-TV Model for HSIs
In this section, we first introduce our new SSC-based model with TV regularization (SSC-TV), to effectively account for the spatial dependencies between the input data points. Next, we incorporate a random sketching technique into this model, leading to our unified Sketch-SSC-TV model for large-scale HSIs. Finally, we develop an efficient optimization algorithm for the resulting model based on ADMM.
The SSC-TV Model
HSIs not only record the spectrum of materials in the spectral domain but also capture the distribution of ground-objects in the spatial domain. As the distribution of ground materials typically shows some continuity, HSIs are composed of various nearly homogeneous regions made of pixels that belong to the same class with a very high probability [34][35][36][37][38][39][40]. For this reason, spectral signatures of pixels within small local regions are typically very similar. Conversely, the pixels belonging to different classes are more likely to occupy different spatial locations and exhibit significantly different spectral characteristics. HSI clustering assigns pixels into distinct groups according to the spectral similarities such that the pixels from the same group are more similar to each other than to those from other groups. Here, each cluster is viewed as a subspace. Thus, by conducting subspace clustering the pixels of HSIs in local homogeneous regions are likely to be grouped together in the same cluster. At the same time, the pixels showing significant spectral differences, which are typically also spatially separated, are assigned to different clusters. This way, subspace clustering results in a meaningful interpretation of the spatial content of HSIs. Thus, idealy, the subspaces in the spectral domain correspond to the cluster structure in the spatial domain. However, due to noise and spectral variability, the actual results of the subspace clustering model differ from the ideal cluster structure, and do not agree perfectly with the spatial content. Such sensitivity to noise and to spectral variability is inherent to all the methods that perform pixel-wise processing and thus also to the SSC model in (1), where sparse coefficients are calculated independently for each pixel. Random variations in the recorded spectral responses affect the solution of the sparse coding problem such that in the resulting sparse representation some data points may be represented by data points from different subspaces. This degrades the construction of the similarity matrix, thereby deteriorating spectral clustering performance. We aim to alleviate this problem by imposing a spatial constraint that makes the model less sensitive to random spectral variations of individual pixels.
To accommodate for the fact that pixels within a local homogeneous region are likely to belong to the same class, we require explicitly that sparse coefficients of nearby pixels likely to be mutually similar, that is, selecting similar sets of pixels in the subspace-sparse representation. Formally, this means that the coefficient matrix C exhibits certain local smoothness. Recall that pixels y i and y j are likely to belong to the same class if C ij = 0. In reality, C ij is rarely exactly 0, but the larger C ij , the more likely it is that y i and y j are from the same class. Ideally, y i as an atom in the dictionary, only contributes to the representation of pixels in the same class. Since neighbouring pixels from a local region of the input image Y usually belong to the same class in the ideal case, all of them are likely to select the same atoms in the subspace representation. Hence, any row of C, c i = [C i1 , C i2 , ..., C iMN ], composed of the coefficients that correspond to an atom y i , will reflect some aspect of the spatial structure of HSI. In other words, the ideal coefficients should reflect the local smoothness and discontinuities that are present in the original HSI, as shown in Figure 1, where each c i is reshaped to a M × N 2-D slice. This motivates us to introduce TV spatial regularization on sparse coefficients, which promotes effectively piece-wise smoothness while preserving sharp transitions among the distinct regions.
Let x ∈ R MN denote a vector of raster scanned pixel values from a grayscale image of size M × N and define the anisotropic TV norm (An alternative isotropic TV norm formulation is where H x and H y are the forward finite-difference operators in the horizontal and vertical directions, respectively, with periodic boundary conditions. For the 2-D matrix Y reshaped from a 3-D HSI Y ∈ R M×N×B in HSI, the corresponding TV norm is formulated as Now we incorporate the spatial constraint into the SSC model. In particular, we impose the TV norm as defined above on the sparse coefficient matrix C, and formulate our SSC-TV model as where λ and λ tv are two penalty parameters corresponding to sparsity and spatial constraint, respectively. Like with the standard SSC, the similarity matrix is obtained from C by applying (2), and fed into the spectral clustering. The TV norm imposed on C promotes the local smoothness of the resulting subspace-sparse representation, which encourages neighbouring pixels to select a common set of pixels from the same class. Since pixels belonging to the same class tend to be spatially clustered as well (within one or multiple local regions), this locally smooth coefficient structure will also lead to an improved agreement of the resulting spectral clustering with the underlying spatial structure.
Figure 1.
A motivation for applying the TV norm in the sparse subspace clustering (SSC) model. In the ideal case, coefficient matrices of pixels in hyperspectral images (HSIs) should be piece-wise smooth in local region and have similar edges to the original HSI, which becomes apparent after reshaping them to a 3-D cube. Observe that each M × N slice in this cube corresponds to one row in the 2-D matrix C and resembles the spatial structures in the original HSI. In order to preserve such smoothness in local regions and edge structure of the coefficient matrix, the TV spatial constraint is employed.
The Sketch-SSC-TV Model for Large-Scale HSIs
The problem at this point is to solve the sparse coefficient matrix C from the cost function in (6). However, as the number of pixels in HSIs, MN, is typically very large, the matrix C ∈ R MN×MN in (6) is huge. The optimization problem of the SSC-TV model actually cannot be efficiently solved in practice due to its prohibitively high computational complexity. The traditional SSC-based methods [11,[20][21][22][23]41,42] also suffer from the same problem. One key obstacle is that they have to calculate and save the inverse of the entire large matrix (Y T Y + µI) ∈ R MN×MN in memory based on the ADMM algorithm, whose time complexity reaches O((MN) 3 ), which is infeasible for large-scale data sets. In addition, for the TV-regularized model in (6), the complexity to solve the subproblem with respect to the TV-norm is O((MN) 2 log(MN)), which further increases the computation burden. Despite the effectiveness of incorporating the TV-norm in the tasks such as HSI unmixing [43,44], superresolution [45] and denoising [46][47][48], the exploitation of TV-regularization in the SSC model is impractical, especially for large-scale HSIs. In the following parts, a sketched SSC (Sketch-SSC) [30] method designed for large-scale data sets will be introduced, and then our Sketch-SSC-TV model is present.
The Sketch-SSC Model
The recently proposed Sketch-SSC method [30], which was explored in the context of computer vision, employs a random projection matrix R ∈ R MN×n to sketch the input data, compressing the self-representation dictionary Y in (1) to a compact one D ∈ R B×n := YR. The objective function of the Sketch-SSC with respect to the sparse coefficient matrix A ∈ R n×MN can be formulated as arg min By using the random sketching matrix R, the number of optimization variables in the sparse matrix is significantly reduced, making the Sketch-SSC model applicable to large-scale data sets. We illustrate this pictorially in Figure 2. After obtaining the sparse matrix A, the similarity matrix W is built via the KNN graph of A for spectral clustering. The random matrix R ∈ R MN×n used here is known as Johnson-Lindenstrauss transform (JLT), which can compress Y to a very small dictionary while preserving major information in Y. The typically used JLTs are matrices with independent and identically distributed (i.i.d.) ±1 entries multiplied by 1/ √ n [49]. It was proved in Reference [30] that with a properly selected sketching matrix R the compressed dictionary D shows an equal expressive capability to Y since it preserves the column space of Y with high probability.
The Sketch-SSC-TV Model
By using the sketching technique in Reference [30], we convert the SSC-TV model in (6) to the following Sketch-SSC-TV model: Compared with (6), the self-representation dictionary Y is replaced with the sketched D, and also the constraint diag(C) = 0 is not necessary any more because I is not the trivial solution of (8). For simplicity, here we also remove the affine subspace constraint 1 T C = 1 T . D serves as the basis to represent the whole data and thus pixels in HSI now lie in the union of subspaces described by D. Similarly to the self-representation method SSC, the coefficients with respect to D should preserve the smoothness of pixel values in local image regions. Since the neighbouring pixels are often in the same class, they ideally select the same or similar set of atoms in D which are constituting together that particular class. As for the computational complexity, the heaviest part in the traditional SSC-based methods for solving the inverse of (Y T Y + µI) ∈ R MN×MN is replaced with the inverse of (D T D + µI) ∈ R n×n in (8), which reduces the complexity from O((MN) 3 ) to O(n 3 ). In addition, for the model in (6) the complexity of the solver to the TV term is also reduced from O((MN) 2 log(MN)) in (6) to O(MNnlog(MN)) in (8). Note that n is much smaller than MN. Our experimental results shown later indicate that when n is larger than 100, there is no obvious performance improvement, and thus n can be empirically set to a value around 100, which can be more than thousand times smaller than MN in large-scale HSIs. Therefore, the computational complexity of the Sketch-SSC-TV model can be significantly reduced.
We solve the resulting model by the ADMM algorithm, as described in the following subsection. After obtaining the sparse coefficient matrix A, we cannot apply it directly in the same way as the traditional SSC-based methods to construct the similarity matrix since the size of A is n × MN and it cannot explicitly indicate the connections between input data points.
Here we use a KNN graph to build the similarity matrix with the sparse matrix A. For each a a a i from the i-th column of A, the first k nearest neighbours in Euclidean distance are located, denoted as N k (a a a i ). Then the similarity matrix W is calculated as , 0 otherwise (9) where w ij is obtained with a Gaussian kernel function: For large-scale HSIs, the construction of the KNN graph may result in a high computation burden. However, various methods [50][51][52] can be used to speed up this procedure. The obtained sparse similarity matrix W serves as an input to the spectral clustering framework to produce the clustering result. The complete procedure of the proposed Sketch-SSC-TV method is summarised in Algorithm 1.
Optimization
In order to solve model (8), three auxiliary variables B, Z ∈ R n×MN and U ∈ R 2MN×n are introduced, and then model (8) becomes arg min where H = [H x ; H y ] is the TV operator in spatial direction of HSIs. Based on the efficient ADMM algorithm, the optimization problem (11) can be solved by minimizing the resulting augmented Lagrangian function as: where Y 1 , Y 2 ∈ R n×MN and Y 3 ∈ R 2MN×n are the Lagrange multipliers, and µ is a weighting parameter.
To this end, the following subproblems can be solved iteratively. In each subproblem, a variable is updated with others being fixed.
Update B
The objective function with respect to B is given by: The solution can be obtained by setting the first-order derivative to zero:
Update A
The objective function with respect to A is given by: By setting the first-order derivative to zero, we can obtain As for each row of A, H is a convolution, the above problem can be efficiently solved by using the fast Fourier transform (FFT) method: where , and F (·) and F −1 (·) denote the FFT and the inverse FFT, respectively.
Update Z
The objective function with respect to Z is given by: By introducing the following soft-thresholding operator: the problem in (18) can be solved by
Update U
The objective function with respect to U is given by: Similarly, U can be updated by
Update Other Parameters
The next step is to update the multipliers Y 1 , Y 2 , Y 3 and µ by The above 5 steps are iteratively updated until the stop criterion is satisfied, that is, where MaxIter is the predefined maximum number of iteration. Algorithm 2 summarizes the whole optimization steps of the Sketch-SSC-TV model. (14). 5: Update A by (17). 6: Update Z by (18). 7: Update U by (22). 8: Update other parameters by (23).
Convergence Analysis
The convergence property of the ADMM algorithm has been theoretically proven when two blocks of variables are alternatively updated [31,53,54]. However, it is difficult to guarantee the convergence of ADMM for the cases with more than two blocks [55]. In our problem (11), there are four variables {B, A, Z, U}. We show a week convergence property of our algorithm by proving that the solution obtained by Algorithm 2 converges to a Karush-Kuhn-Tucker (KKT) point under some mild conditions. We refer to these conditions as "mild", meaning that they are most of the time fulfilled in practice, which is evidenced by the experimental results shown later. This weak convergence property is stated in Theorem 1 given below. We first introduce a lemma from Reference [56] that we will need in proving this theorem.
Lemma 1 ([56]
). Let X be a real Hilbert space endowed with an inner product · , a norm · with its dual norm · dual , and y ∈ ∂ x , where ∂ f (·) is the subgradient of f (·). Then we have y dual = 1 if x = 0, and y dual ≤ 1 if x = 0.
be the sequence that is derived from Algorithm 2. If lim r→∞ µ(Z r+1 − Z r ) = 0 and lim r→∞ µ(U r+1 T − U r T ) = 0, the sequence {Γ r } ∞ r=1 is bounded, and its accumulation point converges to a KKT point.
Proof. We first prove the boundedness of the variable sequences {B r , A r , Z r , U r , Y r 1 , Y r 2 , Y r 3 }. With the definition of L(·) in (12) and the solver to the U-subproblem (22), we obtain where ∂L U (·) is the subgradient of the non-smooth function L(·) with respect to U. Based on the above-stated Lemma 1, we derive that (24), so the sequence {Y r+1 3 } is bounded. In the update of Z, we have where ∇L A denotes the gradient of smooth function L(·) with respect to A. With the updating rules for Y 2 and Y 3 in (23), we reformulate the equation in (26) as follows: When Let r vary from 1 to t and sum both sides of (28), we get Because of the boundedness of Y r 1 , Y r 2 and Y r 3 , we conclude that is also bounded. We can obtain the following equation with (12) Due to the boundedness of L(B r+1 , A r+1 , Z r+1 , U r+1 , Y r 1 , Y r 2 , Y r 3 ), Y r 1 , Y r 2 and Y r 3 , the left side is bounded and thus the right side of (30) is bounded as well, which deduces that each term in the right side of (30) is bounded. Therefore, we conclude that the sequences of {B r }, {A r }, {Z r }, {U r } are bounded. To this end, the proof to the boundedness of the variable sequences {B r , be the sequence that is generated by Algorithm 2. Based on Bolzano-Weierstrass theorem [57], it is known that for a bounded sequence, there exists at least one accumulation point. We denote by Next, we prove that the accumulation point Γ * satisfies the KKT conditions [58], which means {Γ} ∞ r=1 converges to a KKT point. The proof is similar to that in Reference [59,60]. A KKT point of (11) should meet the KKT conditions, including: According to the updating rules in (23), we have: As lim r→∞ (Y r+1 Based on the updating rules in (14), we have: By left multiplying D T D + µI, we obtain that Considering lim r→∞ (B r+1 − B r ) = 0, we drive the following equation: Due to A * = B * , we can infer that D T DB * − D T Y * − Y * 1 = 0, which satisfies the KKT condition (35). Similarly, we have the following equation according to the updating rule (17) Thus, Combining lim r→∞ (A r+1 − A r ) = 0 and the proved conditions (32)-(34), we obtain Y * T 3 H + Y * 1 + Y * 2 = 0, which satisfies the KKT condition (36). To prove the condition (37), we reformulate it as follows: where scalar function Θ λ µ (t) = t + λ µ |t| is applied to Z element-wise. Based on Reference [61], we have the following relation: Now the condition (37) is transformed equivalently to (47). Based on the updating rules in (18), we have As lim r→∞ (Z r+1 − Z r ) = 0 and A * = Z * , we derive that Z * = R λ µ (Z * + Y * 2 µ ), which satisfies the condition (37). The last KKT condition (38) can be proved similarly as (37). We first reformulate it to the following equivalent condition: With the updating rule (22), we have Taking lim r→∞ (U r+1 − U r ) = 0 and HA * T − U * = 0, we conclude that , which proves the condition (38). Overall, the accumulation point Γ * satisfies all the KKT conditions. This completes the proof of Theorem 1.
Theorem 1 assures the theoretical convergence property of our algorithm under mild conditions. In the next Section, we will prove the convergence empirically by conducting experiments on real data sets.
Experimental Settings
We conduct experiments on three widely used bench mark data sets: Indian Pines, Pavia University and Salinas. The results of two classical clustering methods FCM [5] and k-means [7], the random swap clustering (RSC) [62], the original SSC method [11], the SSC-based extensions L2-SSC [22] and JSSC [23], and the state-of-the-art large-scale clustering methods SSSC [28], SSC-OMP [29] and Sketch-SSC [30] are reported and analysed. The clustering methods FCM, k-means, RSC, SSC, SSSC, SSC-OMP and Sketch-SSC yield the results based on the spectral information alone while the L2-SSC, JSSC and Sketch-SSC-TV methods employ both spatial and spectral information.
We conduct four independent experiments using the three data sets. The traditional SSC-based methods [11,22,23] cannot cluster large-scale data sets. For this reason, we first test all the methods on the cropped version of Indian Pines. In order to compare with the methods [28][29][30] designed for large-scale data sets, we also test the performance on the original large-scale HSIs. We refer to the cropped data set as the small HSIs and to the original data sets as the large-scale HSIs.
Two commonly utilized quantitative metrics including overall accuracy (OA) and Kappa coefficient (κ) are employed to evaluate the clustering performance. In addition, we report the running time (t) as well for all the methods. For a dataset Y = [y 1 , y 2 , ..., y N ] ∈ R B×N with N samples, the OA is obtained by ∑ N i=1 δ(map(r i ), l i )/N, where r i and l i are the cluster label obtained by clustering and the true label of y i , respectively, and δ(x, y) equals one if x = y and equals zero otherwise. The map(·) is a pair-wise mapping function that finds the best match between the clustering results and ground truth. We apply here the Hungarian algorithm [63] to derive the best mapping function. For more details about cluster matching, we refer to Reference [64]. The obtained mapping function finds the label for each pixel. Thus, κ can be directly computed from the corresponding confusion matrix [65]. The running time records the whole clustering procedure for each clustering method. The optimal parameters for the traditional SSC-based methods SSC, L2-SSC and JSSC on the small HSI are set according to References [20,22,23]. The parameters of the other analysed methods were tuned to produce the best results in terms of OA to guarantee a fair comparison. The total number of the randomly selected samples in the SSSC method is equal to n for the small HSIs. For simplicity, the number of samples in the large-scale HSIs is set to 10 per class. In order to avoid the biased clustering results caused by randomness, the methods FCM, k-means, SSSC, Sketch-SSC and Sketch-SSC-TV are repeated five times and the averaged performance are reported. In the spectral clustering method, to reduce the computational complexity, we only calculate c eigenvectors of the Laplacian matrix L whose time complexity is O(c(MN) 2 ). For the Sketch-SSC and Sketch-SSC-TV models, the sketching matrices R are shared in each simulation. We set n = 70, σ 2 = ∑ i,j a a a i − a a a j
Pavia University
This data was collected by the Reflective Optics System Imaging Spectrometer (ROSIS) sensor during a flight campaign over Pavia, Northern Italy. The typically used image consists of 610 × 340 pixels, resulting in 207,400 pixels with 103 spectral reflectance bands. The resolution is 1.3 m per pixel and the number of ground-truth classes is 9. The false color image and ground truth are shown in Figure 6a,b.
Salinas
The third image was acquired by the AVIRIS sensor over the Salinas Valley, CA, USA. The geometric resolution is 3.7 m per pixel, and the image size is 512 × 217 × 224. There are 16 ground-truth classes. Twenty bands in 108-112, 154-167 and 224 are removed due to water absorption. The false color image and ground truth are shown in Figure 7a,b.
Experiments on the Small HSI
In this part, the experiments are conducted on the data in Figure 3 that is cropped from the original Indian Pines as indicated by the yellow box in Figure 5a. The specific class names and their corresponding clustering results are shown in Table 1, where the best result is marked in bold and the sub-optimal result is underlined.
The results reveal that our proposed Sketch-SSC-TV model achieves the best performance in terms of clustering accuracy and κ. The optimal parameters of the Sketch-SSC-TV in terms of OA are λ = 10 −3 , λ tv = 10 −2 . Compared with the classical clustering methods FCM and k-means, the SSC-based methods SSC, L2-SSC, JSSC, Sketch-SSC and Sketch-SSC-TV usually yield higher accuracy, showing the superior capability of SSC model in such clustering task. RSC is a k-means based clustering method, which consists of a sequence of centroid swaps and fine-tuning of the exact centroids with k-means. It shows better performance than k-means in Table 1 in terms of OA and κ. The number of iterations in RSC is set by default to 5000 in the source code (http://www.uef.fi/web/machine-learning/software), resulting thereby in longer running time than k-means and FCM. The classification accuracy of k-means for the class "Soybean-notill" is zero, which means that all the pixels belonging to class 3 are wrongly assigned to other classes. Compared with the original SSC model, the extensions L2-SSC and JSSC by incorporating different spatial information obtain the improved performance with the higher accuracy, which indicates the importance of spatial information in HSI clustering. It is very interesting and also surprised to find that the proposed Sketch-SSC-TV method yields higher clustering accuracy than the L2-SSC and JSSC methods which not only use the uncompressed self-representation dictionary but also takes the spatial information into account. Compared with the large-scale clustering methods SSSC, SSC-OMP and Sketch-SSC, our method yields significant accuracy improvement of more than 20%. The SSSC model heavily relies on the initially selected samples. So when the data sets are contaminated by noise or much diverse in each class, the performance may be greatly degraded. Compared with the Sketch-SSC model, our method offers significant improvement as well, which demonstrates the effectiveness of our approach. Figure 4 shows the similarity matrices obtained by different clustering methods. For a better visual comparison, we randomly select 75 samples per class and arrange them in the sequential order by classes. It is known that the ideal similarity matrix should be block-diagonal as only the samples of the same class are connected in the graph [11]. The results in Figure 4 indicate that our method (Figure 4f) preserves such block-diagonal structure best, which is also the main reason why our approach achieves the highest accuracy in the spectral clustering in Table 1. In general, the similarity matrices in Figure 4e,f constructed by KNN are sparser than that by (2) in Figure 4a-c, but surprisingly they achieve comparable or even better spectral clustering performance, demonstrating the efficiency of sparse graph in the spectral clustering. The similarity matrix in Figure 4d is over sparse due to the strict sparsity constraint in the OMP algorithm, leading to the poor clustering performance. It is clearly observed that there are a lot of wrong connections between class 3 and class 4 in Figure 4a-c,e, which consequently results in the low accuracy for class 3 and class 4 as shown in Table 1. While it is less pronounced in Figure 4f, showing much closer block-diagonal structure, which achieves the highest accuracy for class 3 and 4. Such improved graph connectivity mainly benefits from the utilization of TV spatial regularization.
The results in Table 1 show that the SSSC method achieves the shortest computational time. k-means is known to be an efficient clustering algorithm with the complexity of O (IBcMN) where I is the number of iterations. The time complexity of SSSC is O(I 1 Bn 3 + I 2 nc 2 + MNn 2 ), where n is set to 70 in the experiment. k-means took slightly longer running time than SSSC because it needed much more iterations to converge than SSSC on this data set. However, the results on the big data sets such as Pavia University and Salinas shown later indicate that k-means consistently is the fastest algorithm. It is also observed that the computation time t of the SSSC method is increased when the number of initially selected samples becomes larger. Among the clustering methods designed for large-scale data, SSC-OMP takes the longest time. In general, the large-scale clustering methods are much faster than the traditional SSC-based methods, with more than hundred times speed improvement. The reason for the significant speed improvement is that traditional SSC-based methods SSC, L2-SSC and JSSC use the self-representation dictionary Y, which is commonly huge for large-scale data and involves thereby many time-consuming operations of matrix multiplications and inverse calculations on the large dense matrix Y T Y ∈ R MN×MN in the optimization loop, while scalable clustering methods SSSC, Sketch-SSC and Sketch-SSC-TV employ a compressed dictionary, thus enabling a much lower computational complexity. In our method, the column size of the sketched dictionary D is 70 so that the cost of matrix multiplications and inverse calculation on the new matrix D T D ∈ R 70×70 in the optimization algorithm is significantly reduced in comparison with that on the huge matrix Y T Y in the traditional SSC-based methods. That is why our method only uses 6 seconds to obtain clustering result while the traditional SSC-based methods take around 10 minutes. The computation time of our sketching-based method is comparable to that of FCM and k-means, and hence much smaller than the computation time of the traditional SSC-based methods. Compared with other large-scale clustering methods, the computational cost of the Sketch-SSC-TV is similar.
Experiments on the Large-Scale HSIs
In this part, we conduct three more experiments on the entire HSIs. Due to the high computational memory requirement of the traditional SSC-based methods on large-scale HSIs, the SSC, L2-SSC and JSSC methods cannot be run on our computer for the Pavia University and Salinas. We estimate the required memory for only saving the large matrix Y T Y in the three HSIs by MATLAB as in Table 2. The required memory for the Pavia University is 320.5 GB without considering the extra memory cost for the operations including matrix multiplications and inverse calculations, which is unaffordable for normal computational devices. We report the experimental results in Tables 3-5. The clustering maps are shown in Figures 5-7. The optimal parameters of the Sketch-SSC-TV model are λ = 10 −3 , λ tv = 10 −1 for the Indian Pines, λ = 5 × 10 −2 , λ tv = 5 × 10 −1 for the Pavia University and λ = 10 −3 , λ tv = 10 −4 for the Salinas.
The results in Tables 3-5 reveal that our method consistently achieves the highest clustering accuracy in the three HSIs, which confirms its effectiveness. Clustering for the Indian Pines is a very challenging task as some of the spectral signatures from different classes are very close and also parts of the spectrum are highly mixed due to low spatial resolution [20]. As depicted in Table 3 most of the approaches achieve quite low clustering accuracy, while our method yields a much better result with the accuracy of 60.48%. The FCM, k-means and RSC produce similar accuracy in the Indian Pines and Pavia University, but the k-means is more efficient in terms of computation time than FCM and RSC. The traditional SSC-based methods SSC, L2-SSC and JSSC can be run only on the Indian Pines, however, our method is not only capable of running on all the three large-scale HSIs but also improves clustering performance, which mainly benefits from the exploitation of TV-norm spatial constraint and the sketching technique. Also in Table 3 we can see the computation time of the Sketch-SSC-TV method is significantly reduced by at least 600 times compared to the SSC, L2-SSC and JSSC methods, indicating the efficiency of using a compressed dictionary in our method instead of using the large self-representation dictionary. For the clustering methods designed for large-scale data, SSC-OMP uses much longer time than SSSC, Sketch-SSC and our method. The reason is that the sparse coding for each sample is performed in series. The computational time can be reduced by running simulation in parallel. Compared with the SSC clustering map in Figure 5f, the L2-SSC, JSSC and Sketch-SSC-TV methods have less impulse noise in the clustering maps, which is due to the use of spatial information to promote the connectivity between neighbouring pixels, leading to a more robust similarity matrix. The clustering results in Table 4 show that the accuracy of our method on "Self-Blocking-Bricks" is much lower than that of the reference methods. This can mainly be attributed to the over-smoothed clustering results as shown in Figure 6i, where the "Painted Metal Sheets" and the neighbouring "Self-Blocking-Bricks" are merged. However, this can be alleviated by relaxing the spatial constraint with a smaller λ tv . A possible risk is the reduced overall accuracy. The large-scale clustering methods SSSC and SSC-OMP typically yield worse performance in terms of accuracy than the k-means and RSC method in the three large-scale HSIs, which indicates the limitation of their performance in HSI clustering. In Figures 5i, 6f and 7f the SSSC clustering maps are seriously deteriorated by the impulse noise, which is caused by the limited discriminative information in the spectral domain. The SSC-OMP method also suffers from the same problem for the Indian Pines and Pavia University as shown in Figures 5j and 6g. Compared with the Sketch-SSC method, our method achieves significant improvement in terms of accuracy in the three large-scale HSIs, especially in the Indian Pines with the accuracy enhancement of 23.7%. The cost is a slight increase of computational time that comes from the TV-norm regularization. The Sketch-SSC model also suffers from the same impulse noise problem to the SSSC and SSC-OMP approaches in the clustering maps as shown in Figures 5k, 6h and 7h, while in our method such a problem is greatly alleviated as connections between neighbouring pixels are strengthened by the TV-norm constraint.
Analysis of Parameters
In this part, we analyse the effect of the parameters λ, λ tv , n and k on the clustering performance of the Sketch-SSC-TV method in the large-scale HSIs. 4.5.1. Effect of λ and λ tv λ and λ tv in (8) controls the sparsity level and spatial constraint of the sparse matrix, respectively, which are two important parameters in the model. Let λ be varied in the range of {1 × 10 −4 , 5 × 10 −4 , 1 × 10 −3 , 5 × 10 −3 , 1 × 10 −2 , 5 × 10 −2 } and λ tv in the range of {1 × 10 −4 , 5 × 10 −4 , 1 × 10 −3 , 5 × 10 −3 , 1 × 10 −2 , 5 × 10 −2 , 1 × 10 −1 , 5 × 10 −1 }. The clustering results with respect to λ and λ tv are shown in Figure 8 for the three large-scale HSIs. The results indicate that the clustering performance is more stable with respect to λ than λ tv . According to the experimental results, we recommend to set λ = 10 −3 for all the data. For the parameter λ tv , the value may be different for different data sets, but the clustering accuracy is stable and superior over other methods in a wide range, that is, when λ tv ∈ [5 × 10 −3 , 10 −1 ] for the Indian Pines, λ tv ∈ [5 × 10 −3 , 5 × 10 −1 ] for the Pavia University and λ tv ∈ [10 −4 , 5 × 10 −3 ] for the Salinas. The results of Salinas in Figure 8c are quite different with those of the Indian Pines and Pavia University. For the Salinas when the values of λ tv and λ are similar, the results typically show better performance, which means the sparsity constraint and spatial constraint are equally important. In contrast, for the Indian Pines and Pavia University our method achieves better performance when λ tv is larger than λ, indicating the spatial constraint is more important than the sparsity. The reason may lie in different types of HSIs and different levels of data quality. As each crop in the Salinas is planted regularly in block, there are more homogeneous regions and less edge than the Indian Pines and Pavia University, resulting in much smaller value of the TV-norm in Salinas. In addition, due to high data quality of the Salinas spatial information may be less important than that in the other two HSIs. Thus a lager value of λ tv can make the clustering result over smoothing, leading to a lower accuracy. Among the three constraints of Sketch-SSC-TV model, the data fidelity term is most important for the Salinas. Overall, based on the results in Figure 8 our method is robust and stable with respect to λ and λ tv .
Effect of the Parameter n
n is the number of columns of the sketching matrix R, which decides the sketched dictionary size and also the computation efficiency of the proposed method. We vary n in the range of {10, 20, 40, 70, 100, 140} for the three large-scale HSIs. The results are reported in Figure 9, which shows that a larger n typically can result in a better clustering result. The reason is that a larger sketched dictionary can better preserve the original column space of the input data. For the Indian Pines and Salinas, the number of classes is 16. When n = 10, the sketched dictionary cannot well represent the input data space, which results in the drop of accuracy compared to that when n = 20. It is also revealed in Figure 9 that a small value of n (20 for example) is able to obtain satisfying clustering performance in the three HSIs, which coincides with the fact that the data of HSIs actually lies in a low dimensional subspace.
Effect of the Parameter k
We investigate the effect of the number of neighbours k on the clustering performance of our method by varying k in the range of {5, 10,15,20,30, 50} for the three large-scale HSIs. The results shown in Figure 10 reveal that a larger k yields a higher clustering accuracy in general. The accuracy curves with k < 20 rise more significantly than those with k ≥ 20 in the three HSIs. When k ≥ 20, the accuracy becomes much more stable. Based on the results, we set the value of k to 30 in this paper. Figure 11 shows the squared Frobenius norm of the differences in Z and U values in each two subsequent iterations: Z r+1 − Z r 2 F and U r+1 − U r 2 F . We refer to these distances between the values of Z and U in two successive iterations as updating errors. The results show that the updating errors after a sufficient number of iterations tend to a very small value, meaning that the solutions of Z and U become stable eventually. Moreover, on all three datasets, the updating errors decline monotonically after certain iterations. Thus, we have lim r→∞ µ(Z r+1 − Z r ) = 0 and lim r→∞ µ(U r+1 − U r ) = 0, where µ is a constant. This empirically demonstrates that the conditions in Theorem 1 are satisfied for all three analysed datasets, and it is thus reasonably to assume that they will be satisfied in a similar manner for most other HSIs in practice. It can be observed that the updating errors of Z and U in some datasets are zero at the beginning, which is mainly caused by the small values of A r+1 + Y r 2 /µ in (18) and HA (r+1) T + Y r 3 /µ in (22) at the first several iterations, leading to the output of zero matrices in the thresholding operator R (·). After certain number of iterations, their values increase and the output of thresholding operator is no longer zero, resulting in a temporary increase of updating errors, as shown in Figure 11. But finally they tend to a value that is close to zero. The diagrams in Figure 12 show the evolution of the objective function values with respect to the number of iterations. The results reveal that the objective function is monotonically decreasing to a stable level in the three data sets, demonstrating the practical convergence of our optimization algorithm. Especially, the curves in Figure 12a,c drop sharply in the first few iterations and then become saturated. The results coincide with the aforementioned theoretical convergence analysis.
Conclusions
In this paper, the problem of large-scale HSIs clustering based on the SSC model is addressed for the first time, and a novel clustering method, namely Sketch-SSC-TV, is proposed to incorporates a random projection based sketching technique to significantly reduce the number of optimization variables. In addition, a TV-norm constraint on the sparse coefficient matrix promotes the dependencies between neighbouring pixels, which enhances the block-diagonal structure of the similarity matrix, improving thereby the performance of spectral clustering. We derived an efficient solver based on the ADMM algorithm for the resulting model and also we proved its convergence property theoretically. Unlike the traditional SSC-based methods which cannot be applied on large-scale HSIs due to extremely high computational burden, the proposed method is not only applicable on big data sets but also able to achieve a high level of clustering accuracy. The extensive experimental results clearly demonstrate that our method outperforms the state-of-the-art clustering methods.
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2020-03-05T10:17:24.874Z
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2020-02-29T00:00:00.000
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"oa_url": "https://doi.org/10.3390/rs12050775",
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216518275
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pes2o/s2orc
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v3-fos-license
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Control of electron beam energy-spread by beam loading effects in a laser-plasma accelerator
We present experimental results from a laser wakefield electron accelerator driven by 70 TW ultrashort laser pulses in Helium and Helium–Nitrogen gaseous plasmas with two different Nitrogen concentrations, showing distinct electron-beam qualities. In order to get a clear view of the involved phenomenon, two-dimensional particle-in-cell simulations are performed which not only agreed with the experimental results but also provided an investigation on the evolution of accelerating structures. The experimental and simulation results depict that the beam loading effect can strongly modify the longitudinal accelerating electric field of the wake wave, imposing diametrically opposite effects on the final electron-beam qualities, especially the energy-spread, in the Helium–Nitrogen gas mixtures with different Nitrogen concentrations. In the Helium–Nitrogen-mixed plasma with a lower Nitrogen concentration (0.5%), if appropriately controlled, the beam loading effect can be employed to flatten the accelerating electric field for reducing the electron-beam energy spread. In contrast, in the Helium–Nitrogen-mixed plasmas with a higher Nitrogen concentration (5%), the accelerating electric field of the wake is locally reversed by the self-fields of the overloaded electron bunch, and the correspondingly generated negative-slope region of electric field increases the electron-beam energy-spread.
Introduction
Laser wakefield electron acceleration (LWFA), which was first observed in particle-in-cell simulations (PIC) by Tajima and Dawson in 1979 [1], has the potential to be the basis of a non-conventional technology for building ultra-compact, next-generation high-energy accelerators because of its capability of providing accelerating fields more than three orders of magnitude higher than achievable in conventional RF-based particle accelerators [2][3][4][5]. When an ultra-short ultra-intense laser pulse propagates through an optically transparent plasma, the radial pondermotive force of the laser pulse drives a spherical plasma wake by expelling plasma electrons from its path. In this bubble (or blowout) regime [6], the large associated accelerating field goes beyond several hundreds of GV m −1 . If the background plasma electrons are injected [7,8] into the correct phase of the wakefield, they can be accelerated to high energies, generating collimated and quasi-mono-energetic electron beams. To overcome the non-linear evolution of the laser pulse and the challenge to generate stable and reproducible electron beams via the electron self-injection or spontaneous injection into the plasma wave, several injection mechanisms have been tested to control the electron injection such as optical injection [9][10][11], bubble evolution [12], and density-ramp injection [13][14][15]. A simple, yet effective electron injection scheme, which is known as ionization injection and requires a lower laser-intensity threshold as compared to that for triggering the self-injection, was proposed in 2008 and was recently demonstrated [16][17][18][19]. This scheme utilizes the ionization of inner-shell electrons of high-Z gas atoms (like Nitrogen, Oxygen, Argon, Krypton, etc), which are mixed with low-Z gas atoms (such as Helium which was used for the current study), near or at the peak intensity of laser pulse. The leading part of the laser pulse pre-ionizes the background low-Z gas atoms (e.g., Helium) completely along with the outer-shell ionization of high-Z gas atoms (e.g., Nitrogen), to form the plasma wake wave. The inner-shell electrons of high-Z gas atoms slip backward relative to the plasma wake when they are ionized at the peak of the laser pulse intensity. They are then trapped by the wake wave and gain enough energy via longitudinal acceleration. However, an electron beam generated by this scheme typically has a large energy spread due to continuous ionization and trapping of the inner-shell electrons throughout the whole length of the mixed plasma or up to the depletion of the laser pulse. Later, this scheme was revisited by introducing the 'self-truncated ionization injection (STII)' version in 2014 [20][21][22], where the electron injection length could be shortened to a few hundred micrometers, much shorter than the mechanical limits achieved so far. The initiated unmatched laser pulse ( ¹ k w a 2 p 0 0 ) truncates the injection process due to violation of the ionization injection condition (Δψ0.9, where !ψ=!ψ ion −!ψ btm is the potential difference between the electron ionization and trapping positions of the first wake wave [20,22]) because the self-focusing process leads to a very strong evolution of the wakefield and deforms the bubble before the end of the mixed plasma. After that, an additional improvement of the STII scheme [23] was proposed by employing the beam loading effects [24][25][26], and it enabled loading of charges of ∼0.5 nC within a mono-energetic peak [23].
The doping concentration of the high-Z gas in the low-Z gas has a direct influence not only on the injection mechanism but also on the quality of the generated electron beam. Lower concentrations, typically less than 1% of the Nitrogen gas was used for the STII and resulted in electron beams with narrow energy spreads, as shown in [22,27]. Our recent experimental work showed that higher concentrations (5% and 10%) of Nitrogen (or Krypton) gas lead to a dramatic degradation in the energy spectra of the electron beams [28][29][30]. However, the detailed investigation to explain the various physical mechanisms related to the doped concentration of the trace gas has not been explored so far. The current study provides a detailed microscopic perspective on the distinctions of LWFA in the mixed gases of Helium and Nitrogen of different Nitrogen concentrations. It has been observed that, in case of low concentration (0.5%) of the Nitrogen gas, the STII of two K-shell Nitrogen electrons dominates the injection process, and then the appropriately controlled beam loading effect further suppresses the final energy spread of accelerated electron beam; while in case of high concentrations (5%) of the Nitrogen gas, the ionization injection is activated at an early stage and rapidly terminated due to an over-loading of the injected electrons, which locally reverses the accelerating electric field of the wake wave to further increase the final electron-beam energy-spread in the following acceleration. Quantitative analysis via two-dimensional (2D) particlein-cell (PIC) simulations is performed along with the experimental results. An interferogram for the electron density measurement and the corresponding 2D distribution and on-axis profile of the electron density for fully ionized plasma generated by the interaction of intense laser pulse with the mixed gas of 0.5% Nitrogen+99.5% Helium. Figure 1(a) schematically shows the experimental setup for generating electron beams via the LWFA scheme using 70 TW, horizontally (p-) polarized, 800 nm laser pulses with 30 fs pulse duration. Laser pulses were focused on the front edge of a 4 mm long (along the laser propagating direction) slit-shaped supersonic gas jet [31][32][33] via an off-axis parabolic mirror (OAP) having a 200 cm focal length (F number is 20). The measured laser focal spot had a 25 μm radius of 1/e 2 Gaussian intensity distribution. The peak laser intensity I L and corresponding normalized vector potential a 0 were around 6×10 18 W cm −2 and 1.7, respectively. The laser pulses were focused 2 mm above the gas jet to create an underdense plasma which was online probed using a 6 mJ laser pulse, which was split-off the main pulse after the laser compressor, via interferometric techniques [34]. After crossing the plasma perpendicular to the main beam direction, the probe beam generated interferogram via a Fresnel bi-prism which was then imaged by a 16 bit CCD camera. Electron density with a trapezoidal profile of 1 mm linear entrance and exit ramps and 2 mm plateau at (5.4±0.2)×10 18 cm −3 was obtained by utilizing Abel inversion and 2D fast Fourier transformation (FFT) techniques on the interferograms, as shown in figure 1(b). A beryllium (Be) window was used to couple the electron beam from the vacuum chamber into the diagnostic system installed in air. A calibrated integrating current transformer (ICT) [35] was used for monitoring the electron-beam charge. An 8 cm×16 cm dipole magnet having 2 cm gap between the poles and an effective magnetic-field intensity of 1 T was used as an electron beam energy spectrometer. A Gd 2 O 2 S:Tb fluorescent (DRZ) screen monitored by a 16 bit intensified CCD (ICCD) camera was placed at 26.5 cm away from the magnet entrance to obtain the dispersed electron beam with electron energies above 60 MeV. The resolutions of this home-built energy spectrometer were ±2.5% and ±5% at 150 MeV and 300 MeV, respectively. Figure 2 shows a series of energy spectra and the corresponding total charge of the electron beams generated from the LWFA in pure Helium and in mixtures of Helium and Nitrogen gaseous plasmas with different concentrations of 0.5% and 5% Nitrogen, respectively. Except for the concentration difference, all other parameters are kept constant during the experiment. Figure 2(a) shows a multiple of typical electron energy spectra produced by the LWFA in Helium plasma via self-injection into the plasma wave, which exhibits unstable characteristics with respect to energy and charge. In figure 2(a), shots #1, #2, and #5 show quasi-monoenergetic electron beams having broad relative energy spreads (14%-45%), moderate charges (22-87 pC), and an average fullwidth-at-half-maximum (FWHM) angular divergence of 3.7 mrad, whereas shots #3 and #4 shows monoenergetic electron beams having narrower relative energy spreads (9% and 23%, respectively), nominal charges (1 pC and 3 pC, respectively), an average angular divergence of 3.5 mrad, and the particular characteristic of no dark-current background.
Experimental results
On the other hand and for the case of LWFA in 0.5% Nitrogen +99.5% Helium plasma, multiple electron energy spectra are shown in figure 2(b). The energy spectra show that the STII mechanism is the main contributor to the electron injection process in this case , which results in narrow-energyspread electron beams. Both of the STII conditions [22] are fulfilled in this case, namely, the Helium gas is doped with a very small fraction (0.5%) of Nitrogen whose K-shell electrons are fully ionized and subsequently trapped only near the peak intensity of the laser pulse, and the laser-plasma parameters for the current experiment are unmatched, i.e. k p w 0 =6.2> a 2 0 = 2.7. Due to self-focusing of laser pulse and the evolution of wakefield, the injection of electrons is restricted in time, limiting the energy spread. The obtained maximum peak energy is close to 200 MeV and the maximum charge is 500 pC, as shown in shots #7 of figure 2(b). Shot #9 in figure 2(b) also shows a high-quality monoenergetic beam, which has the narrowest relative energy spread of only 3%, a high charge of 329 pC, and a FWHM angular divergence of 3 mrad. It is clear that there is a common characteristic among those five electron energy spectra, i.e. most of the beam charge is concentrated in the monoenergetic peak.
When the Nitrogen concentration increases to 5%, the electron beam quality remarkably decreases, as shown in figure 2(c). Compared with the electron beams generated from the LWFA in the mixed gases of 0.5% Nitrogen+99.5% Helium, the peak energies and relative energy spreads of the accelerated electron beams clearly degrade when mixed gases of 5% Nitrogen+95% Helium are used. The maximum peak energy obtained is around 154 MeV in a two-bunch spectrum as shown in shot #14 of figure 2(c), where the high-energy bunch has a narrow relative energy spread of 4% and the lowenergy bunch has a broad spectrum with another relative energy spread of 23% and a FWHM angular divergence of 3 mrad. Those results show the same trends in terms of energy spectra as our previous works [22, 27−30]. However, what is different from our previous work is that there is a remarkable decrease on the total charges of electron beams measured by the ICT from the mixed gases of 5% Nitrogen+95% Helium as compared with those electron beams from the mixed gas of 0.5% Nitrogen+99.5% Helium. This may be due to the fact that a lot of ionization-injected electrons from Nitrogen atoms could not be accelerated to high energies and they were lost during the propagation to the ICT.
2D-PIC simulations and discussion
To understand and compare the detailed processes of electron beam acceleration in the above three different cases, 2D-PIC simulations were conducted using OSIRIS 4.0 framework with a moving window scheme [36]. In the simulations, all the laser and plasma parameters were kept as close as possible to the experimental parameters. That is, 800 nm p-polarized laser pulses with a power of 70 TW, laser focal spot w 0 of 25 μm, and normalized vector potential a 0 of 1.7, were focused at 1 mm inside 4 mm long gas targets. The gas densities had a trapezoidal profile extended from z=0 to 4 mm (1 mm linear entrance and exit ramps and 2 mm plateau), and the gas densities of plateau were set at 3.2×10 18 cm −3 and 2.75×10 18 cm −3 for Helium and Helium-Nitrogen-mixed gases, respectively. The simulation box, which moves at the speed of light, was 120×160 μm 2 in size and was divided into cells with size of 0.015625×0.25 μm 2 . Each simulation cell contained 14 particles inside. Overall mixed gas densities are the same for the two different Nitrogen concentration cases, i.e. 0.5% and 5% Nitrogen in Helium, except the amount of the doped neutral gas. In the simulations, the ionization of neutral Helium and Nitrogen gases was modeled in based on the Ammosov-Delone-Krainov tunnel ionization model [37]. We specified the maximum number of ionization levels of Helium and Nitrogen to be 2 and 7 respectively, and the ionization rate for each ionization level was defined and given in [38].
In figure 3, the upper-half panels show the longitudinal electric field, the laser fields and the corresponding density distributions of the He 2+ electrons in x-z computational plane for different positions of the acceleration process in the Helium gas at the gas density of 3.2×10 18 cm −3 . In case of Helium gas, we performed multiple simulations using gas densities of 2.75×10 18 , 3×10 18 , 3.1×10 18 , 3.2×10 18 , and 3.3×10 18 cm −3 , respectively. It is found that, for the current laser pulse conditions, 3×10 18 cm −3 is the threshold gas density for triggering self-injection and acceleration of the He 2+ electrons. However, at the gas densities of 3×10 18 cm −3 and 3.1×10 18 cm −3 , self-injection of He 2+ electrons occurs at a very late time (typically, after laser propagation of 3.5 mm), generating electron beams with low energy of <50 MeV. When the gas density was increased to 3.3×10 18 cm −3 , the final electron beam energy reached 250 MeV, which does not agree with our experimental results presented in figure 2(a). The lower-half panels of figure 3 show the corresponding electron distributions in phase space and lineouts of the injected electrons' energy spectra at different stages of LWFA. From figures 3(a) and (b), one can see that there is no injection occurred in the first half length of the Helium gas target; the trailing second and third wake bubbles evolve faster than the first wake bubble, leading to the primary occurrence of self-injection of He 2+ electrons in the second wake bubble [39] after a propagation distance of ∼3 mm. Meanwhile, few electrons inside the second bubble are accelerated to an energy of approximately 100 MeV, as shown in figure 3(c). After 3.5 mm of propagation (see figure 3(d)), more electrons are injected into the second bubble, however, the acceleration of these electrons has been terminated with a maximum energy of ∼100 MeV because of the disappearance of the second bubble; additionally, tiny amounts of electrons are injected into the first wake bubble but the deformation of the first bubble also limits the acceleration of those electrons to a maximum energy of ∼120 MeV. Finally, an electron beam having a continuous energy spectrum up to ∼150 MeV is generated from the LWFA in Helium plasma, as shown in the blue line in figure 3(d), which is in consistence with the experimental result of shot #5 presented in figure 2(a).
In figure 4, the upper-half panels in (a)-(e) show the longitudinal electric fields along with the laser fields and the corresponding density distributions of the background-plasma electrons (the He 2+ electrons and the L-shell N (1-5)+ electrons) and the injected electrons (only the K-shell N 6+,7+ electrons) respectively, in x-z computational plane at different positions of the LWFA acceleration process in the 0.5% Nitrogen+99.5% Helium gas mixture at the gas density of 2.75×10 18 cm −3 . Since such a gas density is lower than the threshold for triggering the He 2+ electrons as mentioned above, the self-injection of He 2+ electrons was not observed over the entire interaction length, as shown in figure 4. The lineouts of the laser intensity profile in terms of a 0 can be also seen in the upper-half panels. The lower-half panels in (a)-(e) show the corresponding electron distributions in phase-space and lineouts of the injected electrons' energy spectra. A very small number of the K-shell N 6+,7+ electrons are ionization-injected into the first bubble of the wake at ∼1.06 mm, which is just at the beginning of the plateau region of the gas density profile, as shown in figure 4(a). It is clear that at this early position of LWFA, figure 4(a), the peak value of a 0 has just reached ≈3 due to self-focusing, which is lower than the value of 3.8 required for self-trapping of electrons into the first bubble of the wake in the blowout regime as demonstrated by previous simulations [40] and an overview of many experiments [41]. Thus, it is also proved that self-injection of electrons into the first bubble of the wake wave cannot occur. Only ionization-induced injection in the first bubble continues for several hundred micrometers and then self-truncates, as shown in figure 4(b). Simultaneously, due to the beam loading effect, these injected electrons partially flatten the longitudinal accelerating electric field [23] near the center of first bubble and is kept nearly constant until the end of the plasma medium, as shown in figures 4(b)-(e). In the tailored longitudinal accelerating electric field, the field near the end of the bubble is obviously higher than that near its center, resulting in a stronger acceleration for the late-trapped low-energy electrons as compared with the acceleration experienced by the early trapped high-energy electrons. Thus, we can see that the energy spread of the accelerated quasimonoenergetic electron beam is reduced gradually in the remaining acceleration process, as shown by the phase-space distributions of the injected electron beam in figures 4(c) and (d). Similar phenomenon of 'wakefield engineering' has been exploited in numerical simulations of electron-beam-driven plasma wakefield accelerator (PWFA) [42]. After a propagation distance of ∼3 mm, the leading quasi-monoenergetic electron bunch with the reduced energy spread enters into the decelerating phase of wake, as shown in figure 4(e). Finally, the energy spectrum of electron beam with a relatively narrow energy spread at acceleration distance of 4 mm is obtained, as shown in figure 4(e) in blue line, which agrees with the experimental results of figure 2(b). In addition, a subsequent ionization injection of a small number of the two K-shell N 6+,7+ electrons in the second bubble is observed at the distance ∼2.44 mm, as shown in figures 4(c)−(e). However, such a second electron bunch could not obtain a significant high energy acceleration, contributing a background noise to the final quasi-monoenergetic energy spectrum of the electron beam, as shown in figure 4(e).
On the other hand, figure 5 shows the evolution of the LWFA in the gas mixture of 5% Nitrogen+95% Helium at the gas density of 2.75×10 18 cm −3 . There is obviously a large number of Nitrogen K-shell electrons trapped in both the first and second bubbles of the wakefield at the beginning of the laser-plasma interaction, as shown in figure 5(a), which differs from the LWFA in 0.5% Nitrogen+99.5% Helium, figure 4. After that, only a small number of these electrons injected into the first bubble are sufficiently accelerated to 100 MeV, whereas majority of these electrons (mainly injected into the second bubble) are accelerated to very lowenergy around < 5 MeV; simultaneously, the ionization injection of electrons in the first bubble also truncates, as shown in figure 5(b). Due to the higher electron charge overloaded in the first bubble, the now pronounced beam loading effect has a significant impact on the longitudinal electric field used for acceleration, generating a negative-slope region in it near the center of bubble. The early-trapped electrons with high energies, which are located in the negative-slope region, further obtain a stronger acceleration than the later-trapped electrons with low energies. Therefore, one can see that the energy spread of accelerated quasi-monoenergetic electron beam in the first bubble further increases gradually in the remaining acceleration process, as shown in the phase-space distributions of the injected electrons in figures 5(c) and (d). Similarly, the two electron bunches injected into the first bubble enter the decelerating phase after a propagation distance of ∼3 mm, as shown in figure 5(e). In the second bubble of the wakefield, the ionization injection of two K-shell N 6+,7+ electrons lasts until the end of the mixed plasma of 5% Nitrogen+95% Helium, as shown in figure 5. Due to the relatively weak accelerating electric field and the mismatching in phase with it, those injected electrons in the second bubble did not receive significant acceleration from the wakefield, contributing to generation of a low-energy tail (<50 MeV) in the spectrum of electron beam. Additionally, according to the phase-space distributions of the injected electrons, one can see that the total number of injected electrons with energies >5 MeV in the simulation in this case of mixed gas of 5% Nitrogen+95% Helium are remarkably lower than those in the simulation of the 0.5% Nitro-gen+99.5% Helium gas case. This result agrees well with the decrease of the experimentally measured beam charge by the ICT (see figure 2) from the LWFA in the gas mixture of 5% Nitrogen+95% Helium as well. Ultimately, an electron beam having a lower energy up to 150 MeV, larger energy spread, and relatively lower charge is generated in this case, as shown in the blue line in figure 5(e), which is consistent with the experimental results in figure 2(c). Figure 6(a) shows the evolution of the laser peak intensity (a 0 ) in the LWFA in Helium gas at the gas density of 3.2 × 10 18 cm −3 and the gas mixtures of 0.5% Nitrogen + 99.5% Helium and 5% Nitrogen + 95% Helium at the gas density of 2.75 × 10 18 cm −3 , respectively. It is seen that the laser peak intensity experiences self-focusing and defocusing and its evolution in the Helium gas case is faster than that in the other cases, due to relatively high gas density. The value of a 0 evolved in the Helium gas reaches 4 after a propagation distance of ∼2.5 mm, meaning that the self-injection could be triggered after this propagation distance at the gas density of 3.2×10 18 cm −3 , as discussed in figure 3. The values of a 0 evolved in the other two cases and reached 4 after a propagation distance of ∼3 mm and then quickly dropped below 4 again. It means that the self-injection (electrons from the Helium) might be triggered at ∼3 mm and then immediately truncated within a short distance, leading to a negligible contribution from the Helium electrons to the total injected charge as compared with the ionization injection which is dominant. Figure 6(a) also shows the wake wave pseudopotential difference (!ψ) between the Nitrogen's K-shell electron ionization position (!ψ ion ) and the end of the first wake wave (!ψ btm ) for the two gas mixture cases, where the dotted blue and red lines represent results from the 0.5% Nitrogen+99.5% Helium and 5% Nitrogen+95% Helium, respectively. One can see that gradually the laser intensity is above the ionization threshold for the Nitrogen's K-shell (which requires E max >1.9) as it is strongly self-focused in both two cases. The ionized K-shell electrons (N 6+,7+ electrons) can be trapped only if they gain enough energy from the wake, which needs y y D D = ( ) [20,22,43], where the normalized transverse momentum p is estimated to be the normalized laser vector at the ionization position, i.e. p=1.9, and the Lorentz factor γ 0 of the wake phase velocity at the gas density of 2.75×10 18 cm −3 is estimated by the linear theory g w w = =18, 0 p as the dotted black line shown in figure 6(a). The ionization-injection process takes place within a limited region in space (from 700 to 1000 μm) for the two mixed gas cases in the first half of the plasma medium. Correspondingly, figure 6(b) shows the total injected electron beam charge as a function of the laser propagation distance from the simulations for the two mixed gas cases. One can notice that the ionization injection of electrons has both occurred after ∼0.7 mm of laser propagation and the injected beam charge saturates almost over the same laser propagation distance, which is around 1.5 mm. However, the mechanism of the truncation of ionization injection is rather distinct for the two cases. For the LWFA in 0.5% Nitro-gen+99.5% Helium, the ionization injection of K-shell Nitrogen electrons is self-truncated at ∼1.5 mm due to the breakdown of ionization-injection condition (!ψ0.9), where about 16 pC μm −1 of charge in 2D slab geometry (or 112 pC if we assume the width of the beam is 7 μm, which is a common beam width) is injected into the wake wave. After that, the growth trend of the injected charge has slowed down significantly and eventually saturated at about 55 pC μm −1 , meaning that there is an additional ionization injection of Kshell Nitrogen electrons in the second bubble of wakefield which contributes to the low-energy tail of the electron energy spectrum, as consistent with the result in figure 4(e). For the LWFA in the 5% Nitrogen+95% Helium, not only the injection rate of electrons is much faster, but also the total injected charge is much higher than those in the other case, and the ionization-injection process has been terminated at the distance of ∼1.5 mm as well, where the total injected charge reaches 83 pC μm −1 . According to the equation of maximum affordable number of electrons in the bubble regime, i.e. l ḿŃ P 2.5 10 m 0.8 TW 100 8], the beam loading should occur when the charge approaches 335 pC (or 48 pC μm −1 of charge in 2D slab geometry if we assume the width of the beam is 7 μm as well). Thereafter, it can be inferred that the ionization injection has been truncated at the distance of ∼1.5 mm due to the beam loading effect in the LWFA of 5% Nitrogen +95% Helium. Similarly, there is also an additional injection during the distance range of around 2.5-3.5 mm because of the ionization injection of Nitrogen electrons in the second bubble of the wakefield as seen in figure 5, and the whole injection process finally terminates at the level of ∼250 pC μm −1 .
If we make a comparison between the present experimental and simulation results and our previous works [22, 27−30], several notable differences are found. Firstly, although the laser power has been nearly doubled in the present experiment, the electron-beam energies have not been remarkably increased or even decreased compared with those obtained by using the mixed gases of 0.3% Nitro-gen+99.7% Helium [27], 0.5% Nitrogen+99.5% Helium [22,28,29], and 0.5% Krypton+99.5% Helium [30] in our earlier work; this is related to a degradation of laser focusing quality in the current experiment. Secondly, PIC simulations in our previous works mainly focused on the control of final electron-beam energy and energy spread by manipulating the injection process of electrons [22,28] and the effect of Nitrogen concentration on the divergence angle (or emittance) of electron beam [28,29]. However, the time evolution of the accelerating structure during the whole LWFA in the Helium-Nitrogen-mixed gases are investigated in a great detail by the PIC simulations in the current work, and it is found that the beam loading effect plays an important role in controlling the final electron-beam energy spread by appropriately manipulating the Nitrogen concentration. Finally, mono-energetic electron beams with very high charge of ∼0.5 nC are generated from the LWFA in the mixed gas of 0.5% Nitrogen+99.5% Helium in the current experiment, which is in good agreement with the previous results [23].
Conclusion
In summary, we present experimental results from a laser wakefield acceleration in Helium gas and mixtures of Helium-Nitrogen gases with low (0.5%) and high (5%) concentrations of Nitrogen, showing very different electronbeam qualities. 2D-PIC simulations are performed to support the experimental results and provide a microscopic view of different mechanisms which influence the injection and acceleration of the electron beams. The STII scheme dominates electron injection of the LWFA and the beam loading effect subsequently suppresses the energy spread of the injected electron bunch in the plasma of 0.5% Nitro-gen+99.5% Helium, resulting in monoenergetic beams with narrow energy spreads. On the contrary, the LWFA in the plasma of 5% Nitrogen+95% Helium, abundant K-shell Nitrogen electrons are ionized and trapped in the wake wave, leading to beam over-loading which terminates the ionizationinjection process and further increases the energy spread of the injected electron bunch by locally reversing the accelerating electric field which leads to generation of low-quality beams in this case. These results can be considered as a reference for the future applications of LWFA-based electron beams or x-ray sources.
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2020-03-05T11:08:14.652Z
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2020-03-23T00:00:00.000
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3896360
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pes2o/s2orc
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v3-fos-license
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Energy dissipation and fluctuations in a driven liquid
Minimal models of active and driven particles have recently been used to elucidate many properties non-equilibrium systems. However, the relation between energy consumption and changes in the structure and transport properties of these non-equilibrium materials remains to be explored. We explore this relation in a minimal model of a driven liquid that settles into a time periodic steady state. Using concepts from stochastic thermodynamics and liquid state theories, we show how the work performed on the system by the various non-conservative, time dependent forces - this quantifies a violation of time reversal symmetry - modifies the structural, transport, and phase transition properties of the driven liquid
INTRODUCTION
Minimal models of active matter have provided an analytically and computationally tractable test bed to study non-equilibrium systems. Phase transitions in some classes of model systems composed of self-propelled particles are beginning to be characterized [1][2][3][4][5]. Recent work has also studied nucleation phenomena [6,7] and obtained expressions for pressure and other mechanical properties of active media [8][9][10][11]. In spite of these advances, understanding the connections between energy consumption and the structural properties of these systems remains a challenging problem [12].
In our work, we explore these connections in a model non-equilibrium liquid driven by time-periodic forces. The rotational dynamics that result from the driving have similarities with a range of systems, including colloids in a periodically changing magnetic or electric field [13][14][15][16], shaken plastic particles and chiral wires [17,18], and chemical and biological microswimmers with active rotational degrees of freedom [19][20][21][22][23][24]. Yet despite their relevance to this wide range of experimental and biological systems, model systems with rotational dynamics have only recently begun to be studied [25][26][27].
The central points of this paper are as follows: First, we describe the class of driven liquids with rotational dynamics considered in this paper and their associated phase diagrams. Second, we show that the density fluctuations in our non-equilibrium system are surprisingly well described by Gaussian statistics [28]. Within this effective description, we are able to derive simple scaling relations for the work performed on the system due to the non-equilibrium driving forces. These analytical predictions are validated by simulation data from the full many-particle systems. Third, we derive a relation that shows how the rate of work done on the model system changes the fluctuations in the conservative forces experienced by the particles. In other words, our relation describes how energy dissipation changes the structural properties of the non-equilibrium material. This relation between work and force fluctuations can be viewed as an instantiation of the Harada-Sasa relation [29,30] that connects work performed in a non-equilibrium system to a breakdown of the fluctuation dissipation relation. We demonstrate this relation numerically for a variety of driven systems to firmly establish its general nature. Finally, using a minimal model we demonstrate how the aforementioned breakdown of the fluctuation dissipation relation can change the diffusion constant of the system. While our driven liquid is substantially more complex than the minimal model, this provides intuition for the interplay between dissipation and transport. Crucially, it provides a model for how the phase transition properties of the driven liquid depend on the magnitude of the driving force. Taken together, our results elucidate how a violation of time reversal symmetry can be used to alter the structural, transport, and phase transition properties of a liquid.
PHASE BEHAVIOR OF A DRIVEN SYSTEM
Our model system is composed of purely repulsive 2dimensional disks whose positions evolve in time according to driven Brownian dynamics: where D 0 is the bare diffusion constant, F c,i is given by the derivative of the Weeks-Chandler-Anderson interaction potential [31], and η i (t) = (η i,x (t), η i,y (t)) are Gaussian-distributed random variables with η i (t) = 0 and η i,µ (t)η j,ν (t ) = 2D 0 δ i,j δ µ,ν δ(t − t ). In all of our simulations and calculations, we set β = 1/k B T = 1. All results reported here are for a number density of ρ = N/L 2 = 0. 45. In addition to the conservative forces, half of the particles are driven by an external force acting on the center of mass of the particle whose direction changes periodically in time according to: For the other half of the particles, F d = 0. Thus, in the zero-temperature limit, a single driven particle will trace a circle in the plane. F d is the same for all driven particles, so that a pure system of driven particles, if we move to a frame of reference that is rotating with F d , will look the same as the equilibrium system. This model was motivated in part by a recent experimental active matter system developed by Luijten, Granick and coworkers [14]. The driving force in Eq. 2 is characterized by a period τ and an amplitude A. The latter is quantified by the Peclet number, a dimensionless measure of the ratio of advective to diffusive velocity in the system. Here we define it as P e = A/γ D0/r0 , where A is the amplitude of the driving force. The bare, single-particle diffusion coefficient (D 0 ), particle radius (r 0 ), and friction coefficient (γ) are all set to 1 in our simulations. The period of the driving force is measured in units of time set by t 0 = r 2 0 /D 0 . We found 3 distinct phases in the region of P e, τ space that we studied here (Fig. 1). We characterized the phases using an order parameter Ω (defined in Methods) that measures the degree of mixing. Briefly, a small value of Ω indicates a mixed system while a large value of Ω indicates a demixed system. At low P e, driven and passive particles remain mixed and the system is homogeneous. As P e is increased, they segregate into regions of purely driven or passive particles. Similar to other strongly damped active systems with rotating dynamics, the interfaces have no particular orientation, and there is a particle current along the interface that decays rapidly in to the bulk [32]. The steady state is timeperiodic with a period τ . As P e is increased further, the system undergoes a transition to a mixed phase characterized by large variations in the local particle number density. Through both transitions, the value of Ω changes smoothly. As τ is increased, phase separation persists over a smaller range of P e. The curve separating the regions a and b in the phase diagram in Fig. 1 (and in phase diagrams generated with other ratios of A x /A y ) are well described by the functional form P e 2 f (τ ) = c, where c is a constant.
We now investigate how the non-equilibrium forces modify the structural and transport properties, in particular the diffusion constant, of this driven liquid. In order to study how these properties change as we approach phase separation, we focus here on the lower mixed region of phase space (Snapshot a, Fig. 1). We then use the relation between diffusion and energy input to explain the dependence of the phase transition between regions a and b in Fig. 1 on P e.
GAUSSIAN DENSITY FLUCTUATIONS IN THE DRIVEN LIQUID
In the context of equilibrium liquids, integral equation theories [33] can be used to demonstrate that, within certain approximations [28,34,35], the coarse-grained field δρ(r, t) = ρ(r, t) −ρ, whereρ is the bulk density of the liquid, can satisfy the following equation of motion: Here, K G is an effective spring constant related to the derivative of a Hamiltonian δH/δ(δρ), γ G is an effective friction, and η G is an effective noise with statistics We provide an expression for K G in the SI (SI Sec. II B ).
Eq. 4 implies that the density fluctuations effectively behave as a particle diffusing in a harmonic potential, and that the probability distribution of δρ values are Gaussian. In many equilibrium liquids ranging from simple hard spheres to water, the statistics of fluctuations in the number of particles inside a small probe volume of the system are Gaussian [28,36]. This property justifies Eq. 4 and has enabled the development of quantitatively accurate theories for the thermodynamics of these liquids [36][37][38].
To determine whether a Gaussian theory can describe the fluctuations out of equilibrium, we measured the statistics of number density fluctuations in the driven liquid. We find that they are indeed Gaussian to a good approximation at points on the phase diagram that are well below the line where the system first phase separates. One example histogram is shown in Fig. 2.
This finding is unexpected for a couple of reasons. First, it is surprising even in equilibrium liquids that density fluctuations in small volumes would be Gaussian. Assuming that the process of particles entering and exiting some small region of the system is Markovian, with a defined entry and exit rate, the naive expectation is that the resulting probability distribution would be a Poisson distribution. It is only in the limit of very large probe volumes that the distribution would tend to be Gaussian, as a result of the central limit theorem. Yet, as noted, Gaussian statistics in small volumes of many equilibrium liquids are well-documented [28,36]. Second, intuition and experience give us little reason to expect that the statistics would remain Gaussian out of equilibrium -for example, 'giant' (i.e. non-Gaussian) number fluctuations have been observed in some active matter systems [39][40][41].
The Gaussian nature of density fluctuations also allows us to predict how the work performed on the nonequilibrium liquid scales with P e and τ . In order to extract this scaling we add an extra driving term F G,d to Eq. 4:δ If F G,d = 0, we recover the equilibrium limit Eq. 4. To mimic the local increase in density as an active particle is driven into surrounding passive particles, we imagine a process where the average density is changed linearly at a rate P e, for a duration τ . This driving protocol corresponds to F G,d = (K G P e)t. We emphasize that we have not derived this driving term from the microscopic equations of motion -rather, we are postulating a minimal protocol based on the physical picture. The average rate of work performed during this Gaussian process can be computed analytically, and is equal to [42]: In Fig. 3, we show thatẇ in the atomistic simulations does indeed scale as predicted in Eq. 6, with fitting parameters γ G and K G . We have checked that the scaling also holds for different driving protocols including cases where the particles are not phase locked or have random phases, and in systems with unequal number fraction of driven and undriven particles (SI Fig. 4). These results strongly suggest that the scaling of the work with P e and τ are consequences of the Gaussian nature of density fluctuations.
DISSIPATION MODIFIES THE STATISTICS OF FORCE FLUCTUATIONS
We now demonstrate how the work done on the system by the non-equilibrium forces affects its microscopic statistics. We begin by noting that according to Floquet theory [43], the non-equilibrium steady state induced by time-periodic driving forces is also time-periodic. For the system to achieve such a time-periodic steady state, the increase in the internal energy of the system due to the total work, w, over each cycle has to be dissipated as heat.
The rate of heat emitted by the driven system (per particle) can be conveniently expressed in terms of the following stochastic integral, where F i and r i denote the conservative force and position vectors for particle i,ṙ i is the rate of change of the position vector of particle i due to forces other than the non-conservative force, the sum is over all the particles in the system, and the integral is performed in the Stratonovich sense [30,44]. The average rate of heat emission q should equal the average rate at which work is performed on the system, ẇ . Before commenting on the effect of dissipative heat and work fluxes in the many body microscopic system, we first apply the definition in Eq. 7 to the simpler Gaussian picture in Eq. 5 above. Specifically, as we show in SI Increasing Pe 2
Increasing Pe
FIG. 3. Rate of work done on the system as a function of P e (top) and τ (bottom) by the driving forces in the full manyparticle simulation, for P e ranging from 2.5-15 and τ ranging from 2.5-30. Lines are fits to Eq. 6. Parameters γG and KG were fit separately to each line; the variation in γG is less than 20% between curves; the variation in KG is less than 5%. Error bars are smaller than the points. Sec. I, the average heat flux of a non-equilibrium system with Gaussian modes can be written as where γ G and F G = −K G δρ are the effective friction co-efficient and conservative force for the Gaussian modes, respectively. The second term in Eq.
The F 2 G term describes the spontaneous fluctuations of the particle in the well, while the F 2 G 0 term describes the relaxation of the system following a random perturbation [29]. In SI Sec. II A, we show that in equilibrium these two terms are equal (a consequence of fluctuation dissipation theorem) and no work is done on the system on average. Out of equilibrium, the work performed is positive and the relation between fluctuations and response breaks down. In this case, the difference between fluctuations and response is predicted exactly by flux of heat, or alternately the rate at which work is performed, as illustrated in Eq. 8. We note that this expression is completely insensitive to the driving protocol; that is, we can use any F G,d we want in Eq. 5.
The forces on the RHS in Eq. 8 are generalized forces that act on the density fluctuations, and not the forces that appear in the individual particle equations of motion. It is reasonable to ask whether a relation similar to Eq. 8 in terms of F, the sum of pair-wise conservative forces acting on a tagged particle in the fluid, could exist. We find that it does. At equilibrium, we show in SI Sec. III that the fluctuations F 2 exactly predict the response F 2 0 (SI Sec. III). Moreover, far from equilibrium, we find in Fig. 4 that the work performed in the many-particle system is indeed related to the breakdown of the equilibrium relation between fluctuations and response, Similar to the interpretation in Eq. 8, F 2 0 captures the response of the non-equilibrium liquid following a random perturbation while F 2 describes the force fluctuations on a tagged particle in its non-equilibrium steady state.
This relation is an instantiation of the Harada-Sasa relation [29,30] and can be obtained from stochastic thermodynamics arguments similar to those used for the Gaussian model. Briefly, the equation of motion for an undriven particle in our liquid is given by Eq. 1 with F d (t) = 0. Multiplying both sides of this equation by F c,i (t), interpreting the resultant equation in the Stratonovich sense [29], and using Eq. 7, we obtain the atomistic equivalent of Eq. 8 connecting the heat flux to statistics of force fluctuations. Since the heat and work fluxes have to balance each other in steady state to ensure a constant average energy, we obtain Eq. 9. We verified Eq. 9 for a wide range of amplitudes and time periods of the driving force in the homogeneous part of the phase diagram. We have also verified that this result holds for driving forces in which the particles are not phase locked, have random phases, and in systems with unequal number fraction of driven and undriven particles (SI Fig. 4). The relation also holds when applied separately to driven and undriven particles in all the cases -in other words, the work performed on average due to the driven particles predicts the change in the force fluctuations of the undriven particles. We now study the implications of this result for the diffusion constant of the non-equilibrium system.
ENHANCED DIFFUSION DUE TO NON-CONSERVATIVE FORCES
Since the diffusion is related as follows to force autocorrelation functions in Brownian dynamics, where d denotes the dimensionality of the system, it is reasonable to ask whether the change in the force correlations that accompanies breakdown of the fluctuation dissipation relation, Eq. 9, affects the diffusion coefficient of the driven liquid. From a physical point of view we may expect the diffusion constant to increase with the driving forces. The driving forces do work on the system by inducing collisions between driven and undriven particles (but not between particles of the same type); these collisions scatter the particles, which can increase the diffusion coefficient.
Indeed, from simulations we find that the diffusion constant increases with the amplitude of the driving force, P e. This increase is well approximated by D − D 0 ∝ P e 2 (Fig. 5). Understanding the basis of the increase of D due to the nonconservative forces is important -in the present context, it can help explain how the phase transition depends on the non-equilibrium forces. We note that non-conservative forces need not necessarily lead to an increase in the diffusion constant: in the self-propelled variety of active matter systems, the diffusion constant eventually decreases as a function of the driving force, leading to self-trapping and phase transitions [45,46].
In order to understand the dependence of D on the non-conservative forces, we consider a minimal model of a tracer particle diffusing in a fluiḋ whereF(r) is spatially dependent force that can be designed to model the forces acting on a tagged particle in the liquid, h is a parameter that tunes the coupling between the fluid and the tracer particle, andη(t) is a Gaussian delta function correlated white noise. In order to ensure no net drift, we constrain F (r)dr = 0. We imagine sampling over many realizations of the forceF(r) from a distribution in order to model the forces exerted by the fluid on a tracer particle. In the liquid considered in the previous sections, the statistics of force fluctuations on a tagged particle satisfy F 2 = F 2 0 when the system is in equilibrium. Equilibrium dynamics in Eq. 11 are achieved wheneverF(r) can be expressed as a gradient of a potential,F(r) = −∇Ũ (r). In such cases, it can be demonstrated that the equivalent relation F 2 = F 2 0 holds, where the averages . . . are taken both over the statistics of the random noiseη(t) and over many realizations of the force. The system can be driven out of equilibrium by ensuring that the force in Eq. 11 has a nonconservative component,F(r) =F c (r) + P eF d (r), wherẽ F c (r) = −∇Ũ (r),F d (r) = ∇ ×Ã(r) and we chooseÃ(r) such that ∇ ·Ã(r) = 0. Like in the previous sections, P e tunes the magnitude of the non-conservative force, F d . In this case, similar to the many-particle driven liquid, a breakdown of the fluctuation dissipation relation is predicted by the total amount of entropy dissipated by the system [30]. Specifically, we use a perturbation theory [47,48] (described in the SI Sec. IV, V, VI) to show that to O(h 2 ), (12) where σ denotes the average rate of entropy dissipation, analogous to ẇ in the many-body liquid. For this minimal model, we obtained expressions for the diffusion constant in Eq. 11 to O(h 2 ): , d is the dimension, and we set Ũ = 0 without loss of generality so that Ũ 2 is simply the variance of energy fluctuations. In instances where the spectrum of force fluctuations is strongly peaked at a particular wave vector q * , the diffusion constant can be simply related to the average entropy dissipation rate: The dynamics of our driven liquid, specified by Eq. 1, are substantially more complicated that the minimal model considered. Nonetheless, the expressions in Eq. 13 and Eq. 14 provide useful insight. By associating the variance Ũ 2 with the sampled variance of energy fluctuations of a tagged particle in the many-body driven liquid, and the entropy production rate σ with the work performed ẇ in the many-body liquid, Eq. 14 demonstrates how the non-conservative forces can modify the diffusion properties of a particle in the fluid. In particular, the minimal model predicts that the diffusion constant can increase as P e 2 in the presence of nonconservative forces and decrease with increasing variance of energy fluctuations. In other words, the diffusion constant need not scale monotonically as P e 2 with driving. Rather, Eq. 13 and Eq. 14 suggest that the increase of the diffusion constant is bounded by terms that scale as P e 2 . The observed scaling from simulation data in Fig. 5 is consistent with this expectation.
Finally, we find that the curve separating the low drive mixed phase from the phase separated region in the phase diagram in Fig. 1 (and in phase diagrams generated with other ratios of A x /A y ) are well described by the functional form P e 2 f (τ ) = c, where c is a constant. This feature is consistent with the relation between diffusion and P e predicted by Eq. 14. Since work is only performed in regions where there is a mixture of driven and undriven particles, Eq. 14 suggests that the diffusion constant can inherit the dependence of the work, w, on P e and the composition f . Note that we are not able to comment on the dependence of D on τ because our minimal model, Eq. 11, doesn't resolve this variable. Rather, the minimal model effectively assumes that τ is much larger than any timescales inherent to the dynamics of the system. A simple linear stability analysis reveals that such a composition dependent diffusion, D(f ), can render the homogeneous phase in region a of Fig. 1 unstable with P e 2 determining the location of the transition. In this manner, our results explain how the renormalization of the diffusion constant due to the non-equilibrium forces can eventually drive the phase separation.
DISCUSSION & CONCLUSIONS
From the rich physics of non-equilibrium materials, and in particular of active particles with rotating dynamics, it is clear that dissipation plays an important role in modifying the structural and dynamical properties of the steady states of these systems. Here, in the context of a class of systems with rotating dynamics, we have identified how the rate of work done by the external forces renormalizes force fluctuations. Using simplified descriptions of density fluctuations, we were also able to model how the work performed in this many body system depends on the non-equilibrium forces. Finally, using a minimal model, we explained the observed enhancement of the diffusion due to the non-equilibrium driving forces and proposed a relation between diffusion and dissipation. The analysis based on the minimal model also helped explain the observed dependence of the phase behavior on the magnitude P e of the driving force. These results demonstrate how the material properties of driven liquids, which are experimentally realizable as colloids, can be tuned simply by violating time reversal symmetry and controlling the amount of energy put in to the system.
where r 0 is the particle radius and is the interaction strength. The time scale is set by t 0 = r 2 0 /D 0 . All results reported are for a driven particle fraction f = 0.5 and number density ρ = N/L 2 = 0.45. LAMMPS [49] was used to integrate the equations of motion. Phase Diagram-To construct the phase diagram, square simulation boxes of size 50r 0 × 50r 0 with periodic boundary conditions were filled with 1125 particles with random initial positions to get ρ = 0.45. We scanned values of P e from 10-160 and τ from 0.05 to 0.3t 0 . Simulations were run with timestep ∆t = 10 −6 t 0 up to t = 1000t 0 . To identify different phases in the system, we defined an order parameter Ω which measures the degree of segregation of driven and passive particles. To compute Ω, we randomly selected a number of small probe areas with radius 1.75r 0 in the steady-state system and computed the normalized difference in the local density of driven and passive particles, and then averaged over many locations and many frames. For a completely mixed system Ω is 0, for a completely separated system Ω is slightly lower than 1 due to the presence of interfaces. We chose a probe volume of radius 1.75r 0 , which is large enough for Ω to take on a range of values, but small enough that inhomogeneities are not all washed out. Since the value of Ω is sensitive to the size of probe volume and the overall density of the system, we defined a phase transition as the location where ∂Ω/∂P e at constant τ is steepest. Density Fluctuations-The number of particles N in a randomly selected probe volume of radius 1.75r 0 was measured in 20 probe volumes per frame in 200 frames of a simulation after the system had reached the steady state. Individual data were binned to construct the histogram. Work-We calculated the work by integratinġ w = F c,i δr i , where the δr i is the displacement of particle i due to the non-conservative forces only in 1 timestep ∆t = 10 −5 t 0 and F c,i is the conservative force. The average is over all particles in the system. Measurements were made for 100τ after the system reached the steady state. Diffusion-Square simulation boxes of size 200r 0 × 200r 0 with PBC were filled with 18000 particles. Trajectories were run for at least 1000τ with a timestep of ∆t = 10 −5 t 0 . The mean-squared-displacement (MSD) of all particles in the simulations was measured after each cycle τ , so that there is no net displacement due to the driving forces. The diffusion coefficient was extracted by fitting the MSD as a function of time according to x 2 (t) = 4Dt.
The University of Chicago, Chicago, IL, 60637
I. RELATION BETWEENq,ẇ AND THE FORCES IN THE DRIVEN SYSTEM
We postulate the following EOM for the driven Gaussian system: In this section all F c , F d , K, γ, D, η shall be understood to be F G,c , F G,d , K G , γ G , D G , η G , the effective conservative force, driving force, spring constant, friction coefficient, diffusion coefficient and noise of the Gaussian density fluctuations, respectively.
We wish to capture the process in which particles are being pushed into each other by the driving forces, thereby changing the average density. This is represented by a time-dependent minimum of the potential given by u(t). We make no assumption here about the form of u(t). To determine the amount of work done on the system, we divide the dynamics in to 2 parts: first, the system is taken from δρ(t) to δρ (t) by the non-conservative forces only, and then it is taken from δρ (t) to δρ(t + ∆t) by the conservative and random forces. In the first step, work is done; no heat is exchanged. In the second step, no work is done; heat is dissipated to the surroundings. If the total energy of the system remains the same, as it does once the system has reached the time-periodic steady state, any work done in the first step must be dissipated as heat in the second step.
The amount of heat dissipated by the system to the bath in the second step is We have 2 options for expanding the stochastic integrand on the RHS: the Stratonovich integral or the Itô integral, and we will use the former. To see why, consider the result of Eq. 2 for an equilibrium system if we expand in the Itô sense: Since terms that are linear in η vanish upon averaging, this gives: Clearly, this cannot be true for an equilibrium system since the net heat flow must be zero. To obtain the correct result it is necessary to interpret the integral in the Stratonovich sense: Since the system is at equilibrium, δρ(t + ∆t) 2 = δρ(t) 2 and the heat flow is 0 as required. Returning to Eq. 2, we now assume that the system is out of equilibrium as a result of the driving force. We have: but now we can no longer assume that δρ(t + ∆t) 2 = δρ (t) 2 . Expanding δρ(t + ∆t) gives: Assuming a vanishing time step, we only keep terms to first order in ∆t. Terms that are linear in η vanish upon averaging. This leaves: By definition, η 2 ∆t 2 = 2D∆t. Since δρ (t) = δρ(t) + u(t)∆t/γ, expanding δρ (t) 2 ∆t gives δρ(t) 2 ∆t + O(∆t 2 ); so, to first order in ∆t we can simply replace δρ (t) with δρ(t) and the form of u(t) is irrelevant. We are left with: Finally, we define (see section II A) In the next section, we show that F 2 0 /γ = − 1 2 lim t→0 η(0)F (t) (see Eq. 18), so that F 2 0 /γ quantifies the response of the system to random perturbations. These considerations give us: and since ẇ = − q :
A. Fluctuations and relaxation in equilibrium
The equation of motion for Gaussian density fluctuations is: In the following, all K, γ, D, F, η shall be understood to be K G , γ G , D G , F G , and η G , the spring constant, friction coefficient, diffusion coefficient, force, and noise of the Gaussian density fluctuations according to Eq. 13, respectively.
The solution of Eq. 13 is: The force-noise correlation is given by: For the Gaussian equation of motion, δρ 2 = 1/(βK), where β = 1/(Dγ). So: The force-force correlation is given by: The diffusion in the many-particle system at equilibrium as a function of particle number density. The bare diffusion coefficient is D0 = 1.
Comparing Eq. 18, 22, and 19 we find that for a system that obeys Eq. 13. In Fig. 1, we show that −2 F(0)F(t) 0 /γ = η(0)F(t) in the many-particle system at equilibrium, where F is the total force on a tagged particle due to the WCA potential, η is the random force on a tagged particle, and γ is the friction coefficient appearing in the individual particle EOM.
B. Expression for the effective spring constants in the Gaussian representation and connection to atomistic simulations
In this section, we provide expressions for the effective spring constant K G using ideas from liquid state theory.
We begin by writing down a Hamiltonian appropriate for Gaussian density fluctuations in a liquid [1]: dr dr δρ(r)χ −1 (r, r )δρ(r) (24) χ −1 is the inverse of the variance, given by: where g(r) is the pair correlation function andρ is the density of the fluid. We approximate the inverse as follows [1,2] To do so we need to compute: The first term is: To find the second term, we first perform a change of variable. The correlation depends only on the magnitude |r − r |. Let r = r − r , then: where in the last line we have renamed r as r.
From the zero frequency limit of the Ornstein-Zernicke equation we have that [3] 1 +ρ dr[g(r) − 1] = 1 1 −ρ drc(r) where g(r) and c(r) are the radial distribution function and direct pair correlation function, respectively. Using this: We can now compute the inverse, χ −1 : Substituting this back in to the Hamiltonian and taking the functional derivative with respect to δρ(r) gives the effective force on δρ(r): Assuming that δρ is not spatially dependent, this looks like a linear restoring force: where K G is the effective spring constant of the Gaussian density fluctuations. Further simplification can be achieved by using the Percus-Yevick closure [1] to write down an approximate expression for c(r), where V (r) denotes the pairwise potential between particles. The term in Eq. 38 proportional to 1/ρ denotes an ideal gas contribution and represents a long wavelength contribution.
Let us first evaluate F 2 0 = − γ 2 lim ∆t→0 η(t) · F(t + ∆t) . Assuming we have fixed a particle with position r 1 at the origin, by Taylor expanding the total force F 1 acting on this particle term by term we get the equation below in the x direction and an analogous equation in the other directions: Taking the dot product with the noise (in 2D to exemplify but easily generalizable to other dimensions) we obtain: Terms of order ∆t 2 or higher in Eq. 41 vanish as ∆t goes to 0. Using the Langevin equationẋ i (t) = 1 γ F i,x (t) + η i,x (t), it follows that: In the above, we have used the fact that dW 2 We also wrote the total potential energy U as a sum of pairwise potentials u(r ij ) and replaced the summation with an integral over the pair correlation function, g(r).
Let us now expand the term F 2 . Again by focusing on a single particle with position r 1 fixed at the origin and doing similar manipulations we obtain: To aid us in evaluating Eq. 46, we assume the system is in equilibrium with a Boltzmann distributed steady state, and use the Yvon-Born-Green hierarchy: [4] g(r)∇u(r) = −K B T ∇g(r) − ρ dr ∇u(r )g 3 (r, r ) Plugging this expression into Eq. 46 cancels the term containing the three-point correlation function, yielding: Integrating by parts and setting boundary terms to zero gives the final expression for F 2 : The expression is equal to that in Eq. 45, completing the proof that F 2 = F 2 0 at equilibrium.
IV. EXPRESSION FOR DIFFUSION FROM PERTURBATION THEORY
The expression for diffusion in terms of force-force and noise-force correlations is: To set up our perturbative approach to computing the quantities in Eq. 50, we start from the overdamped Langevin whereF c (r) = −∇Ũ (r) is the conservative force andF d (r) = ∇ ×Ã(r) the dissipative force.Ũ (r) andÃ(r) obey Ũ (r)dr = 0 and ∇ ·Ã(r) = 0. We will be working in the limit P e , h << 1. The probability distribution for this system obeys the Fokker-Planck equation: When P e = 0, the system is in equilibrium and Eq. 52 is solved simply by the Boltzmann distribution P 0 = e −βhU (r) Z , where Z is the partition function. We construct a perturbation theory in P e by considering the latter term in Eq. 52 as a perturbation to the equilibrium distribution. The first correction to the equilibrium distribution is then given by: where the vectors |u i and v i | are the right and left eigenvectors of W 0 , respectively, and λ i are the eigenvalues. The total probability for our system is then given by: To proceed with evaluating the expression for the diffusion coefficient perturbatively. Defining 1 ≡ h and 2 ≡ P eh, we will evaluate terms to second order in 1,2 . In order for mixed third order terms such as 2 1 2 to be smaller than all quadratic we need h < P e in our setup. We will average the force-force correlations and the noise-force correlations only over the noise in the supplementary information. The averaging over the realizations of the force are implied at the end and shown as angular brackets in the main text. We first consider the expression for Keeping terms to order 2 1,2 we have: The noise-force correlation term is given by: The first term of order 2 1,2 , which we call T 1 , comes from P (r), which we expand in terms of h: The second term quadratic in 1,2 , T 2 , comes from the force present in the exponential in Eq. 60. Ignoring constants, this term can be written as: The term linear in h, let's call it T 3 , can be computed in a similar way, resulting in the following expression: Plugging Eq. 59 and Eq. 65 into the expression 50, and usingF c (q) = −iqŨ (q) andF d (q) = iq ×Ã(q) yields the desired relationship: We can also use this perturbation theory to obtain expressions for the quantities F 2 and F 2 0 : Subtracting Eq. 82 from Eq. 78 yields the relationship for entropy dissipation: In the above we used the fact that dq (2π) dF c (q) ·F d (−q) can be rewritten as drF c (r) · ∇ ×Ã(r) = − drÃ(r) · ∇ ×F c (r) = 0. When P e = 0, entropy production is zero and F 2 = F 2 0 .
We simulated a particle in a 2D periodic force landscape F = (−h cos(x) + hP e cos(y), −h cos(y) − hP ecos(x)). We estimated D eq in such a system by running 5 simulations with P e = 0 and averaging the resulting diffusion constants. Then we varied P e between 0.2 and 0.45, and computed the diffusion constant and the error in each case by averaging over 5 simulations. Next we report how the deviation of the diffusion constant from D eq in our simulations compares to the expected correction due to P e, which is h 2 P e 2 /2. For this specific system, we set F d · F c = 0 thus ensuring that P 1 = 0 (Eq. 53). Hence, although h > P e in these simulations, the correction to the diffusion constant arising from the nonequilibrium forces will scale like P e 2 to leading order.
V. COMPARING OUR DIFFUSION FORMULA TO OTHER ANALYTICAL EXPRESSIONS AT EQUILIBRIUM
At equilibrium, our prediction for the diffusion coefficient is given by: Here we compare our prediction to existing ones in the literature. In 1D, the analytical expression for the diffusion of a tagged particle in a periodic potential is due to [5] and which is in agreement with our results. In 2D the corresponding formula D/D 0 = 1/ e βU is due to [6]. Expanding this expression to order h 2 and recognizing that This result is also in agreement with our prediction.
VI. HIGHER ORDER TERMS IN EXPRESSION FOR DIFFUSION
We now show that higher order terms in h do not diverge. Higher order terms of ∞ 0F (0) ·F(t)dt can be expressed generally as: q i ·F(q 2 ) iq 2 ·F(q 3 ) . . . iq m−1 ·F(q m )Ũ (q m+1 ) . . .Ũ (q n ) |q 2 | 2 (|q 2 | 2 + |q 3 | 2 ) . . . (|q 2 | 2 + |q 3 | 2 + . . . + |q m | 2 ) where n ≥ 3 and n ≥ m ≥ 2. This quantity scales with system size as L (1−n)d+(2−m)+2(m−1) . When m = n, we get a maximum scaling with system size as L (1−n)d+n . For d = 1, higher order terms in n can diverge with system size. For d = 2, the scaling is L 2−n , which can diverge only for terms of order less than or equal to 2. (Therefore, as long as F = 0 holds, then the quadratic terms are well behaved as described in Sec.IV). For d=3, the scaling is L 3−2n , which can not diverge since n ≥ 2.
The higher order terms in ∞ 0 η(0) ·F(t)dt can also be conveniently expressed as: where n ≥ 3 and n − 1 ≥ m ≥ 1. Terms with m = n in this case are zero. This quantity scales with system size as L (1−n)d+(−m)+2m . When m = n, we obtain the maximum scaling with system size but these terms are null so the higher order terms cannot diverge.
VII. SCALING RELATIONS IN OTHER KINDS OF DRIVING
In Fig. 4, we verify the scaling of work, force fluctuations for other kinds of driving forces. This establishes their general nature. and P e 2 (right). Top two rows correspond to in-phase driving like in the main text, but the fraction of active particles is, in this order, 0.9 and 0.1. The bottom three rows correspond to active particles with initial phases drawn from a uniform distribution from 0 to 2π and fractions of active particles 0.9, 0.5 and 0.1, in this order. Error bars are smaller than the points. In the scaling of F 2 − F 2 0 with the work, the τ values sampled were 3, 7, 11, 15 and 19. In the scaling with τ , the KG and γG parameters can vary between the five investigated systems, but for each system the variation in these parameters is small. Maximum variation in KG was ∼ 14% and maximum variation in γG was ∼ 11%.
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2017-08-01T19:44:25.000Z
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2016-11-02T00:00:00.000
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"oa_url": "https://www.pnas.org/content/pnas/115/14/3569.full.pdf",
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207947261
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pes2o/s2orc
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v3-fos-license
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Impact of health education intervention on knowledge and perception of cervical cancer and screening for women in Ghana
Background The burden of cervical cancer continues to rise in developing economies. Women in the sub-Saharan African region have higher chances of developing cervical cancer due to a greater prevalence of related risk factors. The purpose of this study was to determine the effect of health education intervention on cervical cancer and screening perceptions of women in the Komenda, Edina, Eguafo, and Abirem (K.E.E.A) District in the Central Region of Ghana. Methods A non-equivalent control-group design was used to select church women; 396 in the intervention group and 386 in the control group, aged 11 to 70 years in the K.E.E.A District in the Central Region of Ghana. Data was collected via a validated structured interview schedule and analysed using the paired - and independent-samples t-tests, Kruskal-Wallis test, and Mann-Whitney U test. Results A comparison of the mean differences between the pre-post-test scores for the intervention and control groups showed a statistically significant difference for knowledge of cervical cancer (t = 6.22, df = 780, p = 0.001), knowledge of cervical cancer screening (t = 5.96, df = 780, p = 0.001), perceived seriousness (t = 3.36, df = 780, p = 0.001), perceived benefits (t = 9.19, df = 780, p = 0.001), and perceived barriers (t = 3.19, df = 780, p = 0.001). However, perceived susceptibility for the intervention group reduced, evidenced by a decrease in the mean (mean = − 0.12) compared to the control group (mean = 0.93) and this was statistically significant (t = 2.72, df = 780, p = 0.007). Conclusions Health education interventions are critical in improving knowledge and perceptions, and increasing self-efficacy of women about cervical cancer and screening.
Background
Cervical cancer is a disease of concern to women's health worldwide. It is estimated that 8.6 million women above 15 years of age in Ghana are at increased risk of developing cervical cancer [1]. In addition, annually, 3052 women are diagnosed with cancer of the cervix with some 1556 deaths occurring in Ghana [1]. Cancer of the cervix is caused by the Human Papilloma Virus (HPV) which is a sexually transmitted infection (STI) [2]. Infection with the HPV could take about 15 to 20 years before leading to cervical cancer, especially in individuals with normal immune function [2]. The disease can be prevented through vaccination and screening and treatment of precancerous lesions, and Ghana commenced implementation of HPV vaccination in 2013 [3].
Although cervical cancer can be prevented through early screening and treatment of precancerous lesions, cervical cancer screening in Ghana seems to have been restricted to the regional and teaching hospitals as well as some few private health facilities, and most women at the community level lack access to cervical cancer screening services [3]. The low level of awareness and knowledge about the disease and screening are some of the factors impacting cervical cancer screening utilisation [4,5]. A study conducted in Elmina showed that only 6.4% of women had knowledge about cervical cancer and 2.3% had knowledge about Pap smear tests [6].
Therefore, efforts to increase awareness, knowledge and understanding of the perceptions of women about cervical cancer and screening through the provision of an educational intervention will be an important step in promoting the health of women. Health education may enable women to increase their intention to screen. Evidence from a systematic review of studies conducted in developed settings strongly supports the use of health education programmes in increasing cervical cancer screening utilisation [7]. Nonetheless, in Ghana, there seems to be a paucity of data on the impact of health education intervention on cervical cancer screening utilisation. This study is meant to fill a gap in the existing literature in the area of promoting women's health. The study hypothesised that there would be an increase in knowledge about cervical cancer, knowledge about cervical cancer screening, perceived susceptibility, perceived seriousness, perceived benefits, and self-efficacy in the intervention group compared to the control group. This study also hypothesised that perceived barriers about cervical cancer screening would decrease for women in the intervention group compared to the control group.
Research Design
The study was conducted in the Komenda, Edina, Eguafo, Abirem (K.E.E.A.) District in the Central Region of Ghana using a non-equivalent control group design. By using this design, there was a possibility of selection bias which was mitigated by carefully and randomly selecting participants in both the intervention and control groups.
Population and Setting
The population of women was estimated as 22,064 [8]. Women in this study referred to female adolescents and adults. The K.E.E.A District which is located in the southern part of Ghana was selected because most girls indulge in sexual activities before attaining 15 years of age without using a condom, which may put them at risk of HPV and cervical cancer [8,9]. This calls for an urgent need to provide educational intervention about cervical cancer and screening. The indigenes of K.E.E.A are mostly involved in fishing and farming. There are health centres in the district that offer primary health care [10]. However, these health facilities do not provide cervical cancer screening services. The nearest health facilities that offer cervical cancer screening are the Cape Coast Teaching Hospital and a private medical laboratory, Life Sciences Diagnostic Centre, all in the Cape Coast Metropolis which is about 12 km from the capital of the K.E.E.A District, Elmina.
Sample and Sampling
Considering the population of women in the K.E.E.A District (22,064), the study estimated 394 participants using the equation proposed by Glenn [11]. The parameters considered were precision level of plus or minus 5%, confidence level of 95% and a proportion of 50% which reflects the maximum variability in the population [11]. A sample size of 394 was required for this study, as this would be representative of the total population of women in the district. This was increased by 6% to cover any degree of uncertainties such as dropouts. Therefore, the total sample size required was 418 each for intervention and control groups. However, due to attrition and migration, a total sample size of 396 was realized for the intervention group and 386 for the control group.
Although the Ministry of Health's guidelines on noncommunicable diseases stipulate cervical cancer screening for women 25-64 years [12], women aged 11 to 70 years residing in the K.E.E.A District were included in the eligibility criteria for this study. It was assumed that if women as young as 11 years obtain information about cervical cancer and screening, they would be well informed about the risk factors and prevention strategies in reducing their vulnerability.
Out of a total of five major towns in the district -Elmina, Komenda, Eguafo, Abirem and Kissi -two were randomly selected using simple random sampling with replacement technique and these constituted the intervention and control groups. Elmina Township was selected as the intervention group whilst Kissi constituted the control group.
The criteria for selection of churches for inclusion in the study were that the church should have regular congregation and have a branch in Elmina and Kissi. Using this criteria, twelve churches were included in the study. Out of the twelve churches found in both towns, eight were randomly selected to participate in the study using the simple random sampling with replacement technique. It was assumed that selection of the eight churches will provide adequate sample for the study.
The justification for using churches is that Ghanaian women believe in faith for healing and most people rely on supernatural forces for healing, which could affect the rate of detection of diseases and its outcome [13,14]. This strongly suggests that women in churches will be more likely to cooperate and this could also encourage participation in cervical cancer screening [15,16]. The findings of this study may however not be generalised to all women because the characteristics of women involved in this study may differ from those who belong to other religious sects or social gatherings.
The congregation sizes for the churches differed and there was no valid list for all members of the varied churches. Therefore, participants from the selected churches were conveniently sampled based on their consent to participate in the study. In all churches, eligible women interested in participating were enrolled in the study. However, there was a dropout rate of 5.3% in the intervention group and 7.7% in the control group.
Data Collection
A questionnaire was adapted from Ebu et al. [6] and Mupepi et al. [5]. The adaptation was to make it culturally relevant and applicable to the current study based on the hypotheses the study sought to test. The outcome measures of the study were knowledge of cervical cancer, knowledge of cervical cancer screening, perceived susceptibility, perceived seriousness, perceived benefits, perceived barriers, and self-efficacy. Perceived susceptibility was operationally defined as an individual's subjective perception of being at risk of cervical cancer, perceived seriousnesssubjective evaluation of an individual to perceive the seriousness or the possible consequences of cervical cancer, and perceived barriersfactors perceived to be hindering one's ability to engage in cervical cancer screening or overcoming possible factors associated with seeking cervical cancer screening. Perceived benefit was defined as the subjective benefits of engaging in cervical cancer screening to prevent cervical cancer which is influenced by an individual's level of motivation, and self-efficacythe confidence to engage in cervical cancer screening. They were all measured at baseline and 6 weeks. The method of measurement for all the outcome measures was a questionnaire. The instrument had already been validated with the following Cronbach's alphas: knowledge about cervical cancer = .738, knowledge about cervical cancer screening = .704, perceived susceptibility = .824, perceived seriousness = .820, perceived benefits = .798 and perceived barriers = .795. Face and content validity were achieved by showing it to experts in the field of cervical cancer and screening.
The following constructs of the Health Belief Model were used to determine women's knowledge and perception of cervical cancer and screening. Knowledge about cervical cancer consisted of 10 items on the risk factors, signs and symptoms, and prevention. Knowledge about cervical cancer screening consisted of five items. Perceived susceptibility, perceived seriousness, and perceived barriers subscales consisted of eight items each. Perceived benefits subscale consisted of five items. The items on knowledge about cervical cancer and screening were categorical variables with the responses "Yes", "No" and "Don't Know". Perceived susceptibility, perceived seriousness, perceived benefits, and perceived barriers were measured on a four-point Likert scale.
Participants were recruited from churches by making announcements about the project in the respective churches. Six diploma prepared nurses were trained on the use of the standardised educational tool to deliver effective health education on cervical cancer and screening. They also received training on how to administer the questionnaire to both the intervention and the control groups in order to obtain relevant data for the study. This was delivered through face to face interview with the individual respondents. For both the intervention and control groups, an initial pre-test data were collected from eligible participants who volunteered for the study. This was followed by education on cervical cancer and screening in the intervention group.
Intervention
The intervention comprised a comprehensive education on cervical cancer and screening. Women in the selected churches in Elmina constituted the intervention group. Women from the selected churches who consented to participate in the education intervention were grouped into their various churches and assessed before giving health education using a standardised educational tool on cervical cancer and screening. The health education included information on cervical cancer and screening to increase the level of awareness about the disease. The education on cervical cancer focused on the cause, predisposing factors, signs and symptoms, complications and methods of prevention. Regarding cervical cancer screening, participants were introduced to where they could go for testing, persons who carry out the test and the part of the body needed for the test. The benefits of cervical cancer screening and the perceived barriers to screening were addressed. This educational intervention spanned a period of six weeks and included the use of lectures, discussions, videos, and leaflets. The health education was given once every week in the respective churches. Therefore, each participating church had six sessions. Each of the sessions were delivered by a qualified nurse with sufficient knowledge about the disease. On average, each lecture took approximately 1 hour after which participants were given the opportunity to ask questions and any misconceptions clarified. Participants were also given time to reflect and discuss issues concerning cervical cancer and screening amongst themselves. After six weeks of education, the participants were reassessed with the same instrument as used prior to the educational intervention to assess any changes in knowledge and perceptions about the disease and screening. For participants who agreed to participate in the study but due to some reasons were not available at the time of data collection or the intervention, efforts were made to either collect the data or carry out the health education at a later date convenient to them but within the time for the project.
For the control group, initial data were collected from eligible women in the churches constituting the control group who volunteered to participate in the study. Participants from the selected churches were conveniently sampled based on their consent to participate in the study. Data were collected from the same group six weeks after the initial data collection.
Data Analysis
Measurement of knowledge about cervical cancer consisted of 10 items on a dichotomous scale. Each correct answer on the items was assigned a score of one and an incorrect answer attracted a score of zero. The individual scores were computed for pre-and post-tests in both groups and used for the analysis. Knowledge about cervical cancer screening consisted of 5 items. The items were also dichotomous and scored and computed as described for knowledge on cervical cancer. The perception aspect of the questionnaire contained questions on perceptions of susceptibility, seriousness, benefits and barriers. These were scored by participants strongly agreeing, agreeing, disagreeing or strongly disagreeing to each of the statements that constituted the sub-scales. For positive statements on the sub-scale, strongly agree was assigned a score of (4), agree (3), disagree (2) and strongly disagree (1). The reverse score was used for negative statements. The individual scores for perceived susceptibility, seriousness, benefits and barriers were computed for the levels of agreement before and after the intervention in both groups and used for the analysis. Self-efficacy was measured using a single item with a binary outcome of how confident they are in seeking cervical cancer screening.
Data were analysed with the Statistical Package for Social Sciences software version 21.0 (IBM Corporation, Armonk, NY, USA). The paired sample t-test was used to determine knowledge about cervical cancer, knowledge about cervical cancer screening, perceived susceptibility, perceived seriousness, and perceived benefits within the intervention group by comparing the before and after intervention scores. A similar analysis was done for the control group. The independent-sample ttest was used to determine the effect of the intervention by comparing participants scores on knowledge about cervical cancer, knowledge about cervical cancer screening, perceived susceptibility, perceived seriousness, and perceived benefits between the intervention and control groups. Self-efficacy for the various participants before and after the educational intervention were also analysed using the Kruskal-Wallis test with the Post Hoc performed using Mann-Whitney U. Table 1 presents the socio-demographic characteristics of the respondents in both the intervention and control groups. For the intervention group, 21.5% of the women were within the age group 50-59 years. Regarding marital status,44.4% of the women in the intervention group were married, compared 52.6% in the control group. In connection with the educational status of the women, 45.2% of the women in the intervention group had primary education whiles 50.0% of the women in the control group had primary education. Also, 17.7% of women in the intervention group had no formal education. Table 1 further shows that a higher percentage of women in the intervention group, 70.7%, were employed. It is worth mentioning that women in this community are mostly self-employed. They are basically fish mongers, farmers and petty traders. Table 2 presents the results of a paired-samples t-test conducted to compare the effect of cervical cancer and screening education on women's knowledge of cervical cancer, knowledge of cervical cancer screening, perceived susceptibility, perceived seriousness, perceived benefits, and perceived barriers in the intervention group. As can be seen in Table 2, a comparison of the mean for knowledge of cervical cancer before (mean = 3.44) and after (7.12) the intervention shows that there might be some difference. To test whether the difference in mean between the two conditions was statistically significant, a paired samples t-test was conducted. The result of this test revealed that there was a statistically significant difference between the pre and posttest (t = 25.25, df = 395, p = 0.001). Also, comparison of the mean for knowledge of cervical cancer screening, perceived seriousness, perceived benefits and perceived barriers before and after the intervention in Table 2 shows some differences. These were also tested statistically using paired samples t-tests and there was a statistically significant difference between the pre and posttest scores for knowledge of cervical cancer screening (t = -15.62, df = 395, p = 0.001), perceived seriousness (t = 8.93, df = 395, p = 0.001), perceived benefits (t = 8.13, df = 395, p = 0.001), and perceived barriers (t = 3.46, df = 395, p = 0.001). However, there was no statistically significant difference between the pre and posttest scores for perceived susceptibility (t = 0.44, df = 395, p = 0.331). Table 3 presents the results of the paired-samples t-test performed on women's cervical cancer and screening pre and posttest without any intervention. Comparison of the mean for the two conditions for knowledge of cervical cancer, knowledge of cervical cancer screening, perceived susceptibility, perceived seriousness, and perceived benefits shows some differences. The differences were tested statistically, and there was a statistically significant difference between the before and after the test for knowledge of cervical cancer (t = 16.09, df = 385, p = 0.001), knowledge of cervical cancer screening (t = 4.75, df = 385, p = 0.001), perceived susceptibility (t = 3.53, df = 385, p = 0.001), perceived seriousness (t = 3.37, df = 385, p = 0.001), and perceived benefits (t = 4.96, df = 385, p = 0.001). However, there was no statistically significant difference for the two conditions for perceived barrier (t = 0.99, df = 385, p = 0.162).
Independent samples t-test on Pretest Scores on Cervical
Cancer and Screening for the Intervention group (Elmina) and control group (Kissi) Table 4 presents the results of the independent-samples t-test performed on pretest scores for cervical cancer and screening of two independent groups of women in the intervention and control groups. The baseline information of the women about cervical cancer and screening in both groups were assessed. As can be seen in Table 4, comparison of the mean for knowledge of cervical cancer in the two independent groups suggests that women in the intervention group had more information about cervical cancer (mean = 3.44) compared to the control group (mean = 2.53). There was also a difference in the mean for perceived seriousness in the two groups. Women in the intervention group had a higher mean (mean = 23.94) compared to the control group (mean = 22.99). To test whether the difference in mean between the two groups was statistically significant, the independent-samples t-test was performed. The results Table 5 presents the results of the independentsamples t-test performed on posttest scores for cervical cancer and screening of two independent groups of women in the intervention and control groups. Independent-sample t-test between the differences of the pre-post-test of the intervention and control groups Table 6 presents the Independent-sample t-test between the differences of the pre-test-post-test results of the intervention and control groups. The intervention group received education on cervical cancer and screening while the control group did not have any form of education on cervical cancer. A comparison of the mean differences between the pre-post-test of the intervention and control groups suggests a higher mean for knowledge of cervical cancer in the intervention group (mean = 3.67) compared to the control group (mean = 2.38). The mean difference was statistically significant for knowledge of cervical cancer (t = 6.22, df = 780, p = 0.001). Again, the intervention group had higher mean for knowledge of cervical cancer screening (mean = 1.11), perceived seriousness (mean = 2.26), perceived benefits (mean = 1.50) and perceived barriers (mean = 0.96) compared to the control group (0.43, 0.97, -0.97 and -0.26, respectively). These differences were tested using the independent-samples t-test, and findings revealed statistically significant mean difference for knowledge of cervical cancer screening (t = 5.96, df = 780, p = 0.001), perceived seriousness (t = 3.36, df = 780, p = 0.001), perceived benefits (t = 9.19, df = 780, p = 0.001), and perceived barriers (t = 3.19, df = 780, p = 0.001). However, perceived susceptibility for the intervention group reduced, evidenced by a decrease in the mean (mean = -0.12) compared to the control group (mean = 0.93) and this was statistically significant (t = 2.72, df = 780, p = 0.007). Furthermore, the pre-post-test scores for the two groups were assessed on the basis of their self-efficacy. Kruskal-Wallis test performed on the data obtained showed that there was a statistically significant effect of health education on self-efficacy (H = 81.99, df = 3, p = 0.001). Post Hoc comparisons using the Mann-Whitney U test with Bonferronni correction at 0.0167 level of significance showed that women in the intervention group had higher self-efficacy (mean rank = 435.77) compared to the control group (mean rank = 346.08).
Effects of Health Education Intervention on Knowledge of Cervical Cancer and Cervical Cancer Screening
The findings of the study suggest an increase in knowledge about cervical cancer in the intervention group compared to the control group, evidenced by a higher mean for the intervention. A possible explanation of this finding may be that the participants in the intervention group may have gained some knowledge after they had been educated about the disease. This supports the findings of a health education intervention study on cervical cancer conducted in Nigeria, Jamaica and Egypt [17][18][19][20][21].
In Nigeria, the intervention increased the level of knowledge and awareness of cervical cancer and screening [22]. A similar finding was observed in Jamaica as participants had a massive improvement in knowledge about cervical cancer risk factors, symptoms and prevention [20]. Cervical cancer intervention programme for married women in Egypt also reported a significant improvement in knowledge about the disease after the intervention. These studies highlight the important role health education plays in shaping knowledge of health appropriate behaviours. Interestingly, although the control group did not receive any education, there was a marginal increase in knowledge of cervical cancer between the pretest and the posttest scores within this group. This suggests that the pretest might have motivated participants to search for information on cervical cancer. The health education intervention was observed to have impacted knowledge of cervical cancer screening, as the intervention group had higher scores after the intervention compared to the control group. A possible explanation for this observation may be that the participants had comprehensive information about screening during the education sessions. The finding of the present study is consistent with the findings of previous intervention studies [17,20,21]. The similarities in the methodology employed could account for the findings observed. A comparison of the post-test scores for the intervention and control groups strongly suggests that the health education intervention enhanced the knowledge of women about cervical cancer screening. Surprisingly, a critical examination of the pre and post-test scores for knowledge of cervical cancer screening in the control group points to the fact that even without any intervention, there was a slight improvement in knowledge. Since the study was a community-based one, certain factors were difficult to control, so participants might have read or obtained information about cervical cancer screening after the initial assessment.
Effects of Health Education Intervention on Perceptions of Cervical Cancer Screening
The findings further suggest that perceived seriousness about cervical cancer increased for women in the intervention group compared to the control group after the pre-post-test scores for both the intervention and control groups have been compared. This indicates that the health education might have enabled participants to evaluate the complications associated with the disease and how these could impact their health and well-being, as evidenced by a higher mean for the post-test scores for the intervention and the differences between the prepost-test scores for the intervention compared to the control group. An earlier study found health education to have improved perception of the seriousness of cervical cancer [21]. The homogeneity in the methodologies between the current study and that of Ahmed et al. [21] could explain the similarity in findings. Nonetheless, in studies conducted among Turkish and incarcerated women in the United States, perceived susceptibility was low for women in the intervention group [22,23]. Cultural sensitivity and level of literacy among these populations may have accounted for the differences in findings. Again, differences in the health education tool used for the intervention in these studies may have contributed to the observed findings. Furthermore, a critical analysis of the findings suggests that within the intervention group only, the level of perceived susceptibility actually decreased whilst it increased in the control group. A possible explanation could be that health education enabled women in the intervention group to evaluate their level of risk about cervical cancer. It could also be that since they are now equipped with adequate information about the risk factors, they are more prepared to adopt measures that will protect them from getting the disease.
In addition, perceived benefits of cervical cancer screening significantly increased for women in the intervention group compared to the control group after the pre-post-test assessment. This finding indicates that the participants may have understood the benefits of engaging in cervical cancer screening as a result of the health education intervention. Earlier studies affirmed this finding [21,24,25]. A previous study reported that health education changed women's perception of the benefits of preventing cancer of the cervix and modified their beliefs [24]. Similarly, an educational programme conducted for married women in Egypt saw a greater improvement in perceived benefits of cervical cancer screening after the programme [21]. In Nigeria, peer education greatly influenced the perception of the benefits of cervical screening [25]. It is worth mentioning that within the intervention group only, there was an increase in the perception of the benefits of screening after comparing the before and after scores. However, the after scores for the control group significantly decreased. It could be assumed that the education intervention contributed to the difference since the control group did not receive any form of education; the participants in the control group may have provided responses that put them in good light.
The study's findings failed to support the hypothesis that perceived barriers about cervical cancer screening will decrease for women in the intervention group compared to the control group as evidenced by a higher mean for the intervention group after comparing the differences between the pre-post-test scores. Additionally, comparison of the post-test scores for the intervention and control group suggests that the intervention group had more barriers compared to the control group. A possible explanation could be that health education exposed the women in the intervention group to the reality of the problem of cervical cancer by enlightening them on the challenges to seeking cervical cancer screening. Additionally, the intervention took place in a fishing community, and most of the women have low socioeconomic status, as they are mainly into petty trading [10]. The finding of the current study is consistent with that described by Ahmed et al. [21] in which the perception of barriers was found to be high after the implementation of the intervention among married women in Egypt. In contrast, other studies conducted in developed settings have reported fewer barriers post-intervention within the intervention group [22,24]. It is plausible to assume that in developed settings, there may be wellstructured programmes to facilitate cervical cancer screening, so women may not encounter many challenges in an attempt to have a screening test done. However, in resource-constrained settings like Ghana, cervical cancer screening facilities may not be well developed [4].
Furthermore, the findings of the study failed to support the hypothesis that perceived susceptibility to cervical cancer will increase for women in the intervention group compared to the control group. It seems the women were able to evaluate their level of risk as a result of the health education programme and found out to be less at risk. A possible explanation could also be that the participants gave socially desirable responses since they are from religious groups and that may have affected the outcome of the study. Similar findings were reported by Bebis et al. [23] among Turkish women and Ramaswamy et al. [22] among incarcerated women in the United States in which the after-intervention scores demonstrated less susceptibility to cervical cancer. In the present study, perceived susceptibility scores were found to be high for the control group when the posttest scores for the intervention and control groups were compared. Again, the findings suggest that the control group had higher susceptibility perception after comparing pre-post-test scores for both the intervention and control groups. It could be assumed that women in the control group provided responses that put them in good light. It could also be possible that participants in the control group may have searched for information about the disease, which could have influenced the outcome of their responses. Nonetheless, other empirical works strongly suggest that perception of susceptibility significantly increased after intervention in Egypt, Greece, and Nigeria [21,24,25]. Therefore, the effect of health education on perceived susceptibility is unclear in the literature, which requires further evidence.
Additionally, self-efficacy or level of confidence is an important determinant of health behaviour [26,27]. Several studies have examined its impact on cervical cancer screening [27][28][29]. The findings of the present study suggest that women who were educated on cervical cancer and screening had higher self-efficacy. This finding is consistent with the findings of an intervention study conducted in Turkey in which levels of self-efficacy greatly improved in the intervention group compared to the control group [28]. Women with increased selfefficacy may have a higher tendency of engaging in health appropriate behaviours, since they may have been exposed to some information that may have influenced their knowledge status. It is critical to note that direct experience of mastery can greatly increase self-efficacy belief [30]. An earlier study reported a direct relationship between knowledge level [31], health literacy [32], and self-efficacy. Education intervention study among diabetes patients reported increased levels of self-efficacy after the education sessions [33]. Additionally, a randomised controlled trial reported that needs-based client education may significantly improve the level of confidence and health status [34]. This implies that self-efficacy is critical in enabling individuals to successfully perform actions that can potentially improve their health.
Conclusions
The findings of the study suggest that health education, which employed lectures, discussions, videos, and leaflets, may be critical in shaping knowledge of cervical cancer and screening, changing perceptions, and building self-efficacy towards cervical cancer screening in Ghana. In the present study, the intervention group demonstrated high knowledge and positive beliefs towards cervical cancer screening despite the barriers to cervical cancer screening. It was evident that the barriers could hinder eligible women from seeking screening, though they may be well informed and have increased self-efficacy. Measures to reduce the barriers may increase cervical cancer screening uptake among the study population. It is critical to note that education interventions may enable women to evaluate their level of susceptibility and take measures to reduce risks of contracting the disease, as those who received the health education in this study self-reported to be less susceptible to cervical cancer. The findings of the study are important in guiding health education interventions on cervical cancer and screening in Ghana.
|
2019-11-13T01:55:26.335Z
|
2019-11-11T00:00:00.000
|
{
"year": 2019,
"sha1": "57e6a12c11254f30e374b6afe74fbd76b8d3957c",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.1186/s12889-019-7867-x",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
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"Medicine",
"Education"
],
"extfieldsofstudy": [
"Medicine"
]
}
|
246010240
|
pes2o/s2orc
|
v3-fos-license
|
Theoretical description of the motion of a material particle in pan vibrating batchers for agricultural purposes
One of the main tasks of which is to ensure sustainable feed production using highly efficient resource-saving machines and technologies. It is known that it is recommended to feed agricultural animals and poultry in the form of feed mixtures, balanced in composition. Feed grain can be processed into compound feed, developing its compound feed production in places of direct consumption. In the technology of compound feed preparation, one of the most important links is the dosing process, subject to special requirements for the accuracy of the input of components to obtain a homogeneous feed mixture. It is recognized to consider the use of vibration technologies and machines as promising in the field of dosing, allowing to achieve significant results in improving quality indicators. The paper proposes a theoretical description of the motion of a material particle through vibrating batchers, which can be both single-component and multicomponent. A differential equation for the relative motion of a particle along an inclined plane performing longitudinal nonharmonic oscillations is obtained. The article presents theoretical trajectories of changes in the displacement, velocity, and acceleration of a particle for one period of oscillation of the tray by a vibrating batcher at various values of the generalized coefficient K, obtained on a computer from the results of numerical solutions.
Introduction
The national project of Russia in the field of agriculture provides for the intensive development of the livestock sector. One of the main tasks of which is to ensure sustainable feed production using highly efficient resource-saving machines and technologies.
It is known that it is recommended to feed agricultural animals and poultry in the form of feed mixtures, balanced in composition. Feeding grain fodder in the form of turtle is ineffective and economically inexpedient. Complete feed, balanced in basic nutritional elements, microelements, and vitamins are 25-30 % more effective than conventional grain feeds [1].
Feed grain can be processed into compound feed, developing its compound feed production. This allows you to reduce the cost of purchasing raw materials, their transportation, more efficiently use grain fodder, purchased expensive protein and vitamin-mineral components, and continuously provide collective, peasant (farm) farms with their compound feed. Therefore, the production of compound feed in places of direct consumption becomes a condition for the profitable management of the livestock industry. The cost of the prepared compound feed depends on the correct construction of the technological process, the choice of working equipment, its configuration in the line, and the clarity of the work of the constituent mechanisms.
Results
At the Department of Agroengineering of the Omsk State Agrarian University, samples of new feed preparation machines of vibration and shock operating principles have been created [2]. They can be used as separate independent machines and equipment and fit well into the proposed technological scheme of a feed mill for the preparation of bulk feed mixtures (feed). This paper proposes a theoretical description of the movement of a material particle in tray vibrating batchers, which can be both single-component [3][4][5] and multicomponent [6,7]. The peculiarity of the proposed tray vibration batchers is that the working body (tray ), performs longitudinal oscillations according to an inharmonic law, in which the movement of feed particles (ingredients) occurs at a constant average speed, which is the technological basis for obtaining a small error in dosing.
It is fairly noted that "the problem of the motion of a material point on a vibrating rough surface plays no less role in the theory of vibrational displacement than the equations of motion of an oscillator in the theory of linear oscillations" [8]. When determining the law of motion of bulk feed in a tray vibrating meter, the following assumptions were made: • A material particle moves along the load-carrying body (tray) of the vibration dosing device.
• Air resistance has no significant effect on feed movement. The theoretical model is reduced to the analysis of the motion of a material particle lying on a rough surface, which is subjected to kinematic vibration excitation, obeying the law Ф = А(φ): cos sin , at , at . sin The material particle of bulk feed is in equilibrium under the action of the following forces ( Although it is known that the actual dependences of the sliding friction force on the value of the relative velocity observed in practice are of a more complex nature and can differ significantly from the Coulomb approximation [9]. Nevertheless, following the established tradition in solving dynamic problems and considering that the main interest is the averaged parameters of the vibration dosing process, we use the concept of the static friction characteristic in relation to our scheme of a tray (single and multicomponent) vibration batcher, which makes it relatively easy to obtain general and visual results.
From the dynamics of relative motion, it follows that when a material particle of mass m moves along a vibrating plane, a transfer force of inertia acts, which is equal to the product of the mass and the acceleration of the plane (the tray of the vibrating batcher).
The inclined plane performs longitudinal nonharmonic oscillations according to the law described by equation (1). Obviously, in this case, the movement of the feed particle occurs without separation from the surface of the tray of the vibrating meter and coincides with the direction of the oscillations.
Based on the foregoing, we will compose the differential equation of the relative motion of the particle in the projection on the x-axis in general form: where m is the mass of the particle, kg; g is acceleration of gravity, m / s 2 ; N is the force of normal pressure, N; f is coefficient of friction of the particle against the bottom of the vibrating metering tray; α is the angle of inclination of the tray to the horizon, degrees; x & and y & are the current value of the particle velocity along the x and y axes, m/s. In expression (2), considering the above assumptions of the model under consideration, we neglect the gravity component due to its insignificance in comparison with the inertia force (according to our calculations, the value of the gravity component is << 0.05 ΣF).
Since a continuous downhill motion is considered, there is no relative motion of the particle in the vertical direction (y 0). Hence it follows that the relative motion of the particle will be characterized by the x coordinate along the plane of the tray, i.e., you can write: х & >0. Considering that Iin = mA(φ)ω 2 and introducing the designation (х 2 +у 2 ≠0), we can, after reducing the right and left sides by m, write equation (2), like the motion of a particle with viscous friction, in the following form: (4) where φ is the generalized angular coordinate; к is the generalized coefficient of equivalent viscous friction, which takes into account the mechanism of interaction of the particle with the bottom of the tray and the angle of inclination of the tray of the vibrating batcher to the horizon (takes smaller values with increasing particle velocity х & and vice versa); х к & is the dissipative force represented by viscous friction, i.e., the friction force becomes proportional to the particle velocity and depends on the tilt angle of the vibrating metering tray; А(φ)ω 2 is a periodic external action (А(φ) is the amplitude of a variable value at any moment in time according to the system of equations (1).
Expression (4) is a differential equation of forced vibrations of a system with one degree of freedom in the presence of a dissipative force with linear friction, known from the theory of vibrations [10,11] and describing our mechanical system, shown in Figure 1. The resulting differential equation cannot be integrated by quadratures. A numerical method will be used to solve it; the same method that was implemented on a computer using the expansion of functions in a Taylor series, limited by the first three terms: The first term in equation (5) is determined from the kinematics of the chute vibrating batcher, for the initial conditions: Х0=А(φ) cosφ (7) Differentiating expression (7) with respect to time, we find the second term in equation (5): The third term is found from the dynamics of the trough vibrating batcher according to the expression (3): (3) with respect to time, we determine the fourth term in equation (5): is the time derivative of expression (1): Thus, the obtained mathematical model makes it possible to describe the process of moving particles of bulk feed under various operating modes of the trough vibrating batcher. To clarify the physical essence of the vibration displacement process of the obtained mathematical model, a computer program has been compiled.
Based on the results of numerical solutions obtained on a computer, for fixed values of the vibration parameters of the tray (А = 8 mm, ω = 47,1 s -1) graphical dependencies (Fig. 2, 3, 4) are constructed, clearly illustrating the nature of the behavior of movement, speed, and acceleration of a material particle for one period of oscillation of the vibrating batcher tray at different values of the generalized coefficient of equivalent viscous friction k (in what follows we will simply call the generalized coefficient).
The number of curves shown in these figures has been chosen for clarity reasons only. As can be seen from the graphs, regardless of the numerical value of the generalized coefficient, the behavior of the movement, velocity, and acceleration of the particle in one revolution of the cam vibrator has the same functional form. From Figure 2 it follows that at first the particle displacement grows rapidly up to a certain limit, and then monotonically increases. It is also shown here that to obtain large displacements of a particle in a continuous mode, it is necessary to strive for the selection of smaller values of the generalized coefficient.
As can be seen from Figure 4, the acceleration of the particle sharply increases, therefore, it is legitimate to assume that there are large values of the acceleration of the working body. And that is not a desirable outcome from the point of view of the durability and reliability of the operation of the pan vibrating batcher. The nature of the particle velocity change during one period of oscillation of the chute has the form shown in Figure 3, from which the period of unsteady motion in the chute vibrating batcher is a short-term phenomenon. Then it becomes stable with a constant value of the average speed, regardless of the generalized coefficient and vibration parameters (Fig. 3). Therefore, it is necessary to limit the choice of the lower limit of the value of the generalized coefficient.
Conclusion
It should be emphasized that the similar graphs discussed above have the same development tendency for other values of the vibration parameters (A, ω) of the chute vibrating batcher. In other words, for the real process of dosing bulk feeds with the proposed trough vibration dispenser, both single-component and multi-component, there is a range of rational values of the generalized coefficient, which are acceptable both, if possible, to obtain the maximum average speed of particle movement in a continuous mode, and by the values of dynamic loads in the system.
|
2022-01-18T20:08:01.904Z
|
2022-01-01T00:00:00.000
|
{
"year": 2022,
"sha1": "16e25c57b3d65e4dfa89c410cd6d55fb1ff25cf8",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.1088/1755-1315/954/1/012067",
"oa_status": "GOLD",
"pdf_src": "IOP",
"pdf_hash": "16e25c57b3d65e4dfa89c410cd6d55fb1ff25cf8",
"s2fieldsofstudy": [
"Materials Science"
],
"extfieldsofstudy": [
"Physics"
]
}
|
257098947
|
pes2o/s2orc
|
v3-fos-license
|
Glycolysis drives STING signaling to facilitate dendritic cell antitumor function
Activation of STING signaling in DCs promotes antitumor immunity. Aerobic glycolysis is a metabolic hallmark of activated DCs, but how the glycolytic pathway intersects with STING signaling in tumor-infiltrating DCs remains elusive. Here, we show that glycolysis drives STING signaling to facilitate DC-mediated antitumor immune responses. Tumor-infiltrating DCs exhibited elevated glycolysis, and blockade of glycolysis by DC-specific Ldha/Ldhb double deletion resulted in defective antitumor immunity. Mechanistically, glycolysis augmented ATP production to boost STING activation and STING-dependent DC antitumor functions. Moreover, DC-intrinsic STING activation accelerated HIF-1α–mediated glycolysis and established a positive feedback loop. Importantly, glycolysis facilitated STING-dependent DC activity in tissue samples from patients with non–small cell lung cancer. Our results provide mechanistic insight into how the crosstalk of glycolytic metabolism and STING signaling enhances DC antitumor activity and can be harnessed to improve cancer therapies.
Introduction
Tumor-infiltrating DCs present tumor antigens and provide cytokine signals to initiate productive antitumor T cell immunity (1,2). Tumor DNA (Tu-DNA) uptake by intratumoral DCs triggers the activation of the cytoplasmic DNA-sensing cGAS/STING pathway, which is required for type I IFN induction (3)(4)(5). This functional STING-dependent cytosolic DNA sensing is critical for spontaneous and radiation-induced antitumor immunity (6)(7)(8). Intratumoral injection of STING agonists has been shown to be beneficial for antitumor immunity and tumor immunotherapies in preclinical models (5,9,10). However, the intrinsic activation of STING within tumor cells and T cells can promote tumor metastasis and induce T cell death, respectively (11,12). Thus, therapeutic strategies activating DC-intrinsic STING signaling may hold significant potential for cancer immunotherapy.
Metabolic programs are crucial for DC homeostasis and antitumor function (13,14). Glucose metabolism serves as an essential metabolic process that maintains cellular energy and cell mass (15). In the absence of oxygen, aerobic glycolysis converts glucose to lactate via lactate dehydrogenase (LDH). LDH has two common subunits, LDHA and LDHB, which combine to generate 5 isoforms (A 4 B 0 , A 3 B 1 , A 2 B 2 , A 1 B 3 , and A 0 B 4 ) with distinct kinetic properties (16). In the presence of oxygen, glucose is metabolized into pyruvate, which is imported into mitochondria for the tricarboxylic acid cycle and couples with oxidative phosphorylation (OXPHOS) to generate ATP (15). Immature resting DCs rely on OXPHOS to supply their energy demands (17). In contrast, activated DCs quickly induce glycolysis, and this early glycolytic reprogramming provides important metabolic intermediates to sustain the production of DC activation-related molecules (13,18). Aerobic glycolysis is a metabolic hallmark of activated DCs. Glucose restriction is predominant in tumors and causes glycolytic and bioenergetic defects in tumor-infiltrating T cells (19). Notably, aerobic glycolysis promotes IFN-γ expression in CD4 + T cells through an epigenetic mechanism of Ifng transcription (16). Importantly, glycolytic metabolism augments ATP production to drive phosphoinositide 3-kinase signaling, thereby enhancing effector T helper 17 cell and CD8 + T cell immunity (20,21). However, the potential importance of the glycolytic pathway in DC-intrinsic STING signaling and antitumor function in the tumor microenvironment (TME) remains largely unexplored.
Here, we addressed this fundamental question using a combination of metabolomics analysis, conditional gene targeting in mice, and functional cell characterization in mouse tumor models and human cancer tissues. We found that elevated glycolysis augmented glycolytic ATP production in tumor-infiltrating DCs, thus driving STING signaling to facilitate DC-mediated antitumor immune responses. DC-specific deletion of Ldha/Ldhb blunted STING-dependent type I IFN signaling and dampened antitumor immune responses. Blockade of glycolysis decreased ATP production and caused STING signaling defects, and intracellular ATP delivery restored STING signaling in Ldha/Ldhb-knockout DCs. DC-intrinsic STING activation accelerated HIF-1α-mediated glycolysis and established a positive feedback loop. Consistent with this finding, the glycolytic Activation of STING signaling in DCs promotes antitumor immunity. Aerobic glycolysis is a metabolic hallmark of activated DCs, but how the glycolytic pathway intersects with STING signaling in tumor-infiltrating DCs remains elusive. Here, we show that glycolysis drives STING signaling to facilitate DC-mediated antitumor immune responses. Tumor-infiltrating DCs exhibited elevated glycolysis, and blockade of glycolysis by DC-specific Ldha/Ldhb double deletion resulted in defective antitumor immunity. Mechanistically, glycolysis augmented ATP production to boost STING activation and STING-dependent DC antitumor functions. Moreover, DC-intrinsic STING activation accelerated HIF-1α-mediated glycolysis and established a positive feedback loop. Importantly, glycolysis facilitated STING-dependent DC activity in tissue samples from patients with non-small cell lung cancer. Our results provide mechanistic insight into how the crosstalk of glycolytic metabolism and STING signaling enhances DC antitumor activity and can be harnessed to improve cancer therapies.
filtrating CD4 + and CD8 + T cells and decreased frequencies of tumor-infiltrating IFN-γ-producing and granzyme B-producing CD4 + and CD8 + effector T cells (Figure 2, I and J). Furthermore, LDHA/LDHB deficiency also diminished glycolysis and STING activation in tumor-infiltrating DCs and impaired DC antitumor immunity in B16-F10 melanoma model. (Supplemental Figure 3, A-H). We used an animal model of BMDC-based therapy to confirm the importance of LDHA/LDHB in DC antitumor functions. After inoculation of WT mice with MC38 tumor cells, we injected cGAMP-treated WT BMDCs or LDHA/LDHB-deficient BMDCs into the tumor-bearing mice. Compared with the WT BMDCs, the LDHA/LDHB-deficient BMDCs were less potent in suppressing tumor growth and inducing tumor-infiltrating IFN-γ-producing T cells ( Glycolysis drives STING signaling in DCs. To define the importance of glycolysis in STING signaling events, we activated DCs in the presence or absence of the glycolysis inhibitor 2-deoxy-D-glucose (2-DG). After the addition of 2-DG, the induction of type I IFNs was greatly diminished in cGAMP-stimulated DCs (Supplemental Figure 4, A and B). Parallel experiments revealed that inhibition of glycolysis by another glycolysis inhibitor, either sodium oxamate (OXA) or dichloroacetic acid (DCA), also attenuated the induction of type I IFNs in activated DCs (Supplemental Figure 4, C-G). Moreover, cGAMP-induced STING activation, as shown by its phosphorylation at Ser365, was abolished in glycolysis inhibitor-treated DCs (Supplemental Figure 4, H-J). Importantly, upon cGAMP activation, the glycolytic rates were largely decreased in Ldha/b-DKO DCs ( Figure 3A). In addition, the induction of type I IFNs and the phosphorylation of STING were reduced in cGAMP-stimulated Ldha/b-DKO DCs (Figure 3, B-D). Consistent with these results, upon stimulation with Tu-DNA, LDHA/LDHB deficiency reduced the glycolytic rates, type I IFN induction, and STING phosphorylation ( Figure 3, E-H). These data suggest that inhibition of glycolysis in DCs blunts STING-dependent type I IFN responses.
Glycolysis potentiates STING-dependent DC antitumor functions. To validate the contribution of STING to glycolysis-mediated DC antitumor activity, we stimulated WT and STING-deficient BMDCs (STING is encoded by Tmem173) with cGAMP in the presence of 2-DG. The absence of STING abolished cGAMP-induced IFN-β production, and 2-DG treatment did not further suppress IFN-β production in cGAMP-stimulated STING-deficient DCs ( Figure 4A). Similarly, treatment with DCA did not alter IFN-β production in cGAMP-stimulated STING-deficient DCs ( Figure 4B). Using an animal model of BMDC-based therapy, we confirmed the importance of STING activity to the enhancing effect of glycolysis on DC antitumor functions. After inoculating WT mice with MC38 tumor cells, we injected WT BMDCs and STING-deficient BMDCs pretreated with cGAMP in the presence or absence of pathway was involved with STING-dependent antitumor activity of DCs in tissue samples from patients with non-small cell lung cancer (NSCLC). These results delineate a mechanism by which glycolysis promotes STING-mediated DC antitumor immunity and suggest that enhancing DC-intrinsic glycolysis might help improve the efficacy of DC-centric immunotherapies.
Results
STING signaling-activated DCs exhibited enhanced glycolysis. To explore whether glycolytic metabolism involves DC-intrinsic STING signaling, we employed an unbiased, systemic metabolomics approach to examine the glucose metabolic changes during STING agonist cGAMP-induced STING activation in bone marrow-derived DCs (BMDCs) ( Figure 1A). Intriguingly, the levels of glycolytic intermediates, such as pyruvate and lactate, were significantly increased in cGAMP-stimulated DCs (Figure 1, B and C). In addition, the levels of tricarboxylic acid intermediates, such as citric acid, succinic acid, fumaric acid, and malate, were also significantly upregulated in cGAMP-stimulated DCs ( Figure 1, B and C). Indeed, cGAMP-induced DCs had increased baseline and maximum glycolytic rates ( Figure 1D). The OXPHOS rates at baseline and at maximum capacity were also increased in cGAMP-treated DCs ( Figure 1E). Upon stimulation with tumor-derived DNA, which activates the cGAS/STING pathway in DCs, the baseline and maximum glycolytic rates were increased and the maximum OXPHOS rates were decreased (Figure 1, F and G). To investigate whether glucose metabolism is reprogrammed in tumor-infiltrating DCs, we performed RNA-Seq using DCs freshly isolated from the spleens and the tumors of tumor-bearing mice. Notably, intratumoral DCs exhibited enrichment ( Figure 1H). Consistent with this finding, increased glycolytic activity was observed in tumor-infiltrating DCs ( Figure 1I). In contrast, the OXPHOS rates were reduced in tumor-infiltrating DCs ( Figure 1J). These results suggest that STING signaling-activated DCs exhibited enhanced glycolysis.
Blockade Figure 4G). Parallel studies revealed that DCA pretreatment also impaired WT DC-mediated antitumor immunity, and antitumor activities were comparable in mice injected with STING-deficient DCs with or without DCA pretreatment (Figure 4, H-J), suggesting that the defect in STING activity contributes to glycolysis inhibitor-induced impairment of DC antitumor functions. Therefore, glycolysis potentiates STING-dependent DC antitumor functions.
Glycolysis promotes STING signaling via glycolytic ATP production. Given that ATP production is tightly associated with glycolytic metabolism (21), we tested the cellular levels of ATP in activated DCs. Upon stimulation with cGAMP or tumor-derived DNA, the ATP production capacity in activated DCs was enhanced ( Figure 5, A and B). In addition, tumor-infiltrating DCs were associated with elevated cellular ATP levels ( Figure 5C). Treatment with 2-DG or DCA reduced ATP levels in activated DCs ( Figure 5, D and E).
2-DG into the tumor-bearing mice. Compared with the untreated WT DCs, the untreated STING-deficient DCs were less potent in suppressing tumor growth ( Figure 4C). Pretreatment of WT DCs or STING-deficient DCs with 2-DG did not alter the numbers of cGAMP-stimulated DCs in the draining lymph nodes ( Figure 4D). Notably, pretreatment of WT DCs with 2-DG reduced the numbers of tumor-infiltrating cGAMP-stimulated DCs, CD4 + T cells, and CD8 + T cells and decreased the frequencies of tumor-infiltrating IFN-γ-producing and granzyme B-producing CD4 + and CD8 + T cells (Figure 4, D-F). In contrast, mice injected with STING-deficient DCs pretreated with or without 2-DG displayed no apparent differences in the numbers of tumor-infiltrating cGAMP-stimulated DCs, CD4 + T cells, and CD8 + T cells or the frequencies of tumor-infiltrating IFN-γ-producing and granzyme B-producing CD4 + and CD8 + T cells (Figure 4, D-F). Pretreatment of WT DCs or STING-deficient DCs with 2-DG did not alter the frequencies Ldha/b-DKO DCs was greatly reduced under treatment with both ATP and SLO treatment ( Figure 5I). In contrast, treatment with SLO alone did not activate STING signaling or promote glycolysis ( Figure 5, G-I, and Supplemental Figure 5, C and D). These data suggest that glycolysis promotes STING signaling via glycolytic ATP production.
Glycolysis facilitates STING signaling in DCs from patients with NSCLC. Given the potential importance of glycolysis in DC antitumor function, we examined the relationship between LDHA expression and the STING-dependent type I IFN signature. Interestingly, LDHA expression positively correlated with STING signaling, IFNA expression, and IFNB expression ( Figure 6, A-C). Indeed, the levels of glycolytic rates in DCs from NSCLC tissue were higher than those in DCs from paracancerous tissue ( Figure 6D). Consistent with these results, the levels of ATP and STING phosphorylation (at S366 in human STING) were increased in DCs from NSCLC tissue compared with DCs from paracancerous tissue Consistent with these observations, activated Ldha/b-DKO DCs exhibited a weaker ability to produce ATP ( Figure 5F). Because treatment with ATP alone did not activate STING signaling or promote glycolysis (Supplemental Figure 5, A and B), we used streptolysin-O (SLO) to deliver exogenous ATP to DCs to investigate whether reduced ATP production accounts for the decrease in STING signaling. In the presence of ATP and SLO, ATP levels in activated DCs were considerably increased ( Figure 5, D and E). Notably, glycolysis inhibitor treatment did not alter ATP levels in activated DCs under treatment with both ATP and SLO ( Figure 5, D and E). Similarly, treatment with ATP and SLO increased ATP levels in activated Ldha/b-DKO DCs and abolished the difference in ATP levels between WT DCs and Ldha/b-DKO DCs ( Figure 5F). Indeed, SLO-assisted intracellular delivery of ATP restored STING signaling activation in 2-DG-or DCA-treated activated DCs (Figure 5, G and H). Consistent with these results, the difference in cGAMP-induced STING phosphorylation between WT DCs and olism ( Figure 7B). Importantly, cGAMP stimulation upregulated the expression of HIF-1α-related genes and increased the protein level of HIF-1α (Figure 7, B and C, and Supplemental Figure 7A), which was responsible for the induction of glycolytic enzymes. In contrast, cGAMP stimulation did not alter the HIF-1α protein level in STING-deficient DCs (Supplemental Figure 7B), suggesting that HIF-1α-mediated induction of glycolytic enzymes relies on STING signaling. Notably, both PKM2 and HIF-1α accumulated in cGAMP-stimulated WT DCs (Figure 7, A and C). In addition, HIF-1α, HK2, and PKM2 were greatly accumulated in tumor-infiltrating DCs ( Figure 7D). Because PKM2 is a critical modulator that regulates HIF-1α stability and activity (22), we examined the interplay between PKM2 and HIF-1α using the small-molecule drug TEPP-46, which can block the interaction between HIF-1α and PKM2 (22). As expected, TEPP-46 treatment inhibited the association of PKM2 with HIF-1α and attenuated the induction of HIF-1α and PKM2 ( Figure 7C). Furthermore, treatment with TEPP-46 diminished glycolysis, STING phosphorylation, and type I IFN induction in cGAMP-stimulated WT DCs (Figure 7, E-G). Consistent with these results, the absence of HIF-1α abolished PKM2 accumulation, STING phosphorylation, type I IFN induction, and glycolysis in cGAMP-stimulated DCs (Supplemental Figure 7, C-F). HIF-1α-knockdown DCs were less potent in suppressing tumor growth and inducing tumor-infiltrating IFN-γ-producing T cells (Supplemental Figure 7, G and H). Importantly, TEPP-46 treatment attenuated the expression of HIF-1α, HK2, and PKM2, an diminished glycolysis, STING phosphorylation, and type I IFN induction in tumor-infiltrating WT DCs (Figure 7, H-J). However, ( Figure 6, E and F). We next treated freshly isolated NSCLC DCs with 2-DG to confirm the involvement of glycolysis in STING-dependent type I IFN signaling in NSCLC tissue. As expected, STING phosphorylation and type I IFN induction were reduced in NSCLC DCs after 2-DG treatment ( Figure 6, G and H). Importantly, SLO-assisted intracellular delivery of ATP restored STING phosphorylation in 2-DG-treated NSCLC DCs ( Figure 6I). Collectively, these results indicate that the elevated glycolysis in tumor-infiltrating DCs facilitates STING signaling in human NSCLC.
STING signaling promotes glycolysis and establishes a positive feedback loop. We found that key glycolytic enzymes, such as hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2), were greatly accumulated in cGAMP-stimulated WT DCs ( Figure 7A), an effect that was responsible for the extensive enhancement of glycolysis in activated WT DCs. However, STING-deficient DCs stimulated with or without cGAMP exhibited no apparent difference in HK2 and PKM2 protein levels (Supplemental Figure 6A). Consistent with this finding, cGAMP stimulation did not increase the glycolytic rates or ATP levels in STING-deficient DCs (Supplemental Figure 6, B and C). Furthermore, stimulation with tumor-derived DNA did not change the glycolytic rates or ATP levels in STING-deficient DCs (Supplemental Figure 6, D and E). These results suggest that STING signaling controls glycolysis-related gene expression.
To determine the mechanistic link between glycolysis and STING signaling, we performed RNA-Seq using WT BMDCs stimulated with cGAMP. Functional pathway enrichment analysis revealed that cGAMP stimulation significantly affected the pathways involving cytosolic DNA sensing and central carbon metab- TEPP-46 treatment did not alter the expression of HIF-1α and PKM2, the level of glycolytic activity, or the induction of type I IFN in tumor-infiltrating STING-deficient DCs (Figure 7, H-J). Notably, TEPP-46 treatment inhibited the induction of PKM2 and HIF-1α and resulted in defective activation of STING in NSCLC DCs (Figure 7, K and L). These findings suggest that DC-intrinsic STING activation accelerates HIF-1α-mediated glycolysis and establishes a positive feedback loop.
Discussion
Our results demonstrate that glycolysis drives STING signaling to promote DC-mediated antitumor immune responses. The contribution of glycolysis to STING-dependent DC antitumor immunity was best discerned using Ldha/Ldhb DC-conditional knockout mice. LDHA/ LDHB deficiency inhibited glycolytic metabolism and ATP production, thus diminishing the STING-dependent type I IFN response and limiting DC antitumor functions. Consequently, SLO-assisted intracellular delivery of ATP restored STING signaling activation in LDHA/LDHB-deficient DCs. Notably, STING signaling controlled HIF-1α-mediated expression of glycolysis-related genes and established a positive feedback loop. Importantly, glycolysis facilitated STING signaling in DCs from human NSCLC tissues. Our work showed that the glycolytic pathway is essential for the STING-dependent antitumor activity of DCs and thus defines a critical metabolic mechanism of DC-intrinsic STING signaling regulation.
Aerobic glycolysis is a metabolic hallmark of activated DCs, but the connection between glycolytic metabolism and STING signaling remains elusive. Using systematic approaches in con- A, B, D-G, and J) and 2-way ANOVA (C and H); *P < 0.05; **P < 0.01. junction with metabolomics and transcriptomic analysis, we demonstrated that elevated glycolysis-derived ATP production in activated DCs is required for STING phosphorylation and enhances its functions to orchestrate type I IFN production. Prior studies have suggested that glycolytic metabolism augments ATP production, triggering a PI3K-centered positive feedback regulatory circuit and driving effector T cell responses (20,21). Based on these findings, we rationalized that ATP accumulation via DCand T cell-intrinsic glycolysis may be a potential mechanism for host tumor immune surveillance. Therefore, pharmacologically enhancing glycolysis-dependent ATP production is a promising strategy to strengthen antitumor immune responses.
Recent studies have showed interplay between glucose metabolites and innate immune sensing during immune activation in response to viral infections or tumors (15,23). Glycolysis-derived lactate directly binds to the mitochondrial antiviral-signaling (MAVS) protein and suppresses its functions to orchestrate type I IFN production (15), indicating that lactate acts as a natural barrier to impede cytosolic RNA sensing. Lactate also disrupts DC-mediated tumor rejection in the TME (24,25), whereas its potential role in cytosolic DNA sensing remains to be determined. ROS, the important metabolites of glucose metabolism, are involved in STING-dependent immune sensing (4,26). ROS-mediated DNA oxidation enhances immune recognition and potentiates STING signaling in autoimmunity (26). DC-de-rived ROS stabilize SENP3 to boost STING activation in the TME (4). On the other hand, ROS production blocks STING-dependent type I IFN responses in DNA virus-infected macrophages (27,28), indicating that ROS production in different cell types may have different roles in STING activation. In our study, we demonstrated that the glucose metabolite ATP promoted STING phosphorylation to initiate DC-mediated antitumor immunity, suggesting that ATP is indispensable for cytosolic DNA sensing. However, whether and how other glucose metabolites participate in STING signaling is still unclear. Therefore, the functional importance of different glucose metabolites in STING-dependent immune sensing remains to be further studied.
STING agonists and tumor-derived DNA can activate the STING signaling pathway in DCs (4,29). Our results revealed that glucose metabolism was primed in DCs upon STING activation. Elevated aerobic glycolysis contributed to STING signaling activation, although the potential role of other types of glucose metabolism still needs to be defined. DC-intrinsic STING activation accelerated HIF-1α-mediated glycolysis and establishes a positive feedback loop. STING deficiency abolished the increases in the glycolytic rates and ATP production in activated DCs, thereby inhibiting DC antitumor immune responses. Consistent with these findings, STING signaling drives HIF-1α stabilization to increase glycolysis in macrophages during Brucella abortus infection (30). Additionally, STING agonists activate STAT3 to gov- row cells were harvested from tibias and femurs, and red blood cells were lysed with 150 mM NH 4 Cl/10 mM NaHCO 3 /1 mM EDTA. Remaining cells were cultured in RPMI 1640 medium supplemented with 20% FBS, 100 units/mL penicillin plus streptomycin, 20 ng/mL Granulocyte-Macrophage Colony Stimulating Factor (PeproTech) for 7-9 days to obtain BMDCs.
Patients and specimens. Human NSCLC tissues and paracancerous tissues were obtained from patients with NSCLC at Shanghai Chest Hospital, Shanghai Jiao Tong University School of Medicine. Fresh tumor tissues and paracancerous tissues were digested with collagenase VIII and DNase I. Immune cells were enriched by density-gradient centrifugation. DCs were isolated using a Pan-DC Enrichment Kit (130-100-777, Miltenyi) for further experiments.
In summary, our findings elucidate an essential function for glycolysis in DC-mediated antitumor immunity. Our study provides crucial molecular insight into how the crosstalk of energy metabolism and STING signaling regulates DC antitumor activity. These results offer an important paradigm and strategy for the improvement of DC-centric immunotherapies.
Methods
Mice. C57BL/6 background Ldha-floxed mice and Ldhb-floxed mice were generated at Shanghai Model Organisms. Ldha/b-DKO mice were obtained by crossing Ldha-floxed and Ldhb-floxed mice with Cd11c-Cre mice (The Jackson Laboratory). C57BL/6 background Tmem173 -/mice were generated at Shanghai Model Organisms. Ageand sex-matched mice were used in our experiments. All mice were housed in a specific pathogen-free facility. QIAGEN). cGAMP was purchased from InvivoGen Inc. TEPP-46 and DMXAA were purchased from Selleck. Sodium oxamic acid was purchased from Adamas-beta. DCA and cyclodextrin were purchased from Sigma-Aldrich. Human DCs were freshly isolated from NSCLC tissues and paracancerous tissues and purified with a Pan-DC Enrichment Kit (130-100-777, Miltenyi). The purified human DCs were untreated or treated with 2-DG or TEPP-46 for 8 hours and then used for further experiments.
DC stimulation and treatment. BMDCs were harvested for experiments in vitro. For further analysis, BMDCs were counted and seeded in cell culture plates followed by cGAMP or purified Tu-DNA stimulation, respectively. cGAMP and Tu-DNA were transfected using Tenfect DNA transfection reagent (TEYE Corporation) according to the manufacturer's instructions. Tu-DNA was obtained from MC38 Data are shown as the mean ± SEM. Statistical analysis was performed using 1-way ANOVA (I and J) and 2-tailed Student's t test (E, G, and L); *P < 0.05; **P < 0.01.
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2023-02-24T06:18:32.351Z
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2023-02-23T00:00:00.000
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233952041
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pes2o/s2orc
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A Study in the Early Prediction of ICT Literacy Ratings Using Sustainability in Data Mining Techniques
: It would be very beneficial to determine in advance whether a student is likely to succeed or fail within a particular learning area, and it is hypothesized that this can be accomplished by examining student patterns based on the data generated before the learning process begins. Therefore, this article examines the sustainability of data-mining techniques used to predict learning outcomes. Data regarding students’ educational backgrounds and learning processes are analyzed by examining their learning patterns. When such achievement-level patterns are identified, teachers can provide the students with proactive feedback and guidance to help prevent failure. As a practical application, this study investigates students’ perceptions of computer and internet use and predicts their levels of information and communication technology literacy in advance via sustainability-in-data-mining techniques. The technique employed herein applies OneR, J48, bagging, random forest, multilayer perceptron, and sequential minimal optimization (SMO) algorithms. The highest early prediction result of approximately 69% accuracy was yielded for the SMO algorithm when using 47 attributes. Overall, via data-mining techniques, these results will aid the identification of students facing risks early on during the learning process, as well as the creation of customized learning and educational strategies for each of these students.
Introduction
In learning scenarios, it is important for teachers to be able to identify students potentially at risk of faring poorly within a learning area and provide educational intervention proactively. Educational institutions are becoming increasingly concerned with achieving such interventions early on in the learning process [1] because estimating the ratio of positive-to-negative learning outcomes (i.e., succeeding or failing to learn) is critical to strategic planning. Through analysis of the variables from a student's background, it is possible to identify whether or not the student will be likely to succeed prior to immersion in the learning experience. Subsequently, appropriate actions can be taken to facilitate successful outcomes [2]. The capacity to analyze and predict academic performance represents an important milestone in the educational domain, and it is an important factor in building a student's future [3,4]. Therefore, these predictive variables can be used to identify students' learning characteristics to create adaptable methods for providing high-quality education to improve learning outcomes [5,6].
Existing studies have shown that students leverage relevant personal variables and attributes for their academic progress during instruction. These studies focused on the possibility of predicting academic achievement by utilizing student background factors as determined by surveys conducted prior to a class. Such background factors are necessary to analyze students' perceptive capabilities. If automated methods could be employed for
Application of Data-Mining Techniques in Education
Data-mining techniques are used to discover new information hidden within large databases [1,6]. Owing to advances in computing technology, these techniques are increasingly being used to solve problems and make discoveries in various fields of science, medicine, finance, and business [9,10]. In particular, data mining is being used in the field of education to diagnose students' learning factors and provide them with a variety of educational services [11,12].
The education industry leverages data-mining techniques to predict academic performance in advance lessons. The mined data relate to elements of the entire learning course (e.g., midterm, quiz, and activity content). Online and offline programming introductory courses applied similar metrics using neural-network (NN), DT, SVM, and NBC methods. These results showed that the SVM algorithms were the most efficient after 50% completion of a course. Failure rates were predicted with 92% efficiency in online classes and 83% efficiency in offline classes [13]. When predicting early time periods for majors in information-technology (IT)-related areas, seven algorithms (i.e., DT, rule induction, artificial NN, KNN, NBC, and random forest) were used. In this study, year-2007 student data were used for training, and the predicted rate was expressed using similar data from 2008. The results showed that NBC had a prediction rate of 83.7% [14]. University informatics courses used REPTree, J48, and M5P data-mining techniques to predict student performance. The attributes used to create the models included exam conditions, exam points, activities points, and more. The predictive model showed an average of 65% positive results and could reasonably predict a student's academic achievement [1].
However, for the early prediction of overall academic performance, graduation credits, or final-grade ratings, directly relevant attributes (e.g., exam and quiz scores) are commonly used. These related attributes are highly correlated with collected and predicted data, and assessments can be used to early-predict a student's achievement, but only after the learning process begins.
There are two ways to assess success or failure likelihood after the learning process begins. First, the research must ensure early prediction of the overall performance or required credits. Second, the student can express an early prediction rate based on the responses to a personal questionnaire provided prior to the learning process. For highschool students, a DT algorithm was used to predict student achievement, which was divided into five rating categories: "Unsatisfactory" (6%), "Basic" (40%), "Moderate" (38%), Sustainability 2021, 13, 2141 3 of 11 "Good" (14%), and "Excellent" (3%). The data used included measures of self-esteem, self-concept, habits, motivation, cognitive skills, study strategies, and emotional variables representing personal factors related to academic performance. The prediction accuracy in that study was the highest in the "Basic" category with 40% of the student distribution. The remaining categories were in the range of 34-83% [15]. For college students, three algorithms (i.e., DT, NN, and SVM) were used to predict academic performance. The data included measures of online time, frequency of internet connection, amount of internet traffic, and usage behaviors online, which are linked to academic performance. The results showed that the SVM algorithm was the most accurate when predicting passing and failing grades (69-73%), followed by NN (68-71%) and DT (60-62%) [16]. The data used for college students included measures of age, gender, personality, motivation, and learning strategy, and data mining was used to predict the learning outcomes.
The results of that study indicated that SVM (73.3%) was the highest among the six algorithms, followed by KNN (69.4%), NN (69.0%), NBC (69.0%), DT (65.9%), and logistic regression (60.0%). Finally, for college students, the results were more accurate for freshmen than for seniors [17]. In the current paper, the early prediction of academic performance using extant learning processes is precluded, and the attributes directly relevant to predicting final grades are excluded. Additionally, the perception of IT-related students, which constitutes non-grade data focused on predicting final performance, is predicted using six sustainability-in-data-mining algorithms.
ICT Literacy
ICT literacy has been emphasized as an ability to be acquired by all to keep pace with IT development. Such literacy includes the ability to use digital technologies to solve problems, analyze, and generate information based on data, and communicate with others [18,19]. This is the interactions generated by learning to facilitate teacher decisionmaking when big data are generated, these big data are managed, and analyzed by data mining [20]. Since 2007, ICT literacy tests have been employed, and IT-related perceptions have been surveyed among elementary-and middle-school students in Korea [21,22].
The ICT literacy-test questionnaire comprises 36 questions concerning the internet, computer literacy, and IT curricula for daily life. The test results are divided into four levels (i.e., excellent, average, basic, and poor) according to student achievement. The criteria for each level are determined via expert consultation and consideration of the student's ability. The surveys of IT-related students measure the perceptions of their ability to use computers, smart devices, internet tools, and software. Details are shown in Table 1.
Research Method
The proposed method predicted ICT literacy levels using sustainability-in-data-mining techniques based on students' IT-related perceptions. The ICT literacy rating prediction used six algorithms and data mining. A dataset from 2011 was used as the training set, and an attribute selector set of 47, 24, and 17 attributes were used for elementary schools, depending on the information gain ranking and empirical method. Similarly, sets of 47, 22, and 14 attributes were selected for middle-school students.
The data-mining technique selected six sets of algorithms referenced in the preceding studies. Several algorithms were used, including rule-based machine learning, OneR, DT, J48, ensemble listeners, bagging, random forest, neural networks, MLP, SVM, and sequential minimal optimization (SMO) [23]. This study used 10-fold cross-validation to create an optimal method for evaluating model performance [24].
The proposed model applied a dataset from 2012 as the test set, and the model's predictive accuracy was evaluated by measuring accuracy, precision, recall, and F1 score (i.e., F-measure). The flowchart of the ICT literacy evaluation prediction is shown in Figure 1.
Research Subject
The subjects of the study were selected by surveying students corresponding to 1% of the number of elementary-and middle-school students in Korea using stratified random sampling. For the 2011 dataset, 12,373 elementary-school students and 15,556 middleschool students were selected. Similarly, 12,905 elementary and 18,072 middle-school Sustainability 2021, 13, 2141 5 of 11 students were selected for 2012. In total, 25,296 elementary-and 33,628 middle-school students participated in this study for two years.
schools, depending on the information gain ranking and empirical method. Similarly, sets of 47, 22, and 14 attributes were selected for middle-school students.
The data-mining technique selected six sets of algorithms referenced in the preceding studies. Several algorithms were used, including rule-based machine learning, OneR, DT, J48, ensemble listeners, bagging, random forest, neural networks, MLP, SVM, and sequential minimal optimization (SMO) [23]. This study used 10-fold cross-validation to create an optimal method for evaluating model performance [24].
The proposed model applied a dataset from 2012 as the test set, and the model's predictive accuracy was evaluated by measuring accuracy, precision, recall, and F1 score (i.e., F-measure). The flowchart of the ICT literacy evaluation prediction is shown in Figure 1.
Research Subject
The subjects of the study were selected by surveying students corresponding to 1% of the number of elementary-and middle-school students in Korea using stratified random sampling. For the 2011 dataset, 12,373 elementary-school students and 15,556 middle-school students were selected. Similarly, 12,905 elementary and 18,072 middle-school students were selected for 2012. In total, 25,296 elementary-and 33,628 middle-school students participated in this study for two years.
Preprocessing Data
ICT literacy results and IT questionnaire data of the elementary-and middle-school students were collected for the years 2011 and 2012. The data corresponding to 2011 were used as the training set, and those corresponding to 2012 were used as the test set. The criteria for data purification required the selection of missing values and excluded outliers that resulted in unstable or distorted data.
In this study, attribute selection was performed to improve the efficiency of data prediction [5]. The data attributes used in the analysis of data prediction were extracted from the 2011 training dataset using information gain and average merit. Information gain can determine the importance of a given attribute when deciding which attributes in the training dataset are most useful for distinguishing the classes to be learned, including the order In this study, attribute selection was performed to improve the efficiency of data prediction [5]. The data attributes used in the analysis of data prediction were extracted from the 2011 training dataset using information gain and average merit. Information gain can determine the importance of a given attribute when deciding which attributes in the training dataset are most useful for distinguishing the classes to be learned, including the order of attributes. Using the heuristic method, attributes related to the research issues were included, and unnecessary items were deleted (e.g., user ID, student name, and registration number) to finally select the appropriate attributes. The details regarding this procedure are shown in Table 2.
Parameter Setting and Final Model Confirmation
The proposed ICT literacy rating prediction model increased the efficiency of the results when using the six data-mining algorithms. The analysis of the results for prediction was performed using 10-fold cross-validation to change the attributes of the data and basic option parameters and to adjust the highest prediction rate.
The proposed model used data mining to compare actual and predicted data results. As a result, models having higher accuracy were considered to be better.
Data-Mining Techniques Used
Regarding the data-mining techniques, six algorithms were selected by comparing and analyzing their performance accuracies and capabilities based on previous studies. The OneR algorithm is a simple classification rule that is typically applied to a dataset to test a particular attribute. It is a simple and accurate classification algorithm that can create one rule for each predictor and select the rule having the smallest number of errors [25]. The J48 algorithm determines classification criteria based on normalized entropy difference and uses the concept of information entropy to create a DT from the learning data [26]. Bagging is used for statistical classification and regression, and it is an ensemble meta-algorithm designed to improve safety and accuracy. It can reduce the distribution of unstable procedures, such as regression trees, while greatly improving predictive accuracy [27]. Random forest is an ensemble learning method used for the creation, classification, and regression operations of multiple decision trees during training cycles. The benefits of random forest are that it selects one optimal solution, but it randomly selects from the k best options, thereby improving the decision trees [28]. The MLP is a kind of feed-forward artificial NN comprising at least three node hierarchies in which each node, except the input node, is a neuron that uses a nonlinear activation function [6]. The SMO algorithm is sensitive to fine-tuning, but manual fine-tuning is not desirable because it does not guarantee the efficiency of results [13].
Evaluation Criteria
In this study, accuracy, precision, recall (sensitivity), and F1 score were used as criteria for evaluating the six data-mining algorithms [29,30]. Accuracy is the percentage of the measurement that matches the actual and predicted values of the algorithm among the total data (1). Precision is the ratio between actually correct predictions of the positive class (true-positive (TP)) and all predictions of the positive class by the proposed model (TP + false positive (FP)). In other words, it is the ratio of what the algorithm predicted to be the correct answer (2). Recall (sensitivity) is the ratio of actual correct answers (TP + false negative (FN)) when the correct answer was accurately predicted (TP) (3). Precision and recall can be biased if there are many positives or negatives in the data, and the F1 score is used for the performance evaluation of the model using the harmonic mean of precision and recall (4).
Research Results
The proposed method predicted student ICT literacy levels using sustainability-indata-mining techniques based on the perceptions of those IT-related learners. Information gain can be used to transform datasets to determine attribute importance and to distinguish classes. Therefore, the attributes found in the elementary-school results were divided into 47, 24, and 17 based on the average merit value of information gain ranking. The attributes from the middle-school results were divided into 47, 22, and 14.
The early-predicted results for elementary-and middle-school ICT literacy were characteristic of the algorithm used in the sustainability-in-data-mining techniques, indicating normal changes with the number of choices in the attributes.
ICT Literacy-Level Prediction Results for Elementary Students
The results of the ICT literacy-level predictions for the 2012 elementary-school dataset showed that the accuracy corresponded to the number of selected attributes. The lowest accuracy was 62.8%, and the highest was 67.3%. The highest early prediction result of all six algorithms was provided by SMO (67.3%), which used 47 attributes. The lowest prediction result was provided by OneR (62.8%), which used 17 attributes. The details regarding these results are shown in Figure 2. The F1 score uses the harmonic average of precision and recall and is an indicator of test and prediction. In this study, the SMO algorithm scored the highest (0.499) when 47 attributes were used. The lowest prediction result was returned by OneR (0.388) using 17 attributes. The details regarding these results are shown in Table 4.
Prediction Results for Middle-School Students
The results of the ICT literacy grade predictions using the 2012 IT-related middleschool dataset showed varying accuracies according to the number of selected attributes. For this dataset, the accuracy ranged from 63.9% to 68.7%. As noted, the highest prediction score was provided by SMO, which used 47 attributes (68.7%). This was also the highest score achieved when comparing those of the other algorithms. The lowest prediction score was provided by MLP, which used 14 attributes (63.9%). The details regarding these results are shown in Figure 3. The F1 score uses the harmonic average of precision and recall and is an indicator of test and prediction. In this study, the SMO algorithm scored the highest (0.499) when 47 attributes were used. The lowest prediction result was returned by OneR (0.388) using 17 attributes. The details regarding these results are shown in Table 4.
Prediction Results for Middle-School Students
The results of the ICT literacy grade predictions using the 2012 IT-related middleschool dataset showed varying accuracies according to the number of selected attributes. For this dataset, the accuracy ranged from 63.9% to 68.7%. As noted, the highest prediction score was provided by SMO, which used 47 attributes (68.7%). This was also the highest
Prediction Results for Middle-School Students
The results of the ICT literacy grade predictions using the 2012 IT-related middleschool dataset showed varying accuracies according to the number of selected attributes. For this dataset, the accuracy ranged from 63.9% to 68.7%. As noted, the highest prediction score was provided by SMO, which used 47 attributes (68.7%). This was also the highest score achieved when comparing those of the other algorithms. The lowest prediction score was provided by MLP, which used 14 attributes (63.9%). The details regarding these results are shown in Figure 3. The F1 score is an indicator of how well a prediction matches reality, and it uses a harmonic mean of precision and recall. As a result, when 47 attributes were used, SMO (0.541) exhibited the highest score. The lowest prediction score was provided by OneR (0.504) using 14 attributes. The details regarding these results are shown in Table 5.
Conclusions
This paper presented a model for predicting early academic performance-based learning perception using sustainability-in-data-mining techniques. Specifically, ICT literacy levels were predicted using six algorithms based on the students' perception of IT and ICT factors.
The highest SMO algorithm prediction results were 67.3% when using 47 attributes, and the lowest SMO algorithm prediction results were 65.0% when using 24 attributes for the 2012 elementary-school dataset. Therefore, the difference between the two cases was 2.3%. For the 2012 middle-school dataset, the highest and lowest prediction results for the SMO algorithms differed by approximately 4.5%, with accuracy scores of 68.7% for 47 attributes and 64.2% for 14 attributes.
The differences between early prediction results for the elementary-and middle-school datasets using the six-algorithm data-mining technique were 2.3% and 4.5%, respectively. By arranging the attributes affecting these results, similar scores can be achieved without significant changes in early prediction accuracy, even when a small number of features is selected. In particular, the accuracy results of the elementary-and middle-school students were more favorable when 24 and 17 attributes, respectively, were used than when all 47 were used. This was true for the MLP, bagging, and J48 algorithms. Therefore, the accuracy is dependent on both the characteristics of the algorithm and the number of attributes.
These results fully answer the three research questions presented in the introduction. The results of these three data-mining techniques can, therefore, be used to inform teachers, institutions, and students in advance of potential learning successes or failures. Moreover, this innovation has the potential of avoiding or mitigating negative learning outcomes while providing students with important insights into improved educational approaches. In summary, it is possible to sufficiently predict early academic performance using sustainability-in-data-mining techniques based on student perceptions of IT competency and ICT literacy. Moreover, during the process of predicting early achievement by recognition, the SMO and RF algorithms were shown to be most effective. Finally, it was determined that the early prediction accuracy remained close to the highest observed ratio without significant changes when the number of attributes was reduced.
I examined the top five attributes of Information Gain ranking from the analyzed attributes. At the elementary school, the ability to attach data, download search documents, communicate on SNS, music, videos, and search articles using the internet was revealed. At middle school, computer virus prevention, the ability to use the internet, the ability to use the operating system, and the ability to resolve errors were shown. Middle school students used more specialized methods of using a computer than elementary school students. On the other hand, I examined the properties under Information Gain ranking. In general, we've found attributes that are not related to ICT, such as whether to keep smart devices or when to use a computer first boot. Analyzing the attributes indicated by this Information Gain, it can be said that they are related to pursuing the direction of learners' learning and educational strategies.
The significance of this study is its development of a new model for the early prediction of academic performance. This can help identify students facing risks early in the learning process via the application of data-mining techniques and the creation of customized learning and educational strategies for each student. Future research will require improvements to these study results via the extension and integration of the analysis of more diverse data to improve prediction accuracy.
This study has certain limitations, particularly, although the use of sustainability-indata-mining techniques to predict achievement using student perception is interesting, it poses some risks. First, more data are required because, in this study, ICT literacy was only analyzed for 1% of Korea's student population via stratified random sampling. This is insufficient to represent larger populations. Second, only six representative algorithms (i.e., SMO, RF, MLP, bagging, J48, and OneR) were selected and studied. The addition of deep-learning algorithms, wherein sustainability-in-data-mining techniques are rapidly evolving, represents an important consideration for future work.
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2021-05-08T00:03:50.730Z
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2021-02-17T00:00:00.000
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16350826
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pes2o/s2orc
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CT Angiography as a Screening Tool for Dural Arteriovenous Fistula in Patients with Pulsatile Tinnitus: Feasibility and Test Characteristics
BACKGROUND AND PURPOSE: The diagnosis of intracranial DAVF with noninvasive cross-sectional imaging such as CTA is challenging. We sought to determine the sensitivity and specificity of CTA compared with cerebral angiography for DAVF in patients presenting with PT. MATERIALS AND METHODS: Following approval of the institutional review board, we reviewed all patients who underwent CTA for PT from 2004 to 2009 and collected clinical and imaging data. Seven patients with PT and proved DAVF and 7 age- and sex-matched control patients with PT but no DAVF composed the study group. CTA images were blindly interpreted by 2 experienced neuroradiologists for the presence of 5 variables: asymmetric arterial feeding vessels, “shaggy” appearance of a dural venous sinus, transcalvarial venous channels, asymmetric venous collaterals, and abnormal size and number of cortical veins. Asymmetric attenuation of jugular veins was additionally assessed. RESULTS: The presence of arterial feeders showed good test characteristics for screening, with a sensitivity of 86% (95% CI, 42–99) and a specificity of 100% (95% CI, 52–100). A shaggy sinus or tentorium was highly specific: sensitivity of 42% (95% CI, 11–79) and specificity of 100% (95% CI, 56–100). The presence of transcalvarial venous channels demonstrated a poor sensitivity of 29% (95% CI, 5–70) but a high specificity 86% (95% CI, 42–99). CT attenuation of the jugular veins showed statistically significant asymmetry in the DAVF group versus the control group (P < .05). CONCLUSIONS: CTA can be used to screen for DAVF in patients with PT. The presence of asymmetrically visible and enlarged arterial feeding vessels has a high sensitivity and specificity for the diagnosis of DAVF.
P T is a common clinical symptom. [1][2][3][4][5] Its causes are predominantly vascular, either arterial or, more commonly, venous. [5][6][7][8][9] An extensive literature on imaging in PT describes a wide variation in the incidence of structural findings in patients with PT. 10,11 These discrepancies likely reflect heterogeneity in study populations, radiologist expertise, and imaging technique used in the evaluation. The utility of any first imaging test of PT is dependent on the sensitivity with which it detects treatable or worrisome causes of PT and allows appropriate management to be undertaken. Accordingly, the ideal test would demonstrate high sensitivity for such clinically significant causes of PT. 4 A recent review of case series found structural/anatomic causes of PT in 44%-91% of cases. 9 Although a majority of patients may remain "idiopathic" even after extensive workup, the possibility of a morbid and/or treatable cause of PT, such as a DAVF, represents a common indication for imaging. Hence, there is interest in and the necessity for imaging protocols that might accurately diagnose or exclude such pathology. Various imaging strategies have been proposed, including MR imaging, MRA, MRV, carotid sonography, CTA/CTV, and DSA. [12][13][14][15][16][17] None of these studies have well-defined test characteristics. Conventional angiography remains the most sensitive and specific technique for diagnosing DAVF. Thus, patients routinely undergo DSA if DAVF remains in the differential diagnosis for a patient with PT following noninvasive imaging.
Findings of DAVF on DSA are well-described, and the technique is accurate in experienced hands. 18 The risk of neuroangiography is not negligible, however, and access to experienced neuroangiographers remains limited. 24 CT and CTA, on the other hand, are efficient, well-tolerated, widely available, and low risk if performed by using appropriate parameters.
Recently, multidetector CTA has successfully supplanted diagnostic DSA in the evaluation of a variety of vascular lesions. What is not surprising, several authors have suggested its use as a means of detecting DAVF and have proposed imaging findings that might suggest the diagnosis. 15,25 CT also carries the advantage of identifying vascular neoplasms as well as bony/developmental abnormalities of the middle ear and otic capsule that may lead to PT. Furthermore, it allows assessment of the dural venous sinuses for stenoses or diverticula unrelated to the presence of a dural fistula. To our knowledge, however, only 2 studies have evaluated CTA/CTV in patients with PT. Using CTA/CTV, Krishnan et al 16 evaluated 16 patients with PT and, somewhat surprising, did not identify a single DAVF. More recently, in a retrospective review of patients with DAVF, Cohen et al 21 found a low sensitivity (15.4%) of CTA for DAVF, but these authors did not describe how the CTAs were evaluated. In this study, we propose and depict a set of CTA findings that suggests the presence of DAVF in patients with PT, and we evaluate the diagnostic performance of these findings as a screening tool for DAVF in patients with PT.
Patient Population
The study was approved by our institutional review board. The data base of our PACS was searched from January 2003 to September 2009 for patients diagnosed with DAVF. Only patients with both CT and CTA as part of their initial work-up and subsequent confirmatory DSA were included in this analysis. A similar search of our hospital information system was performed to identify patients presenting with PT who also had CTA performed but who were not diagnosed with dural fistula. Electronic medical records were used to gather demographic and clinical information. These search results yielded sets of patients with PT who either did (patients) or did not have (controls) DAVFs. All patients underwent confirmatory conventional angiography. Controls with clinically worrisome PT underwent diagnostic conventional angiography, and those with clinically benign PT underwent CTA and confirmatory MR imaging/MRA. Patients with DAVF without PT as a first symptom were excluded. Patients with PT without DAVF (cryptogenic or other venous abnormality) were used as controls. Additionally, all patients classified as having PT demonstrated pulse-synchronous tinnitus defined by otologic clinical evaluation.
Seven patients with PT and angiographically proved DAVF and 7 age-and sex-matched controls with PT but no DAVF composed the study group.
CT/CTA/CTV Parameters
We performed multidetector CT/CTA/CTV on a 16-detector scanner (LightSpeed; GE Healthcare, Milwaukee, Wisconsin). Noncontrast images were obtained from the vertex to the skull base (5-mm section thickness, 120 kV, 400 mA). This was followed by CTA (0.625-mm section thickness, 140 kV, 250 mA). Triggering of CTA acquisition was done manually by a technologist based on peak contrast enhancement in the aortic arch following administration of 120 mL of iohexol with a concentration of 350 mg I/mL (Omnipaque 350; GE Healthcare, Princeton, New Jersey) at a rate of 4 -5 mL/s. Images acquired in this manner demonstrated arterial phase opacification but with both arterial and venous vasculature well-opacified, so both arterial and venous structures could be assessed. The CT source images were postprocessed to create contiguous 1-mm coronal and sagittal reformations, as well as axial, coronal, and sagittal MIP images with both 3and 5-mm section thickness. Volume-rendered 3D images and curved planar reformatted images of the bilateral common and inter-nal carotid arteries and vertebral arteries were also created, as well as 3D images of the vessels of the circle of Willis.
Image Evaluation
Imaging studies were reviewed independently on a PACS workstation by 2 experienced neuroradiologists who were blinded to the final diagnosis. Images of patients and controls were presented to the blinded reviewers in random order. Only the lead author was aware of the final diagnosis based on DSA. Imaging studies contained source data as well as multiplanar and processed series, and each patient's entire examination was evaluated in the same sequence. Before assessment of findings associated with DAVF, the images were evaluated for the presence of mass lesions, an aberrant internal carotid artery or persistent stapedial artery, and abnormalities of the temporal bone such as dehiscence of the superior semicircular canal.
Imaging studies were evaluated for the presence or absence of 5 variables that are associated with DAVF: numerous, asymmetric, and/or dilated feeding arteries; numerous, asymmetric, and/or dilated venous collaterals; "shaggy" appearance of a dural venous sinus or the tentorium cerebelli; transcalvarial venous channels; and increase in the size and number of cortical veins. These variables were scored as "present" or "absent." Images were additionally analyzed for measurable asymmetry in jugular venous attenuation. Finally, images were also comprehensively evaluated for the presence of any pathology that could explain PT at the time of clinical interpretation as well as subsequently during dedicated research evaluation.
Cervical vessels were evaluated specifically to identify usually asymmetric prominent and/or numerous branches of the external carotid arteries that might serve as DAVF feeders. The arterial nature of these channels was confirmed by assessment of their course, inspection of their origin from identifiable arteries (eg, external carotid branches), and also via depiction of equivalent attenuation to arterial sources. When these criteria were met, the vessels were declared positive for arterial feeders. Conversely, numerous and prominent cervical vessels that appeared venous in course, opacification, and origin were labeled as such.
A shaggy appearance of the sinus or tentorium was determined by inspection of these structures for the presence of irregularity along their margins because venous sinuses are typically smoothly marginated. Irregularity was subjectively assessed and thought to suggest the presence of innumerable small venous efferents and/or intimal thickening associated with venous sinus hypertension.
Because fistulizing arterial feeders from the occipital artery often take a transosseous path at the mastoid foramen alongside emissary veins located within or adjacent to the occipitomastoid suture, we evaluated the bone around the occipitomastoid suture for significant asymmetry in the size and number of regional vascular grooves or channels.
Asymmetric attenuation of jugular veins was assessed in a fashion similar to that of previously reported methodology. 25 Hounsfield measurements were made on both patients and controls in 4-mm regions of interest centered within both jugular veins at a level 1 cm below the jugular foramen. Three measurements of mean attenuation were made, and the average value was used for analysis. All measurements were made below the level of stenosis in patients with DAVFrelated dural venous sinus or jugular stenosis.
After evaluation of CT/CTA datasets, the participating neuroradiologists were unblinded. Neuroangiographic datasets were evaluated on the same PACS workstation used for the prior analysis. The presence of abnormal connections between arterial feeders and a dural venous sinus or leptomeningeal vein constituted diagnostic criteria for DAVF. All DAVFs were classified according to the Cognard stage on the basis of conventional angiography: type I representing fistula confined to the sinus wall with antegrade flow; type IIA, a fistula with retrograde flow within the sinus; type IIB, a fistula with retrograde reflux into cortical veins; type IIAϩB, a fistula with retrograde flow within the sinus and reflux into cortical veins; type III, a fistula with direct drainage into cortical veins; type IV, a fistula with reflux into ectatic cortical veins; and type V, a fistula with spinal perimedullary drainage.
Statistical Analysis
The 5 imaging features described above were used as discriminant criteria. We thus based the performance of CT/CTA on its ability to discriminate patients from controls as measured by sensitivity and specificity. 26 Five of the discriminant criteria relied on the judgment of neuroradiologists, while the sixth imaging feature was based on objective measurements of CT attenuation of the draining jugular veins. These latter measurements were subject to standard paired 2-tailed Student t test to assess asymmetry in jugular opacification in both groups. A P value Յ .05 was considered a significant difference. We calculated 95% CIs according to the efficient score method.
Results
Seven patients underwent CT/CTA for diagnostic work-up of PT and were found to have DAVF on subsequent DSA ( Table 1). The average age was 48.9 years (range, 40 -79 years). There were 5 women and 2 men. Six of the 7 had PT on the right and were found to have predominantly a right-sided supply and right-sided drainage. One patient had left-sided supply and drainage. One patient had a bilateral supply that drained into the right sigmoid sinus. Drainage patterns were variable, with 3 patients demonstrating a fistula at the sigmoid sinus, 3 patients demonstrating fistula at the marginal sinus, and 1 patient demonstrating a fistula to an occipital sinus. In comparison and in keeping with the general population, 6 of 7 patients demonstrated right-sided dominant drainage with 1 demonstrating codominant transverse sinus/jugular drainage. All fistulas were evaluated for Cognard stage and venous drainage. Four of 7 patients demonstrated venous sinus stenosis. All stenoses were downstream of the fistula in either the high jugular vein (3/4) or sigmoid sinus (1/4). These stenoses did not correlate with other signs of venous hypertension such as cortical venous drainage. Four of 7 patients demonstrated Cognard stage IIa with some retrograde flow through the venous sinuses. Two patients showed Cognard stage I fistula with antegrade venous flow. One patient showed stage IIaϩb with evidence of retrograde venous sinus flow in addition to cortical venous drainage. This patient reported a long-standing his-tory of right-sided PT before hospitalization for intracerebral hemorrhage.
Age-and sex-matched controls averaged 41.3 years of age (range, 30 -75 years). All presented with PT (Table 2), with predominantly right-sided (5/7) symptoms as in our patients (6/7). Most controls (5/7) had no evidence of pathology demonstrated on CTA images. Two patients had findings on CTA suggesting a possible etiology of symptoms. One patient had a venous diverticulum of the sigmoid sinus ipsilateral to the PT, and the other patient had moderate stenosis of the ipsilateral transverse sinus.
Of 7 patients with DAVFs, 6/7 demonstrated asymmetrically numerous and enlarged arterial feeders surrounding the ipsilateral carotid artery and its external branches (Table 3 and Fig 1). None of the controls demonstrated this finding. The presence of asymmetrically increased arterial feeders (Table 3) showed good test characteristics for screening, with a sensitivity of 86% (95% CI, 42-99), a specificity of 100% (95% CI, 52-100), PPV of 100% (95% CI, 56 -100), and NPV of 88% (95% CI, 67-88). The appearance of asymmetric feeders at the neck and skull base on CTA matched the pattern of arterial supply from external carotid artery branches on angiography. For example, an enlarged occipital artery seen on CTA would also be seen on conventional angiography. Nevertheless, 3/7 patients had angiographic evidence of supply from the MHT (ie, a small vessel that was not consistently identifiable on CTA).
Although the presence of cortical venous drainage is a critical prognostic finding on angiography, CTA does not permit subtle distinction regarding flow directionality. We hypothesized that anatomic depiction of cortical venous dilation may represent a CTA correlate. Indeed, 2 patients demonstrated striking venous collateralization on CTA. Nevertheless, only 2/7 cases demonstrated this finding, generating a sensitivity of 29% (95% CI, [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29], specificity of 100% (95% CI, 82-100), PPV of 100% (95% CI, 38 -90), and NPV of 58% (95% CI, 49 -58). CT attenuation of the jugular veins, measured in Hounsfield units 1 cm below the skull base (Fig 3), showed a statistically significant asymmetry in the DAVF group versus the control group (P Ͻ .05). 1. A, Axial 3-mm-slab MIP from CTA. A clearly abnormal "ring" of arterial feeders is seen around the right distal cervical carotid artery at the C1-2 level (white arrow) in a patient with a right sigmoid DAVF (patient 1). B, Large transosseous venous channels (white arrows) are present on the right in patient 1. This is an uncommon but specific finding for DAVF. These channels do not track to the occipitomastoid suture, a structure from which they must be distinguished in some cases by viewing serial sections. C, Markedly asymmetric venous collaterals along the right occipital bone, both intracranially (white arrow) and extracranially (white arrowhead), as well as asymmetrically enlarged and numerous veins throughout the infratemporal fossa (patient 2). This patient also had large transosseous collaterals (not shown). D, DSA, arterial phase, right external carotid injection in a lateral projection in patient 2, shows arterial feeders from the markedly enlarged occipital artery (short black arrow), as well as from the middle meningeal artery (black arrowheads), the correlates of the arterial feeders shown on the CTA in C, with a sigmoid DAVF (long black arrow).
Discussion
The initial evaluation of the patient with PT begins with a careful history and otoscopic evaluation. The presence of a mass on otoscopy warrants temporal bone CT for further evaluation. PT in the face of normal otoscopic findings, however, represents a common otologic diagnostic dilemma. The differential diagnosis remains broad and unfortunately includes potentially serious vascular etiologies such as DAVF and more benign vascular etiologies such as venous stenoses and diverticula. Moreover, the literature has many statements such as "if DAVF is likely, then catheter-angiography is warranted," 16 which presume some clinical symptom or sign unique to this diagnosis. The otology literature has described this dilemma, 18 with some advocating liberal use of angiography, while others advance MR imaging and CT-based approaches. 4,6 Clinical evaluation clarifies the presence of subjective "whooshing" Fig 3. A, Coronal 3-mm-slab MIP from CTA in patient 3 with a right transverse sinus DAVF, predominantly supplied by right occipital, middle meningeal, and marginal tentorial branches; some supply from the left internal carotid is also seen, as described below. This case illustrates the "asymmetric jugular attenuation" sign, which is easily appreciated when the R and L IJVs are compared. The attenuation of the R IJV is 402 HU, and the L IJV is 318 HU. B, DSA, late arterial phase, left internal carotid injection in an anteroposterior projection in same patient as in A. Fistula supply from small left internal carotid artery dural and tentorial branches (black arrows) contributes to early opacification of the right sigmoid sinus and IJV (note the catheter near the jugular bulb). Due to the arteriovenous shunt surgery and drainage pattern, iodinated contrast preferentially fills the right jugular vein (black arrowheads) and leads to an asymmetric right-sided increase in CT attenuation, which is seen in A.
pulsations that extinguish with gentle neck pressure or altered head position, a finding that supports more benign venous origins of PT. Alternatively, the presence of a bruit raises concern for an arterial vascular abnormality, which indicates complete imaging including DSA. Moreover, the impact of PT on the well-being of the patient is a significant factor in determining how aggressive the work-up should be. The present data provide guidance for most patients who present with neither obvious benignity/low impact nor a clear and clearly worrisome audible bruit.
CTA analysis in the current study was based on cross-sectional correlates of classic angiographic findings of DAVF. These findings are based on the sine qua non of DAVF: arteriovenous shunting. Angiographically, anomalous connections between dural arteries and venous sinuses can be seen directly. Ancillary findings relate to the presence of angiogenic activity and the end results of arterial pressure on venous structures. These findings include multiple arterial feeders, venous intimal thickening, stenosis and/or thrombosis, and dilation of and reflux within venous efferents. 19,20,27 The hypothesis that additional potential CTA findings may demonstrate improved test characteristics derives from the above-mentioned angiographic experience. Two indirect characteristics have been previously reported to be associated with DAVF on CT or CTA: transcalvarial venous channels and asymmetric attenuation of jugular veins. We also chose to assess numerous asymmetric and/or dilated arteries; numerous asymmetric and/or dilated venous collaterals; shaggy appearance of a dural venous sinus or the tentorium cerebelli; and increased number and/or enlargement of cortical veins, all of which derive from angiographic findings.
MR imagingϪbased approaches have demonstrated some success in evaluating DAVF. 21,28,29 Studies have used a variety of techniques and sequences and have shown variable diagnostic performance. De Marco et al 28 presented some of the earliest data, finding MR imaging cortical vein dilation and venous occlusion in 8 of 12 patients with angiographically proved DAVF by using basic spin-and gradient-echo sequences. This description is contrary to the present experience in which the presence of cortical venous dilation was relatively rare, 2/7. Such discrepancies can be accounted for by differ-ences in the study population, a small cohort of DAVF not limited to those presenting with PT, and differences in technique.
More recent MR angiographic analyses have yielded a remarkably wide range of results with sensitivities from 50% to 100%. 21,29 Time-resolved MR imaging and 4D flow MR imaging have shown promise in providing angiography-like images 30,31 and in providing the time resolution that routine CTA/CTV and MR imaging/MRA/MRV lack. Yet these studies represent the results of highly specialized and often proprietary MR images, which, as such, remain in limited use. As shown in the current study, CTA can provide equivalent diagnostic performance but with improved osseous. Its additional merits in cost, efficiency, and widespread availability hardly need emphasis. Recent data on the comparative strength of CTA in evaluating alternative etiologies to PT, such as glomus tumors, buttress the argument that CTA can provide an efficient and comprehensive first-step assessment of patients with PT. 38 The additional radiation associated with CT angiography in comparison with noncontrast CT of the head has been recently investigated. 39 Standard parameters yield mean effective doses of 2.7 mSv for nonenhanced CT, while CTA delivers 5.4 mSv. As a comparison, however, both studies fall below the relatively commonplace 8-to 9-mSv dosage of an abdominal CT. Moreover, cerebral angiography commonly averages 10.6 mSv in effective dose equivalent. 40 The detailed anatomic data provided by CTA and the possible avoidance of DSA dosage must be balanced against the increased dose delivered by CTA.
CTA demonstrates a number of direct and indirect signs that are suggestive of intracranial DAVF. These signs can be used to screen for DAVF in patients with PT. In particular, the presence of abnormally enlarged arterial feeding vessels, a finding not emphasized in prior literature, has high sensitivity, specificity, and PPV for the diagnosis of DAVF.
The discrepancy between the current results and the findings of Cohen et al, 21 however, does require further discussion. In that series of 46 patients, 13 patients underwent CTA, only 2 of whom had findings positive for DAVF. Yet on what basis DAVF was excluded remains speculative because this study was a chart review in which images were not assessed by using a standard set of criteria. It may be that this low sensitivity (15%) represents only a "standard" evaluation without specific focus on potential diagnostic features of DAVF. Because this is often what referring physicians receive, they are then left to wonder whether CTA is worth ordering at all.
With regard to the limitations of our study, our findings may not be easily replicated in all practice settings. The process of formalized evaluation in the academic setting may represent an idealized evaluation. Regardless, the objectives of this study are not only the evaluation of diagnostic performance of CTA in DAVF diagnosis but also the hypothesis-driven depiction of radiographic signs. Here, the appearance of abnormally numerous and enlarged arterial feeders, shaggy sinuses and tentorium, and asymmetric sinus attenuation helps provide keys to improved radiologic evaluation. These signs, especially if confirmed by other investigators, will allow radiologists, neuroradiologists, and experienced nonradiologist physicians to increase their diagnostic sensitivity and specificity for DAVF. Our limited CTA depiction of cortical venous collaterals is likely an artifact of selection bias. In choosing to evaluate patients with PT rather than intracranial hemorrhage, we accordingly selected a cohort with lower grade DAVFs that, by nature, less frequently demonstrate cortical venous collaterals.
A potential additional limitation involves the use of PPV because it depends on the prevalence of disease within the study group. In a case-control study such as this one, the population is necessarily enriched with disease. Nevertheless, as noted by Madani and Connor, 9 the prevalence of DAVF in patients with PT is variably reported to be as high as 20%. A study population with a prevalence of 50% DAVF necessarily will accentuate the PPV.
Interest in cross-sectional correlates of angiographic findings has grown with the advent of increased CT angiographic gantry speed and pitch, improved section profile, ever-advancing tools for postprocessing and image manipulation, and conebeam technologies. In fact, experience in evaluating DAVF with C-arm CT, as recently described by Hiu et al 17 and Lasjaunias et al, 33 helps generate confidence in evaluating CTA correlates of classic angiographic findings. 32 The current series supports findings of earlier reports that suggest that DAVFs associated with PT often involve the sigmoid sinus. 5,33,34 McDougall et al 35 provided a specific description of a cohort of patients with DAVF at the marginal sinus, in which 11 of 14 presented with PT. The marginal sinus is located between layers of dura that encircle the foramen magnum, communicating with the basal venous plexus of the clivus anteriorly and the occipital sinus posteriorly. Our series confirms the association of sigmoid sinus and marginal sinus DAVF with PT.
Recent data on the prognosis of nonhemorrhagic DAVF have highlighted the reassuring risk profile of Cognard I and II fistulas. 22,36,37 Awad et al, 34 in an early meta-analysis, found 203 patients with PT-associated DAVF showing a nonaggressive natural history. Controversy exists as to the rate at which low-grade fistulas convert to aggressive phenotypes. 23,37 CTA evaluation of cortical venous reflux in the current study was limited for several reasons. The cohort of patients with PT does not, in general, have high-grade fistulas. In fact, angiography confirmed no fistula above Cognard grade IIaϩb, implying absence of any venous ectasia. Neuroradiologists could not distinguish the presence or absence of cortical venous reflux by using only static anatomic CTA data, given the known lack of marked cortical venous dilation. Thus, a PPV as low as 50% reflects this limitation. In addition, our series identifies a single case of a patient who personally recalled months of ipsilateral PT symptoms before presenting with intracranial hemorrhage. This single case serves as a reminder that the presence of PT is no guarantee of benign prognosis.
Conclusions
The patient with PT frequently undergoes cross-sectional imaging evaluation, but there is a wide variation in the imaging methods used and in reported performance of these modalities for the diagnosis of DAVF. CTA provides a convenient low-risk method for additional evaluation of vascular causes. The present data suggest that the combined signs of abnormally prominent arterial feeders, shaggy sinus/tentorium, and asymmetric jugular venous attenuation produce useful diagnostic information with a combined sensitivity and specificity Ն90%. The ability of conventional angiography to evaluate venous efferents remains an important diagnostic benefit of DSA because it carries prognostic significance. DSA accordingly is still indicated if CTA is negative but clinical suspicion of DAVF is high. Furthermore, angiographic analysis allows assessment of the feasibility of endovascular therapeutic approaches and the mechanism to accomplish this therapy once a DAVF has been diagnosed noninvasively. We suggest that screening CTA, assessed systematically for the above-discussed variables, should be considered as the first-choice imaging technique to assess structural, neoplastic, and venous causes of PT and to screen for DAVF.
|
2017-04-06T09:37:44.447Z
|
2011-03-01T00:00:00.000
|
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41929070
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pes2o/s2orc
|
v3-fos-license
|
DNAJB6 Induces Degradation of β-Catenin and Causes Partial Reversal of Mesenchymal Phenotype*
We showed that expression of MRJ (DNAJB6) protein is lost in invasive ductal carcinoma, and restoration of MRJ(L) restricts malignant behavior of breast cancer and melanoma cells. However, the signaling pathways influenced by MRJ(L) are largely unknown. Our observations revealed that MRJ(L) expression causes changes in cell morphology concomitant with down-regulation of several mesenchymal markers, viz. vimentin, N-cadherin, Twist, and Slug, and up-regulation of epithelial marker keratin 18. Importantly, MRJ(L) expression led to reduced levels of β-catenin, an epithelial mesenchymal transition marker, and a critical player in the Wnt pathway. We found that MRJ(L) up-regulates expression of DKK1, a well known Wnt/β-catenin signaling inhibitor, that causes degradation of β-catenin. Re-expression of DNAJB6 alters the Wnt/β-catenin signaling in cancer cells, leading to partial reversal of the mesenchymal phenotype. Thus, MRJ(L) may play a role in maintaining an epithelial phenotype, and inhibition of the Wnt/β-catenin pathway may be one of the potential mechanisms contributing to the restriction of malignant behavior by MRJ(L).
The Hsp40/DnaJ family of proteins is known to be a specific client protein provider for the Hsp70/DnaK family of chaperones (1). The important role of the Hsp40 family in a variety of aspects of tumor biology is being described only very recently (2-6). DNAJB6 is a member of the class II Hsp40/DnaJ family of proteins comprising two differentially spliced variants (7). MRJ(S), the shorter variant of DNAJB6, has been widely studied and has been reported to regulate keratin8/k18 organization, assist in NFATc3-mediated transcriptional repression, and play a critical role in Huntington disease (8 -10). On the contrary, the role of the long variant, MRJ(L), in various pathophysiologies has still to be revealed with the exception of a potential role in the nuclear import of human immunodeficiency virus type-2 vpx (11). We and others have reported loss of DNAJB6 in advanced breast cancer patients (7,12). A detailed functional characterization of DNAJB6 performed by us revealed that MRJ(L) can act as a nuclear protein by virtue of its nuclear localization signal and can considerably reduce the expression of several secreted proteins that promote invasion, migration, and metastasis of cancer cells. Functionally, MRJ(L) was found to reduce tumorigenicity and metastasis of melanoma and breast cancer cells (7). But the mechanistic clues to the downstream signaling regulated by MRJ(L) are still largely unknown.
In the course of our studies, we observed distinct morphological changes in the cells that were restored for MRJ(L) expression. The cells were more epithelial like in appearance. In this study, we describe the role of MRJ(L) in causing partial reversal of epithelial mesenchymal transition (EMT). 3 Our studies show that MRJ(L) inhibits Wnt/-catenin signaling by up-regulating DKK1, a secreted inhibitor of Wnt signaling, and reverts certain aspects of EMT. We also demonstrate that this inhibition of Wnt/-catenin signaling is responsible, in part, for the ability of MRJ(L) to restrict malignant behavior.
MDA-MB-435 has now been confirmed to be of melanoma origin (13)(14)(15). However., there still are convincing arguments about the lineage infidelity of this cell line (16). However, the work presented here uses this cell line as a model system for signaling studies.
SUM159 cells were cultured in Ham's F-12 with 5% fetal bovine serum supplemented with insulin (5 mg/ml) and hydrocortisone (1 mg/ml). The cell line SUM1315 was cultured in Ham's F-12 with 5% fetal bovine serum supplemented with insulin (5 mg/ml) and epidermal growth factor (10 ng/ml). Both SUM lines were bought from Asterand plc, Detroit, MI.
RNA Isolation and Real Time Quantitative RT-PCR-TRIzol reagent (Invitrogen) was used to isolate total RNA from cultured cells. RNA was treated with DNase I (Promega, Madison, WI). cDNA synthesis was carried out using a cDNA synthesis kit (Applied Biosystems Inc., Foster City, CA) with 1 g of total RNA as the template and random primers. Real time quantitative RT-PCR (QRT-PCR) analysis was performed on the experimental mRNAs. The PCR primers and probes for Dickkopf1 (DKK1), DNAJB6 (MRJ(L)), c-myc, E-cadherin, and keratin 18 and normalization control gene glyceraldehyde-3-phosphate dehydrogenase were purchased from Applied Biosystems Inc. QRT-PCR was performed on an ABI 7500 HT instrument (Applied Biosystems). The gene expression ⌬Ct values of mRNAs from each sample were calculated by normalizing with normalization control, glyceraldehyde-3-phosphate dehydrogenase, and relative quantitation values were plotted using GraphPad Prism (La Jolla, CA). QRT-PCR analysis was performed on the experimental mRNAs in triplicate, and the experiment was repeated once from an independent passage to confirm the findings.
Plasmids and Constructs-pDKK-707, a luciferase construct encompassing Ϫ707 to ϩ43 bp of human DKK1 promoter was a gift from the Varmus laboratory (21). M50 TOPFlash luciferase construct was a generous gift from Dr. Randall Moon (22).
shRNA Knockdown-Oligo Design Tool TM (Oligoengine, Seattle, WA) was used to design two shRNAs, NM_807 and NM_808, predicted to silence the expression of MRJ(L). The heteroduplexes supplied as asymmetric oligomers were annealed following the manufacturer's instructions and cloned into pSUPERIOR.neoϩGFP (Oligoengine) at the BglII and HindIII sites. Recombinants were analyzed by restriction diges-tion using EcoRI and HindIII (Promega), and recombinant DNA was prepared using the QIA Maxiprep kit (Qiagen, Valencia, CA).
MCF10A cells were transfected with shRNA targeting MRJ(L) (NM_ 808) or a control nontargeting shRNA using Lipofectamine 2000 according to the manufacturer's instructions. The extent of knockdown was assessed using QRT-PCR, using RNA isolated 32 h after transfection.
siRNA Knockdown-Cells were transiently transfected with SMARTpool siRNA (Dharmacon, Chicago) designed to target DKK1 or nontargeting siRNA control using Dharmafect1 (Dharmacon) following the manufacturer's protocol. Conditioned serum-free medium and lysates were collected from both control and DKK1-siRNA-treated cells 38 h post-transfection. The extent of knockdown was assessed using Western blot analysis.
Immunocytochemistry-Cells cultured on coverslips were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature and then permeabilized in PBS containing 0.1% Triton X-100 and 3% bovine serum albumin. F-actin was stained with phalloidin-TRITC (1:100, Molecular Probes) for 1 h at room temperature and observed after mounting the coverslip in VECTASHIELD (Vector Laboratories, Burlingame, CA). Cells were viewed using a Zeiss Axiovert 200 M microscope equipped with an Axiocam camera and Axiovision software (Carl Zeiss, Inc., Thornwood, NY).
Luciferase Assay-Cells were transfected using Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions. Total protein was harvested and luciferase activity measured using a Turner 20/20 luminometer (Turner Biosystems, Sunnyvale, CA). The luciferase reading was normalized to the total protein concentration. Data are expressed as relative luciferase activity, where control is 100%.
Western Blot-The cell monolayer was washed twice with calcium-and magnesium-free PBS and lysed with cold lysis buffer (150 mM NaCl, 50 mM Tris, 1% Nonidet P-40, and protease and phosphatase inhibitors). The lysates were kept on ice for 1 h and centrifuged at 10,000 rpm for 30 min at 4°C. Protein concentration was measured using Bradford reagent (Bio-Rad). Proteins were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (0.2 m). The membranes were blocked with 5% skimmed milk in TBST (1 M Tris, pH 7.5, 9% NaCl, 0.5% Tween 20) and incubated with primary antibodies overnight at 4°C. The membranes were then washed three times with TBST, incubated with respective secondary antibodies for 1 h at room temperature, and then developed using SuperSignal TM (Pierce) following washes for 1 h with three changes of TBST.
Colony Formation Assay-MDA-MB-435 cells stably expressing MRJ(L) were plated in 6-well plates and transfected with DKK1 siRNA and control siRNA using Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions. After 24 h of transfection, the cells were plated into three 10-cm plates in selection medium and were allowed to grow for 12 days. Crystal violet staining was used for visualization of foci. Six different fields were counted for the number of foci, and data are represented as average Ϯ S.E.
Soft Agar Colonization Assay-MDA-MB-435 cells (2 ϫ 10 3 ) treated with DKK1siRNA or control siRNA suspended in 0.35% agar were plated onto a layer of 0.75% BactoAgar in DMEM/ F-12 (10% fetal bovine serum) in 6-well tissue culture dishes. Visible colonies (Ͼ50 cells) were counted after 15 days with the aid of a dissecting microscope.
Wound Healing Assay-Cells were cultured to confluence on 6-well plates, which were previously externally marked with parallel lines to use as guides for the subsequent photography. The cells were transfected with DKK1 siRNA and control siRNA using Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions. After 24 h of transfection, a central linear wound (perpendicular to the guide lines) was made with a 200-l sterile pipette tip. Media were changed gently to remove any floating cells. Phase micrographs of the wound cultures were taken at 0 and 16 h (average doubling time is about 20 h). The photographs were analyzed by measuring the distance from the wound edge of the cell sheet at the end of 16 h to the original wound site. Wound healing activity was calculated as the mean distance between edges in 12 independent fields per well. Each test group was assayed in triplicate, and the results are expressed relative to vector control cell migration.
Migration Assay-Migration assays were conducted using 8-m polyethylene terephthalate filters (BD Pharmingen), as described previously (23). Cells (treated with DKK1siRNA or control siRNA), which migrated to the lower sides of the transwell, were stained using 0.05% crystal violet, and the cell number was counted. Each test group was assayed in triplicate. Four different fields of each insert were photographed at 10ϫ magnification using a Zeiss Axiocam 200 M microscope (Zeiss Axiocam, Carl Zeiss Microimaging Inc.). Each field was divided into quadrants, and cells in diagonally opposite quadrants were counted.
MRJ(L) Expression Leads to Change in Cell
Shape-We compared the morphology of the cells that were engineered to constitutively express MRJ(L) to the corresponding vector control. As seen in Fig. 1, A and B, MRJ(L) stable transfectants of MDA- MB-435 and MCF7 cells show a well spread/pavement-like appearance and a differential actin staining when compared with the respective controls that look elongated and spindleshaped. The discrete morphological difference is highlighted by the extended filopodia very distinctly present on the surface of MRJ(L) expressors (Fig. 1, A Loss of -catenin and gain of keratin 18 were consistent in all three cell lines (Fig. 2).
Degradation of -Catenin by MRJ(L) Is Proteasome-dependent-Stabilized -catenin characteristically represents activated Wnt/-catenin signaling. MRJ(L) expressors showed distinctly reduced levels of -catenin protein. We saw no sig-nificant change in the steady state mRNA levels for -catenin (data not shown); hence, we tested if this reduction was due to enhanced degradation of -catenin. As seen in Fig. 3, A and B 1 We observed greater than 85% reduction in the TOPFlash activity in all the cell lines, indicating a significant inhibition of the Wnt/-catenin signaling (Fig. 4, A-C). MRJ(L) Up-regulates DKK1-DKK1 is a secreted inhibitor of Wnt/-catenin signaling that blocks Wnt signaling by binding to the Wnt co-receptor Lrp5/6. QRT-PCR analysis of different breast cancer cell lines showed that all of them express lower levels of DKK1 mRNA as compared with nontumorigenic MCF10A cells. SUM1315, SUM159, and MCF10.DCIS.com express very low levels of DKK1, whereas MCF7 and MDA-MB-231 cells seem to express slightly higher levels. The levels of DKK1 in MCF10CA1d.cl.1 cells were intermediate (Fig. 5A). We have also evaluated DKK1 mRNA levels in MDA-MB-435 and found it to be very low. Hence, we examined the MRJ(L) expressors for expression of DKK1. As seen in Fig. 5B, we observed a substantial increase in the levels of secreted DKK1 in serum-free conditioned medium from 435-MRJ(L) and MCF10CA1d.cl.1-MRJ(L) cells. These results corroborate with the increased DKK1 transcript by QRT-PCR (supplemental Fig. 1).
To determine whether the increase in DKK1 is at a transcriptional level, we assessed the activity of the luciferase reporter construct pDKK1-707 in MCF10CA1d.cl Silencing of DKK1 from MRJ(L) Expressors Leads to Restoration of -Catenin-DKK1 binding to the Lrp5/6 receptors directs -catenin to phosphorylation and subsequent proteosome-mediated degradation. If the degradation of -catenin in MRJ(L) expressing cells is due to the elevated DKK1 levels, 6A). MCF10CA1d.cl.1 MRJ(L) expressors also show comparable restoration of -catenin levels upon DKK1 silencing (Fig. 6B). For reasons not known to us, we always noticed a slight elevation of -catenin in the control siRNA transfectants of MCF10CA1d.cl.1MRJ(L). Because most of the elevated mesenchymal markers observed by us in Fig. 2 are downstream targets of -catenin transcription, we hypothesized that DKK1 silencing will increase the -catenin protein levels and result in changes in the transcript levels of the mesenchymal markers. We explored the effect of DKK1 silencing, in MDA-MB-435-MRJ(L), on the EMT markers (supplemental Fig. 2). We observed that there was a noticeable up-regulation of mesen- Silencing of DKK1 from MRJ(L) Expressors Increases Their Malignant Activity-We had previously reported that MRJ(L) expression reduced malignant activity of cancer cell lines as measured by in vitro assays. Cells ectopically expressing MRJ(L) showed reduced invasion through Matrigel TM and reduced migration using modified Boyden chamber assay, decreased scratch motility (wound healing) assay, and less anchorage independence as measured by soft agar colony formation. If the reduced malignant behavior of MRJ(L) expressor cells was due to the inhibition of the Wnt/-catenin signaling by the elevated DKK1 levels, silencing DKK1 expression will revert their phenotype and make them more malignant. As observed from the data in Fig. 7, silencing of DKK1 from 435-MRJ(L) increased the foci formation ability by about 3-fold (Fig. 7A), its motility (wound healing ability) by about 2-fold (Fig. 7B), and anchorage-independent growth as measured by soft agar colony formation by about 2.5-fold (Fig. 7C).
Abrogating of MRJ(L) from MCF10A Leads to Activation of Wnt/-Catenin Signaling-MCF10A (immortalized breast epithelial line), expresses noticeably high levels of MRJ(L) as compared with breast cancer cell lines (7). Based on our observations described thus far, we hypothesized that knockdown of MRJ(L) from MCF10A would activate Wnt/-catenin signaling. As seen in Fig. 8A, knockdown of MRJ(L) using shRNA NM_808 increases the -catenin levels. We independently tested the activity of Wnt/-catenin signaling in the MCF10A-MRJ(L) knockdown cells using M50-TOPFlash reporter assays. We saw a significant and reproducible activation of the reporter in the MRJ(L) knockdown cells, indicative of active Wnt/catenin signaling (Fig. 8A). We tested the effect of MRJ(L) knockdown on invasion and motility. We saw a significant (p Ͻ 0.05) 1.5 times increase in number of cells migrated. The invasion ability of the knockdown cells was doubled. These observations indicated a shift toward a malignant phenotype. To test if the MCF10A cells, which show a mostly epithelial phenotype, will show a shift toward a mesenchymal phenotype, we evaluated the changes in transcript levels of some key epithelial markers upon MRJ(L) knockdown from MCF10A cells. As seen in Fig. 8C, we observed that the shRNA knockdown of MRJ(L) was accompanied by a concomitant reduction in DKK1 transcript and down-regulation of CDH1 (E-cadherin) transcript indicating loss of epithelial phenotype. c-Myc is a key oncogenic target of the Wnt/-catenin signaling. We observed that MRJ(L) knockdown increased c-Myc transcript in MCF10A by 2.5-fold, suggesting activation of Wnt/-catenin signaling. However, we did not see any change in the levels of KRT18 (keratin 18), an epithelial marker that gets up-regulated at protein levels upon MRJ(L) overexpression.
DISCUSSION
Heat shock proteins (HSPs) are a group of proteins whose expression is increased when cells are exposed to stress; however, HSPs are also present in cells under normal conditions (26 -28). They are popularly called "chaperones" and are implicated in protein folding and destruction. However, they also have roles in immunological processes, cell cycle regulation, transcriptional activation, and signal transduction (29 -31). Heat shock proteins are overexpressed in a wide range of human cancers, conferring resistance to cytotoxic therapies (29,31). HSP90 and HSP70 are widely studied for their involvement in molecular signaling events that have direct bearing on understanding cancer biology (32)(33)(34)(35)(36). However, the role of HSP40 family members is only recently described (2, 3, 37-42). DNAJB6 is an HSP40 family member whose involvement in cancer has been recently realized (2, 7, 12). Expression of this protein is lost in aggressive breast cancer. The large spliced variant of DNAJB6, MRJ(L) shows reduced expression in aggressive cancer cells. Restoration of MRJ(L) in aggressive breast cancer cells reduces tumorigenicity and metastasis (7). However, the signaling pathways impacted by MRJ(L) were not known.
We observed that MRJ(L) expressors have a distinct epithelial-like appearance and increased expression of filopodia on the surface (Fig. 1). Recent studies have indicated that distinctive filopodia differing in length, positioning, and dynamics are seen in different cells (43). Filopodia can act as sensors of the environment and change to lamellipodia when the cell decides to migrate (44). Besides their role in cell migration, filopodia also have been reported to play a role in formation of adherens junctions between epithelial cells (45). MRJ(L)-expressing cells show numerous filopodia indicative of epithelial transformation that may be a prerequisite for the cells to search for an adjacent cell with extended filopodia leading to interdigitation of filopodia and formation of adherens junctions. Based on known phenotypes, MCF7 is more epithelial-like and MCF10CA1d.cl.1 is much more mesenchyme-like than MCF7, but MDA-MB-435 is the most mesenchyme-like. At the molecular level, we observed that protein levels of mesenchymal markers like CTNNB1 (-catenin), Vim (vimentin), CDH2 (N-cadherin), Twist1, and Slug (SNAI2) were down-regulated in MRJ(L) expressors, whereas the epithelial marker, KRT18 (keratin 18) was up-regulated (Fig. 2). We did not see any noticeable changes in E-cadherin levels (tested in MCF7 and MCF10CA1d.cl.1), but interestingly, MCF10A cells did show a loss of E-cadherin transcript (CDH1) upon MRJ(L) knockdown (Fig. 8). N-cadherin expression has been reported to increase motility of breast cancer cells regardless of their E-cadherin status (46). Vimentin is an important cytoskeletal protein that controls the formation of focal adhesions for efficient migration and invasion (47,48). Buhler and Schaller (49) have shown that forced expression of keratin 18 in aggressive breast cancer cells reduces vimentin expression, alters cell shape, and reduces invasion. Thus, down-regulation of vimentin, N-cadherin levels, and up-regulation of keratin 18 in MRJ(L) cells suggests a shift toward a less malignant phenotype. Taken together, MRJ(L) is capable of down-regulating mesenchymal markers and upregulating the expression of epithelial marker keratin 18. MRJ(L) overexpression led to increased expression of keratin 18 consistently in three different cell lines as observed in Fig. 2. However, this change may be independent of DKK1; when we silenced DKK1 from MDA-MB-435-MRJ(L) cells, we did not see an effect on keratin 18 (data not shown). This could mean that keratin 18 up-regulation by MRJ(L) may be independent of the suppression of the Wnt/-catenin pathway. Also, we did not see keratin 18 transcript levels down concomitant with MRJ(L) knockdown from MCF10A (Fig. 8C). We think that keratin 18, an epithelial marker, may be under multiple checks and controls, and merely silencing MRJ(L) may not be changing its levels. miR-200 family and Zeb1/2 have a critical role in regulating EMT through Slug and Twist levels (50 -53). To see if these signaling pathways affecting mesenchymal phenotype may also be altered by MRJ(L), we tested miR-200 and Zeb1/2 levels in MDA-MB-435-MRJ(L) and found a 10-fold up-regulation of miR-200b and a several fold (C t value Ͼ40, practically not detected) reduction in Zeb2 levels (supplemental Fig. 4). Zeb1 levels were unaltered. Effectively, expression of MRJ(L) appears to be necessary, in part, to maintain the epithelial phenotype. It is tempting to speculate that loss of MRJ(L) may be one of the triggers in tipping cells toward the metastable phenotype (54,55).
EMT is one of the important series of molecular changes in cancer progression, which may affect prognosis of cancers such as breast cancer (56 -58). Several signaling pathways have been implicated to play a role in EMT of cancer cells, and Wnt/catenin is one of the important ones (59,60). Of all the EMTrelated signaling changes that accompanied MRJ(L) overexpression, reduced levels of -catenin protein were most consistent across the different cell types examined by us. This reduction led to significant inhibition of the Wnt/-catenin signaling in the MRJ(L) expressors (Fig. 4). -Catenin is a major player in the Wnt signaling pathway, as well as an important was transiently transfected using NM_808 cloned in pSuperior.neo ϩ GFP. Total protein (35 g) was immunoblotted and analyzed for the levels of MRJ(L) and -catenin. -Tubulin was used to verify equal loading. Activity of TCF/Lef reporter, TOPFlash (50 g), evaluated in MCF10A was transiently transfected using NM_808 compared with their corresponding scrambled shRNA controls. The experiment was performed in triplicate and repeated two times. The error bars represent means Ϯ S.E. Statistical significance was determined if it reached 95% confidence. * indicates p Ͻ 0.05. B, knockdown of MRJ(L) from MCF10A shows increased malignant activity as measured by invasion through Matrigel TM and migration using modified Boyden chamber assay. The experiments were performed in triplicate and repeated one time. The error bars represent means Ϯ S.E. Statistical significance was determined if it reached 95% confidence. * indicates p Ͻ 0.05. C, quantitative RT-PCR analysis was performed to measure transcript levels of epithelial markers E-cadherin and keratin 18 using RNA from MCF10A transiently transfected using NM_808 and compared with the corresponding scrambled (scr) shRNA control. The knockdown of MRJ(L) and simultaneous down-regulation of DKK1 was also monitored as a control. c-Myc levels were evaluated as an indicator of Wnt/-catenin pathway activity. Analysis was performed on the experimental mRNAs in triplicate, and the experiment was repeated once. Statistical significance was determined if it reached 95% confidence. E-cadherin, MRJ(L) and DKK1 had significant changes compared with the scrambled shRNA control p Ͻ 0.05. regulator of EMT in cancer cells and possibly in maintaining a metastable phenotype (54,61,62). It is important to note that -catenin is a molecule that has multiple functions based on its location and partners. At the cell membrane, it binds to ␣-catenin and E-cadherin contributing to the formation of cadherincatenin junctions. These junctions are contributors to cell polarity and the epithelial phenotype, whereas nuclear -catenin binds to Tcf/Lef transcription factors and promotes transcription of target genes that among other phenotypes are responsible for regulating mesenchymal markers (63). Our observations indicate that MRJ(L) expression promotes the degradation of -catenin in a proteasome-dependent manner (Fig. 3). The known pathways that drive the degradation of -catenin are initiated by inhibition of Wnt ligands or their receptors by their inhibitors like sFRPs, DKKs, etc. (64,65). We observed that breast cancer cells show relatively lower mRNA levels of DKK1 as compared with nontumorigenic MCF10A cells (Fig. 5A); moreover, the expression profiles of DKK1 paralleled that of MRJ(L) (supplemental Fig. 3). Analysis of MRJ(L) overexpressors showed increased levels of secreted DKK1, due to the transcriptional up-regulation of DKK1 (Fig. 5C), which possibly caused reduced -catenin protein levels (Fig. 5B). Knockdown of up-regulated endogenous DKK1 by treatment of 435-MRJ(L) with DKK1-siRNA restored -catenin levels in MRJ(L). This confirmed the role of MRJ(L) up-regulated DKK1 in degrading -catenin (Fig. 6). Enhancement in motility, increased foci formation, as well as increased soft agar colonization were seen in DKK1-silenced MDA-MB-435 cells indicating that the MRJ(L) up-regulated DKK1 contributed to the reduction of malignant activity. Importance of MRJ(L) and DKK1 is also evident in Fig. 8B and supplemental Fig. 5 as silencing the expression of either of these genes leads to increased invasion and motility in MCF10A cells. DKK1 loss has been correlated with aggressive breast cancer and is reported to be frequently inactivated by epigenetic inactivation (66). DKK1 is implicated as a player in inhibiting malignant behavior of breast cancer (67)(68)(69)(70)(71). Stable expression of DKK1 in breast cancer cells has been reported to increase expression of keratin 18 levels concomitantly with a decrease in -catenin levels (72). Recently, interesting studies by DiMeo et al. (70) showed that silencing LRP6, a Wnt co-receptor that is blocked by DKK1, reduced the capacity of cancer cells to selfrenew and seed tumors in vivo and also resulted in the re-expression of breast epithelial differentiation markers and repression of EMT transcription factors Slug and Twist.
The Wnt signaling pathway has been reported to be associated with the invasive ductal carcinoma of the breast (73). Stabilized -catenin induces hyperplasia and mammary tumors in mice (74). Activation of Wnt/-catenin signaling components in mammary epithelium induces trans-differentiation (75). In melanoma as well, Wnt signaling has been shown to be involved in inhibition of metastasis suppressors and activation of EMT (76). Our observations demonstrate a role for MRJ(L) in up-regulating DKK1, leading to inhibition of the Wnt/-catenin signaling pathway. This inhibition could be one of the ways by which MRJ(L) reduces tumorigenicity and metastasis of aggressive cancer cells.
|
2018-04-03T05:10:57.210Z
|
2010-06-03T00:00:00.000
|
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"year": 2010,
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"Biology",
"Medicine"
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|
201838888
|
pes2o/s2orc
|
v3-fos-license
|
Dizziness and its association with walking speed and falls efficacy among older men and women in an urban population
Background Dizziness is common among older people and falling is a feared complication. Aim The purpose of this study was to investigate the presence of dizziness and its association with falls, walking speed and fear of falling, including sex differences, among 79-year-olds. Secondary purposes were to describe the relationship between dizziness and falls to number of medications and diseases. Method The study consisted of the fifth cohort of Gothenburg’s H70 birth cohort studies. A sample of 662 79-year-olds (404 women, 258 men) were investigated with questions regarding dizziness, previous falls and falls efficacy [estimated according to the falls efficacy scale Swedish version (FES (S))]. Functional tests included self-selected and maximal walking speed over 20 m. Results Dizziness was reported among 51% of the women and by 58% of the men (p = 0.12). Approximately, 40% had fallen during the past 12 months (41% women, 38% of the men, p = 0.48). Dizziness was related to a higher risk of falls among women (OR 2.63 (95% CI 1.67−4.14, p < 0.0001), but not among men (OR 1.07, 95% CI 0.63−1.82, p = 0.8). Dizzy individuals had lower scores on FES (S) (p < 0.01), more medications (p < 0.001) and diseases (p < 0.001) than those without dizziness. Participants who reported dizziness walked 10% slower than participants without dizziness (p < 0.001). Conclusion Women with dizziness more often reported falls compared to women without dizziness—a trend that was not seen among men. Persons with dizziness walked slower. Many medications increased risk of falling; hence, number of medications alone might help pinpoint risk groups for falling.
Introduction
Dizziness and imbalance are common complaints among older people and increase with advancing age [1]. Dizziness is often reported to be more common among women than among men with approximately 40% of women and 30% of men at age 75 suffering from dizziness or unsteadiness of gait [2,3]. In fact, as many as 45% of older people living at home report having dizziness [4].
Dizziness among older people can have several etiological factors [5] and is sometimes viewed as a geriatric syndrome: a condition caused by degeneration of multiple functions (vestibular, proprioceptal, visual), also referred to as age-dependent dizziness [6,7]. However, dizziness might not strictly be age-dependent but rather considered a complex geriatric condition, also associated with depressive symptoms and fatigue [4]. Dizziness and impaired 1 3 balance have been shown to be more common among those simultaneously suffering from chronic diseases [8]. Other known risk factors for dizziness include locomotor disorders, knee or hip problems, impaired vision, number of medications, benign paroxysmal positional vertigo (BPPV) or vestibular hypofunction [6,[8][9][10]. Dizzy older people tend to be less active than the non-dizzy [8,11,12], potentially due to dizziness and poor balance increasing risk of falling [13]. In Sweden, over 1700 older adults die each year due to fall-related accidents, where stumbling or hitting an obstacle is among the most common causes of falling, followed by dizziness and imbalance [13,14]. Falls and fall-related injuries cost large amounts annually-a figure that in 2012 reached 25 billion Swedish crowns (SEK) [14]. Women tend to fall and seek medical care due to falls more often than men, but fall-related mortality is higher among men [14]. Reasons for this gender difference are not known nor why women, at least in young ages, more often report dizziness compared to men.
Being dizzy can affect walking speed, which is a potential tool when evaluating health, fitness and mobility. Normal function of gait gives a valuable illustration of multi-systemic function and general wellbeing [15,16], and low walking speed may predict morbidity and mortality [15][16][17][18].
Fear of falling and low fall-related self-efficacy have been shown to influence quality of life in a negative way [19] and to be associated with activity restriction [12], reduced social activity and a high occurrence of depressive disorders, especially among women [20][21][22]. Fear of falling has also been associated with frailty among older adults [22]; however, the association between fear of falling and walking speed is not well documented, nor is walking speed in relation to dizziness and falls.
Many medications can cause dizziness and exposure to polypharmacy has been associated with dizziness [3] as well as risk of falling [3,23]. The use of medications has increased remarkably during the past 20 years, where the number of medications taken has increased 100% among older adults [24]. There are some gender differences in drug prescription where women tend to use more medications and also being prescribed inappropriate drugs-like medications with anticholinergic drugs and benzodiazepines with can cause dizziness among older adults [25]. Increased prevalence of medications use also enhances the risks of polypharmacy, side effects and inter-drug interactions.
As the association between dizziness, walking speed and falls is not well documented in larger cohorts of older adults, this study aims to investigate the presence of dizziness, including sex differences, and its association with falls, walking speed and fear of falling among 79-year-olds. Additional aims include analyzing the relationship between dizziness, falls to number of medications and diseases.
Methods
The study comprised the fifth cohort in the Gothenburg H70 birth cohort studies, Sweden and was conducted in 2009. The study participants were selected according to specific birth dates from an urban population born in 1930. Information regarding date of birth and address was obtained from the Swedish national registration register covering all persons registered in Sweden. Invitation for the study was first sent by letter including consent form and the participants were then contacted by telephone. Participants were considered eligible for the study regardless of the place of living (privet household, sheltered living, etc.) A total of 1063 (659 women, 404 men), all aged 79, were invited and 662 (404 women and 258 men, response rate 62%) agreed to participate in the multidisciplinary study. Reasons for not choosing to participate could not be registered due to lack of consent. Measurements were performed by trained nurses and physiotherapists at the neuropsychiatric outpatient clinic at the Sahlgrenska University Hospital. The design, procedures and methods for data collection have been reported elsewhere [26].
Study-specific questions
The participants answered the question regarding dizziness: "Do you have any problems with dizziness, unsteadiness or impaired balance?" (yes/no) which divided the participants into groups of having dizziness/imbalance (answering "yes") and no dizziness imbalance (answering "no"). Specific questions about dizziness were then posed only to the participants reporting dizziness, i.e., about occurrence and duration of dizziness, as well as dizziness according to different positionings/movements. The response alternatives were yes/no. The questions concerning falls were "Have you fallen during the last year? (yes/no)", "How many times have you fallen?" and "If you have fallen, did you get any injury? (no/fracture/soft tissue damage/other)".
Walking speed
Average walking speed among 70-year-olds is 1.10−1.25 m/s and walking speed < 1 m/s is considered a risk of poorer health [27,28]. Walking speed is a highly valid and reliable test [16]. Walking speed was tested over a distance of 20 m. The time consumed for the distance was measured twice: at selfselected/normal walking and when walking as fast as possible (maximal walking speed).
Falls efficacy
Fear of falling was evaluated using The falls efficacy scale Swedish version (FES (S)). The falls efficacy scale, originally developed by Tinetti et al. [29], measures selfperceived confidence in task performance in common activities of daily living without falling. The Swedish version, (FES (S)) [30], is a modification of the original FES which includes 13 items. The respondents rated their confidence on a visual 0−10-point scale (0 = not confident at all and 10 = completely confident). The scale includes three parts: six items measuring personal activities of daily living (ADLs), one question regarding walking up and down stairs (question number 7) and six items measuring instrumental activities of daily life (iADLs). A higher score (of a maximum of 130 points) indicates better confidence/self-efficacy in performing the different activities without falling.
Comorbidity and intake of medication
Regarding diseases, the participants were systematically asked about current conditions and diseases; "Have you been told by a medical doctor or nurse that you have or have had, any of the following diseases or disorders?" and were asked according to a list of conditions including diabetes, high blood pressure, chronic bronchitis, asthma, angina pectoris, cardiac infarction, intermittent claudication, heart failure, sciatic/backpain, stroke, vision and hearing impairment, joint problems. Additionally, participants were asked to list their medications.
Statistical analyses
The software used for the statistical analyses was a statistical program package developed at the Department of Geriatrics at Gothenburg University (GIDSS for Windows). Differences in dichotomized variables were analyzed with logistic regression or Fischer's exact test (difference of occurrence of dizziness and falls for men and women). Significance was always reported for two-tailed tests, and the significance level used was 5%. Means were compared between groups using t test. Linear regression was used for analyzing association between the number of medication and walking speed.
Results
In this cohort comprising 662 (404 women, 258 men) mainly home dwelling 79-year-olds, dizziness and/ or impaired balance was reported by more than half of the individuals with no significant sex differences (51% women, 58% men, p = 0.12). In the majority of the participants (82%), symptoms of dizziness had been present for more than 6 months. Approximately, one-third of the dizzy participants had daily problems with dizziness and more than one quarter (29% of the women and 26% of the men) reported that dizziness had an impact on their daily activities. Dizziness lasting for only seconds was the most commonly reported duration of dizziness and was mainly triggered by rising up from supine position followed by positional changes (Table 1).
Walking speed
Both women and men with dizziness/imbalance had a lower walking speed compared to those without dizziness at both self-selected and maximal speed ( Table 2). Lower normal walking speed was also seen among men with previous falls (mean 1.11 m/s standard deviation (SD) 0.18 without falls compared to 1.05 m/s SD 0.19 if previously fallen, p = 0.05). This observation was not seen among women (mean 1.08 m/s SD 0.18 without falls compared to 1.07 m/s SD 0.20 if previously fallen p = 0.63).
Falls efficacy
Individuals with dizziness had lower total scores on FES (S) than non-dizzy individuals; mean 112 (SD 26) compared to 120 (SD 20) for women (p < 0.01), and 111 (SD 25) compared to 122 (SD 16) for men (p < 0.001), indicating less fall-related self-efficacy and higher fear of falling in task performing for dizzy individuals. For women with prior falls, the mean score was 110 (SD 30) compared to 118 (SD 19) among those without prior falls (p < 0.01). For men, the corresponding figures were 111 (SD 29) compared to 118 (SD 19), (p < 0.01). Both men and women showed relatively high overall self-confidence in performing the different tasks. Nevertheless, the proportion of individuals with maximum scores (128−130p) differed among those with and without dizziness/imbalance (43% compared to 65%, p < 0.01). The proportion of individuals with lowest scores (< 112p) also differed among those with and without dizziness/imbalance (34% compared to 14%, p < 0.01). The question "Do you have any problem with dizziness, unsteadiness or impaired balance" was used to categorize participants as having dizziness or not. Only participants answering "yes" to the first questions were asked the following questions about specific symptoms of dizziness a Total number of participants answering the question
Comorbidity and intake of medication
Almost half of the individuals used five or more medications daily (49% of women and 43% of men, p = 0.23). Number of medications was categorized into four classes 0, 1−4, 5−9 and 10 and more. We found an association between number of medications and dizziness as well as between number of medication and falls ( Among those taking 10 or more medications, more than half of the persons had fallen and 10% of the women and 28% of the men had fallen three times or more during the past year. There was a linear trend between number of medications and lower walking speed (normal and maximal speed,) among both men and women (p < 0.0001).
More than half of the study participants (59%) suffered from high blood pressure, which was the most common condition and more common among men with dizziness than among men without dizziness. Joint problems and vision impairment were more commonly found in both genders among persons reporting dizziness compared to those without (Table 4). Heart failure and self-reported hearing impairment were more common among women with dizziness than women without dizziness. No gender differences were seen regarding number of diseases, mean 3.6 (SD 2.25) for men and 3.5 (SD 2.25) for women, p = 0.5. People with dizziness suffered from a higher number of diseases; mean 4.0 (SD 2.38) compared to 3.0 (SD 2.0) for those without dizziness, p < 0.001. The odds ratio of having dizziness was 1.30 (95% CI 1.17−1.43, p < 0.001) for every added disease. Corresponding figure for having experienced falls was 1.15 (95% CI 1.04−1.28, p < 0.01) for every added disease.
Discussion
Over 50% of 79-year-old men and women reported dizziness and/or impaired balance, with no gender differences. Persons with dizziness walked slower, and women tended to report more falls than men. At age 75, when the cohort was previously investigated, only 30% of the men and 40% of the women reported problems with dizziness [12], where the increase especially among the men at the age of 79 is a bit hard to explain, but could be due to the higher age of our participants. An explanation why dizziness has increased more among the men could be that the men suffered from a higher burden of disease compared to women in this age. However, the number of diagnosed diseases or medications taken was not higher among the men. Men are generally considered healthier and perform better in physical tests, but still mortality rate is higher in males compared to females especially in high ages, and life expectancy is higher among women. Oksuzyan et al. [31] proposed the theory that women go from "healthy" to "unhealthy" in younger ages than men, but tend to live longer as "unhealthy". Possibly, this shift might be in the age around 79, targeting the life expectancy of 80 among Swedish men [32].
However, comorbidity and disease burden may be more complex than just diagnoses, with great variations and severity of the conditions creating an impact on the individual not quantified in this study. Tamber et al. [3] has shown that associations between number of medications, diseases and dizziness are at the same level among young and elderly persons in multivariate analyses, when controlling for dizziness [14]. This supports the theory of equal comorbidity between men and woman in this study. Dizziness is considered more common among women and female predominance is reported in almost every survey investigating dizziness. This study reflects the frequency of dizziness/unsteadiness in a general older adult population and not merely patients seeking medical care due to symptoms of dizziness. Physicians meeting older people should be aware of the fact that dizziness is equally common among both genders in higher ages and ask for symptoms of dizziness, even if this is not the main reason for the consultation, as dizziness and impaired balance may have impact on overall wellbeing and quality of life.
From a clinical standpoint, the most commonly reported duration of dizziness was seconds and the most common triggers of dizziness symptoms (after rising from supine to sitting) were positional changes and head movements like turning or tilting the head backwards (Table 1). These symptoms are often prominent in benign paroxysmal positional vertigo (BPPV), which is common among older people and may create a sense of unsteadiness [10]. Since BPPV is a cause of dizziness that can be cured if treated, physicians should actively search for BPPV among older people reporting dizziness by positional changes [33].
Approximately, 40% of the participants had experienced at least one fall during the past year which is in line with previous reports regarding falls [34]. Women experiencing dizziness reported more falls compared to non-dizzy women, but this trend was not observed among men. One possible explanation may be the reported comorbidity, where dizzy females to a greater extent, when compared to non-dizzy females, reported problems with locomotion (i.e., joint problems) proprioceptal and visual impairment-two feedback systems important for maintaining and controlling balance ( Table 4). This reduction in locomotion and vision may lead to women being more prone to falling. These comorbidities were not present to the same extent among men, possibly explaining the discrepancy observed in terms of fall tendency.
A low walking speed can be a sign of morbidity and poor physical performance [15] and dizzy participants showed a reduction of walking speed by more than 10%. There was a clear relation between a low walking speed and an increasing number of medications, possibly due to multiple comorbidities that physically limited walking. However, the lower walking speed in persons with dizziness/imbalance could also be caused by a voluntary restriction of speed, stemming from fear of movement to reduce the risk of impaired balance function and falls [12]. Nevertheless, experiencing a previous fall was only moderately associated with decreased walking speed among men. A single fall, if for example induced by slipping or stumbling, may possibly be externalized by the patient, deemed out of their control and independent from their health status, thereby not associating it with a fear of risking repetitive falls.
Both men and women with dizziness had higher fear of falling measured with FES (S). Many of the subscales reached ceiling effects, thereby making it difficult to capture true variance to monitor fall-related risks since the balance confidence overall is high even in this high age group. Instability of gait, impaired fall-related fall efficacy and fear of falling is still not well understood. Herman et al. argue that gait variability as a measure of instability is connected with fear of falling rather than muscle strength and previous falls [35]. Even if lower fall-related efficacy indicating fear of falling was found among persons with dizziness/imbalance and those with previous falls, it is doubtful how valuable the (FES (S)) is as a tool to measure balance confidence or to capture individuals with risk for falls among older adults in general.
Number of medications as a single measure has prior been shown to predict mortality and unplanned hospitalization, especially among men [36,37]. We found that dizziness as well as falls was associated with increasing number of medications and conditions, which might support the theory that dizziness, as a symptom, is a part of comorbidity and fragility but also that number of medications alone is associated with falls. Polypharmacy itself also may enhance the risk for adverse drug interaction, potentially causing dizziness and fall tendency which is why the reduction of medication is always desirable. The number of medications alone might, therefore, help pinpoint risk groups needing investigation for physical functioning as well as balance enhancing treatment for fall avoidance.
Limitations
The dual nature of asking about both dizziness and imbalance in the same question poses some limitations. Due to the original design of the longitudinally cross-sectional study, opportunities to change questions does not exist but should be kept in mind for future studies.
More participants completed the questions than those who underwent the walking tests, and not all filled out the Falls Efficacy Scale (S) nor all the questions regarding dizziness. Concerning questions about falls, it may be difficult to recall over a period of one year, thus leading to recall bias. The study has a cross-sectional design making direction of causality difficult. Finally, more women than men were invited in this population-based sample and, therefore, more women than men participated in the survey, which might affect the study's external validity and reduce the overall generalizability. The selection for participating in the study was based on specific birth dates from an urban population.
Conclusion
Dizziness at age 79 was reported among more than half of the participants with no gender differences. Women with dizziness more often reported falls compared to women without dizziness-a trend that was not seen among men. Persons with dizziness/imbalance walked slower than those without. Dizziness was associated with increasing number of medications and increased comorbidity. Many medications increased the risk of falling as well as being associated with decreased walking speed; hence, number of medications alone might help pinpoint risk groups for falling. Acknowledgement Open access funding provided by University of Gothenburg. The authors thank Valter Sundh for statistical support.
Author contributions Ellen Lindell, main author, responsible for design of the work and analyzing the data and writing of the manuscript. Caterina Finizia, responsible for design of the work, interpretation of the data, drafting and critically revising the work including statistical analyses. Therese Karlsson, writing author, responsible for design of the work, interpretation of the data and critically revising the manuscript. Mia Johansson, writing author, responsible for design of the work and critically revising the manuscript. Lena Kollén, writing author, responsible analyzing and interpretation of the data and critically revising the manuscript. Lina Rydén, responsible for study setup, data collection and analyzing the data and critically revising the manuscript. Anna Zettergren, responsible for study setup, data collection and analyzing the data and critically revising the manuscript. Kerstin Frändin, responsible for study setup, data collection and analyzing the data and critically revising the manuscript. Ingmar Skoog, responsible for study setup, data collection and analyzing the data and critically revising the manuscript. All authors have approved the final version of the manuscript to be published.
Compliance with ethical standards
Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest.
Ethical approval
The study was conducted according to the Declaration of Helsinki and was approved by the Regional Ethic Review Board in Gothenburg, Sweden.
Statement of human and animal rights
This article does not contain any studies with animals performed by any of the authors.
Informed consent All participants gave their informed consent before inclusion in the study.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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2019-09-06T14:41:29.431Z
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2019-09-05T00:00:00.000
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The Effectiveness of Rehabilitation after Open Surgical Release for Trigger Finger: A Prospective, Randomized, Controlled Study
Background: It is not clear whether rehabilitation after surgery for trigger finger is effective. The aim of this study was to reveal its effectiveness for trigger finger. Methods: This study was a randomized, controlled trial that included patients who underwent operations for trigger fingers. The patients in the rehabilitation group had postoperative occupational therapy (OT) for 3 months, while the patients in the control group were not referred for rehabilitation but received advice for a range of motion exercises. We evaluated the severity of trigger finger, Disability of Arm-Shoulder-Hand (DASH) score, pain-visual analogue scale (VAS), grip strength, whether they gained a full range of motion (ROM), and complications before and after surgery. Results: Finally, 29 and 28 patients were included in the control and rehabilitation groups, respectively. At final follow-up, the DASH score, grip strength, and ROM were significantly improved in the rehabilitation group compared to that preoperatively. At final follow-up, pain was significantly improved in both groups from that preoperatively. There were no significant differences in the results, including the DASH score, grip strength, ROM and pain-VAS between the control and rehabilitation groups at the final follow-up. Subgroup analysis showed that there is a significant difference in the DASH score of patients doing housework or light work and those with a duration of symptoms >12 months between the control and rehabilitation groups at the final follow-up.
Introduction
Trigger finger is one of the most common conditions treated by hand surgeons.The prevalence in the general population was reported to be 3% [1].Several management approaches have been reported for the treatment of trigger finger [2,3].Corticosteroid injection is a common management strategy in the initial treatment of symptomatic trigger digits.It was reported that 40-80% of patients had a resolution of symptoms following corticosteroid injection [4].If conservative interventions or corticosteroid injections are unsuccessful, open surgery is generally conducted.Though success rates of open surgical release of the A1 pulley were reported to be around 90%, some patients suffer from postoperative adverse outcomes, including finger stiffness or pain [5,6].Everding et al. reported that 4.9% of patients had remaining pain, swelling, or stiffness and that 2.5% had residual contracture after open surgery [5].
Some studies revealed the effectiveness of rehabilitation after some kinds of surgeries, including the fixation of extremity fractures and joint replacement [7][8][9].Rehabilitation is also effective after hand surgeries [10][11][12].As for trigger finger, it was reported that patients with open surgical release and rehabilitation therapy achieved good results and a low rate of complications [13].However, this study is not a comparative study but a case series; thus, it includes many biases.Additionally, it is not clear which type of patients should undergo rehabilitation after open surgery.Therefore, we conducted a randomized controlled study Participants were assigned to the intervention or control group.An independent investigator (R.N.) made a computer-randomized list for each hospital to conceal the treatment allocation.The randomization list was stratified with a block size of four.Based on the lists, the investigator prepared sequentially numbered and sealed envelopes containing the assigned postoperative schedule.After the participants agreed to participation in this study and underwent surgery for trigger finger, the researcher opened the consecutive envelope to figure out the assigned schedule.Blinding of the participants was precluded because of referral to postoperative rehabilitation.
Treatments
All participants underwent open surgery under local anesthesia by one surgeon.After an incision was made at the level of the A1 pulley, blunt dissection was performed on the A1 pulley.A small dissection scissor was used to make an incision longitudinally and open the A1 pulley.Free movement of the flexor tendon was ensured without triggering.Finally, the skin was closed with interrupted 5-0 nylon sutures.The patients took non-steroidal anti-inflammatory drugs (NSAIDS) on demand after surgery.
Intervention
Participants in the rehabilitation group received a referral for postoperative occupational therapy (OT), such as stretching and an active and passive range of motion exercises within a week after surgery.They also received scar treatment, including friction massage and taping, and strengthening, including the use of grippers, weights, balls with different degrees of firmness, or other aides.
The exact type of exercise was left to the hand therapist's discretion in accordance with the condition of participants' hands and their preferences.Additionally, therapists gave appropriate individual advice on activities of daily living and lifestyle for each participant.
Participants in the control group were not referred for rehabilitation after surgery.They received only the orientation and advice for active and passive ROM exercises at distal interphalangeal, proximal interphalangeal, and metacarpophalangeal joints from the primary surgeon during the clinical phase, to be performed by themselves.They were explained that the heel of the hand on the healthy side presses on the back of the hand on the affected side to reach full flexion during passive ROM exercises and that passive extension is also performed using the hand on the healthy side.
Follow-Up
Participants were examined and interviewed before and 1, 3, and 6 months after surgery, and the primary time point was 6 months.The follow-up period was determined by referring to other RCTs of treatments for trigger finger [14,15].We evaluated subjective symptoms and physical findings, including tenderness at the A1 pulley, maximal grip strength, snapping phenomenon, and whether patients gained full range of motion (ROM) of the treated digit.The Disability of Arm-Shoulder-Hand (DASH) score, pain-visual analogue scale (VAS) of the treated digit, and complications were also examined.
Statistical Analysis
The primary endpoints were the effect of postoperative rehabilitation on hand function, such as DASH and grip power.An estimation of the sample size was performed to determine the number of patients who reached an alpha value of 0.05 and a power of 80%.This sample size was based on the assumed primary outcome (DASH score) differences in population means of 6.0, a within-group standard deviation of 8.0, and an approximately equal number of cases in each group.Finally, the calculated sample size with an anticipated loss to follow-up of 15% was 68 patients.
We performed an intention-to-treat analysis among participants.A participant in the rehabilitation group who converted to the no rehabilitation group was analyzed within the rehabilitation group.The independent investor (R.N.) performed a statistical analysis.Fisher's exact test and the Student t-test were used to compare dichotomous and continuous data, respectively.The group differences were analyzed by one-way ANOVA followed by Bonferroni post hoc testing.Statistical analyses were performed using R for Windows (Ver.4.0 accessed on 24 April 2020).The two-sided significance level was set at p < 0.05.
Patient Characteristics
Figure 1 shows the flow of the participants through the trial.Initially, we included 68 patients in this study.Thirty-four of them were randomized into the control and rehabilitation groups, respectively.Four patients in the control group and three patients in the rehabilitation group were lost to follow-up by the final examination.One patient in the control group and three patients in the rehabilitation group withdrew by the final examination.
Patient characteristics are shown in Table 1.There were no significant differences between the control and rehabilitation groups in relation to demographic variables, including gender, age, duration of symptoms, preoperative severity of the trigger finger, dominant hand affected, and affected digits.The preoperative severity was classified using the Quinnell grading [16].The prevalence of diabetes mellitus (DM) and restricted range of motion (ROM) also did not differ significantly between these two groups.DM was well-controlled by oral drugs without insulin treatment in both groups.Patient characteristics are shown in Table 1.There were no significant differences between the control and rehabilitation groups in relation to demographic variables, including gender, age, duration of symptoms, preoperative severity of the trigger finger, domi-
Effect of Postoperative Rehabilitation
Figure 2 shows the results for the DASH score, pain-VAS, grip strength, and range of motion in the control and rehabilitation groups.Improvements of the DASH score, grip strength, and ROM were noted significantly in the rehabilitation group at 6 months after surgery (DASH score; p < 0.001, grip strength; p = 0.004, restriction of ROM; p = 0.008).In both groups, the pain was significantly improved between that occurring pre atively and 6 months postoperatively.We summarized the differences between pre 6-month-postoperative outcomes in Table 2.The grip strength and DASH in the reh tation group were improved significantly when compared to those in the control g However, there were no statistically significant differences in terms of post-operative comes, including DASH scores, pain-VAS, grip strength, and ROM restriction, bet the control and rehabilitation groups (Supplementary Data, Table S1).
Subanalysis
Rehabilitation made the DASH score in patients performing housework or light In both groups, the pain was significantly improved between that occurring preoperatively and 6 months postoperatively.We summarized the differences between pre-and 6-month-postoperative outcomes in Table 2.The grip strength and DASH in the rehabilitation group were improved significantly when compared to those in the control group.However, there were no statistically significant differences in terms of post-operative outcomes, including DASH scores, pain-VAS, grip strength, and ROM restriction, between the control and rehabilitation groups (Supplementary Data, Table S1).
Subanalysis
Rehabilitation made the DASH score in patients performing housework or light work improve significantly 6 months postoperatively (Table 3, 14.2 vs. 6.1, p = 0.04).In the patients with a duration of symptoms > 12 months, the postoperative DASH score of the rehabilitation group was also significantly better than that of the control group (Table 4, 19.6 vs. 0.7, p = 0.005).There are no statistically significant differences in the subanalysis of other factors, such as age, gender, and DM, between the rehabilitation and control groups.
Complications
One patient in the rehabilitation group had a superficial incision infection that was treated with debridement and an antibacterial drug.There were no other adverse events during this study.
Discussion
This study showed that the outcomes, including the DASH score, grip power, and restriction of ROM, at 6 months were only significantly improved after surgery in the rehabilitation group.Pain-VAS was significantly improved in both groups.However, there are no significant differences in the outcomes between the control and rehabilitation groups.In the patients performing housework or light work or the patients with a duration of symptoms over 12 months, there were significant differences in the DASH score 6 months after surgery between the two groups.
The effect of postoperative rehabilitation was revealed in some kinds of surgeries [8,11,17].In hip fracture patients, hospital rehabilitation was significantly related with a lower risk of mortality compared to no rehabilitation [8].It was also reported that rehabilitation improved mobility for veterans with major lower extremity amputation [17].As for hand function, rehabilitation after carpal tunnel release improves hand function one month after surgery and accelerates recovery [11].This study revealed that rehabilitation after surgery significantly improved PRO and hand function, as with other surgeries.
Early motion after upper limb surgery is effective.Many studies demonstrated benefits of early motion, including preventing restriction of ROM, faster healing, decreased disability time, and decreased risk of reflex sympathetic dystrophy [18][19][20].Additionally, early motion helps edema to decrease, while decreased edema also helps with increased ROM [21].A meta-analysis that evaluated the effect of rehabilitation following arthroscopic rotator cuff tear repair revealed that early passive motion results in superior ROM recovery [22].In this study, postoperative rehabilitation was also started within a week, and this early motion by occupational therapists might help improve functional and subjective outcomes.
There were no significant differences in the results, including the DASH score, pain, grip strength, and ROM, between the control and rehabilitation groups in this study.However, both groups could improve these results after surgery.These results imply that simple advice about range of motion exercises may be sufficient for patients after open surgical release.
Some studies have revealed the relationship between long-standing symptoms and postoperative outcomes in relation to various kinds of surgeries [23][24][25].Inderhaug et al. reported that long-standing symptoms (over 12 months) were identified as one of the predictors of inferior long-term outcome after rotator cuff repair [25].This study revealed that patients with long-standing symptoms (over 12 months) tended to show worse postoperative DASH scores.Among such patients, the patients with postoperative rehabilitation could show significantly improved DASH scores compared to the patients without rehabilitation.Degenerative thickening of the flexor tendons causes a persistent flexed flexion deformity of the PIP joint [26].Therefore, postoperative rehabilitation is more important and recommended for patients with long-standing symptoms.
This study also revealed that postoperative rehabilitation for trigger finger was effective for patients performing housework or light work, while there was no significant difference in the postoperative DASH score between those doing heavy manual work with rehabilitation and without it.The postoperative passive and active motion of fingers resulting from doing heavy manual work may act as an adequate range of motion exercises for patients with trigger finger.
DM has an influence on the development of trigger finger [27,28].It was reported that the incidence of trigger finger in patients with DM was about four times higher than in the general population [29] and that approximately 20% of patients with trigger finger had DM [30].This study included 15.8% of patients with DM, consistent with previous reports.As for the impact of DM on surgical outcomes, it was revealed that there were no differences in functional and subjective outcomes between diabetic and nondiabetic patients [31].This study also showed no differences between them.Additionally, rehabilitation had no influence on outcomes for patients with DM in this study.The conditions of all patients with DM were well controlled by oral drugs, which may have had an effect on the outcomes.
This study has some limitations.First, participants could not be blinded due to the nature of the rehabilitation intervention.However, we performed allocation concealment of the participants and minimized the bias in the randomization process.Second, we could not control participants' uncertainty about the use of their hands after surgery in daily life and pain medication because the patients could take NSAIDS on demand for pain control.We could also not perfectly standardize physiotherapy because the type of exercise was left to the different therapists' discretion in accordance with the condition of hands.These factors may have influenced the outcomes.Third, this was not a long-term follow-up study.However, it was revealed that there were no statistical differences in clinical outcomes, including VAS and Quick DASH, at 6 months and 12 months after percutaneous or open release for trigger finger [32].Additionally, several other studies on treatments for trigger finger also set the follow-up period to under 6 months, similarly to this study [14,[33][34][35].In terms of PRO, the DASH score was improved, but not significantly, in the control group.However, the DASH score might be unsuitable to evaluate such a limited hand pathology because the score evaluates the whole upper extremity.
Figure 1 .
Figure 1. and flow of participants through the trial.
Figure 1 .
Figure 1.Design and flow of participants through the trial.
JFigure 2 .
Figure 2. (a) DASH score, (b) pain-VAS, (c) grip power, and (d) proportion of ROM restr DASH score, The Disability of Arm-Shoulder-Hand score; VAS, visual analogue scale; ROM, of motion.The bar and error bar in the graphs show the mean and 95% confidence interva group differences were analyzed by one-way ANOVA followed by Bonferroni post hoc testi values < 0.05 were considered statistically significant.
Figure 2 .
Figure 2. (a) DASH score, (b) pain-VAS, (c) grip power, and (d) proportion of ROM restriction.DASH score, The Disability of Arm-Shoulder-Hand score; VAS, visual analogue scale; ROM, range of motion.The bar and error bar in the graphs show the mean and 95% confidence interval.The group differences were analyzed by one-way ANOVA followed by Bonferroni post hoc testing.p-values < 0.05 were considered statistically significant.
Table 3 .
Distribution of DASH scores categorized by type of occupation at 6 months after surgery.
Table 4 .
Distribution of DASH scores categorized by duration of symptoms at 6 months after surgery.
|
2023-10-27T13:06:36.418Z
|
2023-11-01T00:00:00.000
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133596576
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pes2o/s2orc
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v3-fos-license
|
A Numerical Solution Model for the Heat Transfer in Octagonal Billets
In the quest for high-quality steel products, the need of cast billets with minimum surface and internal defects is of paramount importance. On the other hand, productivity is required to be as high as possible in order to reduce production cost. Different billet shapes have been applied with emphasis upon square, rectangular, and circular cross-sections. It is obvious that the best billet shape that minimizes surface and subsurface defects is the circular one. Nevertheless, this shape creates some problems with respect to handling and safety reasons. One recent attempt is to produce normal octagonal-shaped billets that appear to approach the circular shape albeit easier to handle. In this study, a numerical solution for the heat transfer during solidification in the continuous casting of octagonal billets has been carried out. The developed model deploys an implicit scheme in order to solve the differential equations of heat transfer under the appropriate boundary conditions in a section of an octagonal billet, assuming fully axisymmetric cooling of the bloom. The geometry of the octagonal billet plays an interesting role in the development of the heat transfer analysis. Based upon fundamental principles, a computer program has been developed for this purpose. Consequently, results from the numerical solution are presented and discussed.
Introduction
The mathematical analysis of heat transfer in a billet during the continuous casting process has been investigated from the early stages of the development of continuous casting machines. In this study, some fundamental works related to the numerical solution of heat transfer in solidifying billets are presented. A pioneer work is mentioned in [1] in which a mathematical model of heat transfer in continuously cast steel slabs is described. A computer program was developed to model the problem using a 1D transient conduction equation with the appropriate boundary conditions. Some years later, the design of the mold and spray sections of a continuous casting machine were examined in detail with the aid of a 2D heat-flow mathematical model [2]. Experimental data obtained from commercial casters were used for the validation of the deduced results. In that monumental work, the various aspects of heat transfer in the various sections of a casting machine were analyzed in detail. Later on, surface and subsurface defects in the cast billets became apparent, and research work included mechanical phenomena (like stress and strain) together with the heat transfer analysis; an early typical work on the subject was presented in [3]. It is worth mentioning that the caster automation includes most of the heat transfer formulation and analysis, albeit not published. Consequently, the present literature survey mostly presents indicative published works upon heat transfer analysis. In the 1990s, the continuing casting of steel started maturing, and this was reflected by two studies with practical implementations [4,5]. In a review paper [6], the evolution of heat transfer and mechanical studies for the continuous casting were presented. Furthermore, an analysis on the ideal taper prediction for billet casting and a thorough analysis of thermomechanical behavior in billet casting were also presented [7,8]. The salient features of heat transfer in the secondary cooling zone during the continuous casting of steel were examined in [9]. Primary cooling is considered the start-up of steel solidification process in a water-cooled copper mold, and secondary cooling zone is considered the region just after the mold until the end of the caster in which the billet is cooled by spray nozzles or air-mist systems. Radiation heat exchange between the billet surface and the environment plays also an important role in the secondary cooling zone. Fluid-flow phenomena in the mold captured the interest, and as of that, heat transfer studies were also reported on the subject later on; a typical work is given in [10]. In most similar works, general-purpose heat-transfer software has been deployed either as a direct prediction tool or as a verification one. A typical work is given in [11] in which a 1D heat-transfer simulation program (CON1D) was successfully verified against a 3D finite element model developed in Abaqus. Near-netshape cast sections appeared in industrial practice in the early 2000, and therefore, their thermal-mechanical modeling during casting was developed as well [12]; in this type of analysis, the use of a package like Abaqus has been indispensable. In a relatively recent work [13], the 1D differential equation for heat transfer was solved in order to study the evolved microstructures during the solidification of round billets (so, the radial direction for heat transfer prevails). In the cases under study, the billet diameters were in the range from 210 up to 410 mm; the validation of their model was performed by deploying a semi-analytical model [14] for the prediction of the surface temperature of a cast round billet. The numerical model was used to calculate the local solidification time, the local gradient, and the local solidification time as a function of the distance from the round billet surface; furthermore, a simplified relation for the prediction of the columnar to equaxial transition was proposed. Deploying the finite volume method, a real-time 2D heat transfer and solidification model for continuous casting [15] were developed and tested online. The behavior was satisfactory with less than 10°C deviations on surface central temperatures. A dynamic heat transfer model was developed [16] in order to study the effect of the casting speed on the temperature field of continuously cast billets; various steel grades were taken under consideration. A real-time heat-transfer model and a heat-transfer coefficient identification method [17] were developed. The model validation was achieved by the measurement of surface temperatures by a CCD system that appeared to eliminate the impact of scales on the temperature measurement and keep the measured surface temperature fluctuation within the range of AE10°C. A finite volume method and the alternating direction implicit algorithm (ADI) were selected in order to develop a real-time heat transfer model [18] for the dynamic control of continuous cast billets. The system was applied online to control the electric current of the final electromagnetic stirring (FEMS) system. A simulation model of solidification and heat transfer of 150-mm-square billets was developed [19] for the continuous casting of grade 40 Cr. Nailing tests and temperature measurements were applied in order to fine-tune the model with maximum errors of less than 2%. In another study [20], different micro-segregation models coupled with fluid flow and heat transfer were run to study macrosegregation phenomena in a round billet with a 165-mm diameter. It was predicted that heavy centerline segregation occurs at the end of solidification when the central solid fraction reaches the value of 0.7. The need for high-speed casting under very controllable conditions has led the researchers to seek billet sections that can succeed in the demanding steelmaking environment. Recently, one of the leading manufacturers in the steelmaking sector, Danieli, has announced [21] the octagonal billet as a proven potential shape for successful high-speed casting. Consequently, the present author has taken the opportunity to study the heat transfer in a solidifying billet based on fundamental principles. The work has been carried out for a larger billet size than the one tested in [21] and for two special steel grades, the peritectic grade S355 J2 and the medium-high-grade 42CrMo4, that are both important for many end-user applications.
Formulation of the heat-transfer problem
The general three-dimensional (3D) heat-transfer equation that describes the temperature distribution inside a solidifying object is given by Eq. (1) according to [22] ρc The source term S may be considered [23] to be of the form: Furthermore, T is the temperature, and ρ, c, and k are the density, heat capacity, and thermal conductivity, respectively. The 2D heat-transfer equation in Cartesian coordinates can be written as [22] ρc The boundary conditions that apply in the octagonal billet case are very similar to the ones for the rectangular billet that have been presented in detail in [24] and will not be repeated here. Nevertheless, two important boundary conditions apply in the case under study, which follow: • Due to symmetry, the upper diagonal side ( Figure 1, segment OB) of the domain of interest is supposed to be adiabatic, as well as the lower side ( Figure 1, segment OK) is. In this way, the following formulation holds: ∂T ∂s ¼ 0, where s is the vertical axis on the line • The primary (mold) and secondary cooling systems are the ones applied in Stomana; this is presented in detail in a previous publication [24] and will not be repeated here. However, due to the potential of the octagonal mold, a surplus of water was used in favor of an enhanced cooling behavior for the solidifying billet; this was expressed as an extra percentage of water flow deployed for the octagonal billet.
Theoretical solution
There is no published work on the theoretical solution for the octagonal billet until now. However, a theoretical solution can be approximated by solving the DE of heat transfer on an equivalent circular geometry. According to Figure 1, the selected radius of that circle was R e = 156.6 mm, as given by Eq. (5): This radius is actually the mean value between the circumradius and in-radius of the octagon [25]. The circumradius R being equal to 162.84 mm makes the currently studied octagonal billet equivalent in size to a 250 Â 300 mm 2 billet produced in the Stomana plant, at Pernik, Bulgaria. Now the original problem can be converted into a heat-transfer problem of cylindrical geometry, and the DE together with the boundary conditions is formulated according to Eq. (6): By variable transformation using Eq. (7) the boundary condition at r = R e becomes: In order to fulfill the boundary condition at r = R e according to Eq. (8), Eq. (9) has to be solved for β: where J 0 and J 1 are the Bessel functions of zero and first order, respectively. This theoretical solution is presented in the book of Carslaw and Jaeger [26] and is followed here. There are an infinite number of values for β, which solve Eq. (9); nevertheless, the first six roots [26] are enough for the computations. In this way, the derived solution arrives in the form of Eq. (10): Eq. (10) was applied in order to compute the temperature distribution T(r,t) inside the equivalent to the octagonal geometry cylindrical billet; some results are depicted in Figure 2. In fact, the following parameters were used in these computations: thermal conductivity, k = 50 W/m/K; density, ρ = 7600 kg/m 3 ; heat capacity, c = 400 J/kg/K; thermal diffusivity, α = k/(ρc) = 1.645 Â 10 À5 m 2 /s; heat transfer coefficient, h = 200 W/m/K; cooling water temperature, T f = 30°C, and initial billet temperature, T 0 = 500°C. Figure 1 illustrates the important geometrical features of the octagonal-shaped billet. Considering that the flow of heat takes place only in the radial direction toward the center of the octagon, and certainly toward the center of its circumscribed circle, it appears that due to symmetry, only a small trigonal part becomes the important region (the shaded area in Figure 1) for the solution of the DE of heat transfer. Actually, when uniform cooling takes place around the solidifying billet, then symmetrical conditions prevail in the relevant heat transfer. Consequently, heat flow takes place only in the radial direction. The strongly implicit scheme as presented by Patankar [23] was deployed for the solution of the DE of heat transfer. Figure 3 illustrates the control-volume selection for a small number of nodal points.
Numerical solution
The five-point formulation for the discretization equations was applied. Special attention was paid in the boundary condition formulation of the discretization equations, and a more detailed analysis on this is presented in Appendix I. One may realize that once the boundary conditions are properly written, then a finer grid will ultimately permit the convergence to the engineering solution. In Figure 3, only the lower trigonal part is necessary to be examined. The vertical side, MZ, is the boundary from which the main heat flow (cooling) takes place; the horizontal side OZ and the diagonal side OM, with an inclination of π/8, or 22.5°, are considered to be adiabatic to heat flow due to the aforementioned symmetry reasons. This study is part of a series of published works with respect to the numerical solution of the heat transfer equation in 2D (square and rectangular) and 3D domains [24,[27][28][29]. Although the core of the existing program remained intact, due to the nature of the present trigonal domain, the part of the program related to the boundary conditions had to be developed from scratch. The octagonal billet with R = 162.84 mm can be discretized with a fine grid of 200 Â 200 nodal points. In this way, the space intervals (δx, δy) were about δx = R cos(π/8)/199 = 0.756 mm and δy = R sin(π/8)/199 = 0.313 mm, respectively. Actually, keeping the ratio, δy/ δx = 0.313/0.756 = 0.414 = tan(π/8), exhibited a relatively good convergence; the time interval was Δt = 1 sec.
Running the computer program
The Gauss-Seidel algorithm was applied for the iterative solution of the matrix equations in the case of the 42CrMo4 grade. Over-relaxation was used for the fastest possible convergence, and the over-relaxation parameter used was ω = 1.870, which has exhibited good behavior for this type of studies [30]. The applied maximum error (tolerance) for the computed temperature at each nodal point was 5 Â 10 À3 . In addition to this, the Strongly Implicit Procedure (SIP) [31] was deployed for the iterative solution of the matrix equations in the case of the S355 J2 grade. In this case, however, the same tolerance (5 Â 10 À3 ) was used for the maximum residual value and not the temperature difference at each nodal point.
Results and discussion
In order to validate the new developed program, a solution against the aforementioned theoretical one had to be sought. For this reason, an octagonal billet initially at 500°C, cooled afterwards by an hypothetical fluid at 30°C and with a heat transfer coefficient h = 200 W/m 2 /K, was simulated. The values for the properties of density, thermal conductivity, and heat capacity were the same with the ones used in the theoretical solution. In total, 38 sets of data were randomly selected at various positions inside the cylindrical billet and were compared with similar results deduced from the numerical solution described here in order to verify its validity. Figure 4 presents the compared results of these two sets of computed data. An analysis of variance (ANOVA) was performed for these two sets of results, and the following statistical results were derived: residual standard error, 3.133 on 36 degrees of freedom (DF); multiple-R squared, 0.9957; and F-statistic, 8333 on 1 and 35 DF, with a p-value <2.2 Â 10 À16 . Consequently, a standard error of 3.1°C was found to represent the mean statistical error between the results obtained from the theoretical and current numerical solutions, respectively.
The advantage of studying the heat transfer in an octagonal billet marks the importance of such an experiment: the potential to test some results against theoretical ones gives the confidence about attaining the proper numerical solution; in fact, the octagonal geometry approximates the circular cross-section better than a square one for this comparison to be accomplished. Table 1 presents the chemical analysis for the two selected grades under examination: 42CrMo4 and S355 J2. In the bloom caster of Stomana, in Pernik, the billet size of 250 Â 300 mm 2 is normally used for the production of special steels. The equivalent billet to this size in octagonal shape has a circumradius of R = 162.84 mm, as aforementioned. An interesting idea is that putting the octagonal billet in practice, there is a potential to increase productivity by using more cooling water, an advantage that has been tested in practice [21] for normal rebar grades. Consequently, in this study a surplus of mold water by 12% for both of the two grades 42CrMo4 and S355 J2 was used compared to the normal water-cooling process applied in Stomana for these grades, respectively. Furthermore, a surplus of water in the air-mist region by 3% for the high-carbon grade 42CrMo4 and 4% for the peritectic grade S355 J2 was applied, respectively. Figure 5 illustrates the shell thickness (curve 3) increase as an octagonal billet of grade 42CrMo4 travels down Stomana's continuous casting machine. One may notice that the solidification completes at about 32.4 m from the liquid-steel meniscus in the mold.
At the same time, the centerline temperature (curve 2) drops appreciably at that point revealing the complete solidification at that point, as well; the surface temperature is also presented by curve 1. The casting speed was 0.70 m/min, and the superheat (SPH) was 30°C in the computation. The SPH is actually the excess temperature above liquidus temperature; the liquidus temperature is exclusively a function of the chemical composition of a steel grade. steels normally solidify gradually from liquid to solid phase. The degree of solidification is generally described by a parameter that is called solid fraction (f s ) and represents the percentage of the solid phase in the mixture of solid and liquid phases. When f s = 0, then we have 100% liquid phase, and the steel temperature at this point is the liquidus temperature, T l . When f s = 1, then we have 100% solid phase, and the steel temperature at that point is the solidus temperature, T s . Although the liquidus temperature is always a function of the chemical composition of a steel grade, this is not the case with the solidus temperature. The solidus temperature is also affected by the local cooling rate at solidification (C R ), which is expressed as Eq. (11) shows that the temperature difference between a temperature T P and the previous one T P 0 at point P inside a solidifying billet within a time interval Δt, divided by this time interval, defines that local cooling rate. Consequently, the solidus temperature may be given by a formula of the type: On the other hand, the solid fraction may be considered a function of the local cooling rate and temperature: Therefore, during solidification of carbon steels, there is always a range between solidus and liquidus temperatures in which the solid fraction is in the range from 0 to 1; this physical range inside a solidifying steel body is called mushy zone, and the whole phenomenon is related to micro-segregation. The simple micro-segregation model [32] has been adopted in this work, and the analysis has been described in similar previous studies [24,29,33]. It appears that the larger the carbon content in a steel grade, the larger the mushy zone gets, and central porosity becomes inevitable in the final stages of solidification.
The local cooling rate distribution for the case of a 42CrMo4-grade at the caster position presented in Figure 6 is illustrated in Figure 7.
A short analysis showed that for the data presented in Figure 7, the C R (cooling rate in°C/s) has the following statistics: average value μ = 0.106, standard deviation σ = 0.128, minimum = 0.023, and maximum = 0.494. More than 99% of the C R population is within μ + 3*σ = 0.490. Figure 8 depicts the surface (1) and centerline (2) temperatures in an octagonal billet as the billet solidifies downstream the Stomana billet caster; the shell thickness (3) is presented as a function of the distance from the meniscus of liquid steel in the mold. It is worth mentioning that a S355 J2-billet cast at a speed of 0.85 m/min solidifies even faster than a similar 42CrMo4-grade billet at a lower speed. illustrates the temperature distribution in the critical geometrical region of a S355 J2-grade octagonal billet at a distance of 18.4 m from the meniscus, cast at a speed of 0.85 m/min and a SPH = 30°C.
Peritectic grades have the tendency to crystallize in a mixture of delta (δ) and gamma (γ) phases of iron in an intermediate temperature range, before continuing to a 100% γ-phase solidification. For this reason, the percentages of δand γ-phases are also presented in Figure 9 at the selected solid fractions. All these calculations come also from the simple micro-segregation model [32] that is adopted in the developed program. Figure 10 depicts the local cooling rates at the same position from the meniscus as for the aforementioned temperature distribution presented in Figure 9.
For the cooling rate distribution (C R in°C/s) presented in Figure 10, a short statistical analysis derived the following results: average value μ = 0.138, standard deviation σ = 0.156, minimum = 0.027, and maximum = 0.490. Almost 99% of the population is within μ + 3*σ = 0.606.
The minimum average number of iterations required for convergence by the Strongly Implicit Procedure (SIP) was attained at the value of 0.95 for the iteration parameter α as explained by Stone [31] in order to speed up the convergence process. Actually, this value (α = 0.95) was used in order to solve the derived system of matrix equations in the case of the S355 J2 grade. Figure 11 illustrates the effect of this iteration parameter on the ratio of the required average number of iterations to the minimum number of iterations required at α equal to 0.95. In general, the convergence behavior improved as the selected iteration parameter increased. It is worth noting that convergence succeeded within an extensive range of the iteration parameter although above the limiting value of α = 0.95, the solution procedure started to diverge. For verification purposes, two sets of temperature results were generated by the Gauss-Seidel and Strongly Implicit Procedure, respectively, taken after a long simulation time (at the same time instant t = 2460 sec, equivalent to a distance of 34.85 m from meniscus). Using R [34], a Pearson's product-moment correlation test gave a coefficient of 0.9999344 for these two sets of results with a t-Student value equal to 17,455, 39,998 degrees of freedom and a p-value < 2.2 Â 10 À16 .
Conclusions
The 2D differential equation of heat transfer was numerically solved for the derivation of the temperature distribution inside an octagonal billet. The deduced model was applied for the casting case of octagonal billets for an equivalent shape to the size of 250 Â 300 mm 2 billets that are normally cast at the Stomana billet caster. Furthermore, the simulated octagonal billets were considered to be from the two special grades of 42CrMo4 and S355 J2. Two different iterative methods were applied for the solution of the resulting system of equations, the Gauss-Seidel method and the strongly implicit procedure, both exhibiting satisfactory behavior. A surplus of cooling water especially at the mold may result in a potential productivity increase. Computational results from the simple micro-segregation model were also included in the present study.
A. Appendix I
The applied solution is based on the implicit scheme as described by Patankar in 1980 [23]. Figure 3 shows the control volumes and the nodal points upon which the numerical solution was based; in the graph, only a very rough discretization was considered with 6 Â 6 nodal points just for illustrative purposes. Normally, for nodal points in the interior of the geometry under investigation, the implicit scheme works on five adjacent points; in this way, for every internal point P, the four adjacent nodal points of interest are nominated as E (east), W (west), N (north), and S (south). The numerical discretization equation for the heat transfer equation at P is then given by Eq. (14) a P T P ¼ a E T E þ a W T W þ a N T N þ a S T S þ b P (14) where the temperature coefficients and the constant term are given by Eq. (15): , a W ¼ k w Δy δx ð Þ w , a N ¼ k n Δx δy ð Þ n , a S ¼ k s Δx δy ð Þ s , a P 0 ¼ ρc Δx Δy Δt , b P ¼ S C Δx Δy þ a P 0 T P 0 , a P ¼ a E þ a W þ a N þ a S þ a P 0 À S P Δx Δy The described formulation is proven to satisfy the energy balance within the control volume that is included by the sides "n," "e," "w," and "s." Extra care is required in the derivation of the discretization equations at the borders of the studied geometry. For example, Eq. (16) describes the formulation for the temperature of a typical point on the outer area of the octagonal billet that is cooled by the water-cooled copper mold; the heat transfer is driven by a lower water temperature (T f ), by a rate given by a heat transfer coefficient h: Similarly, another very interesting point of analysis on the derivation of the discretization equation is at a diagonal point A; the formulation is presented by Eq. (17); in this case, there is no heat transfer (due to symmetry) in the area above the diagonal (adiabatic formulation):
|
2019-04-18T07:28:02.031Z
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2019-02-08T00:00:00.000
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247259371
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pes2o/s2orc
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v3-fos-license
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The Relationship between Emotional Stability, Psychological Well-Being and Life Satisfaction of Romanian Medical Doctors during COVID-19 Period: A Cross-Sectional Study
Due to the COVID-19 pandemic, as well as the fast progression of modern society, occupational stress has recently reached alarming levels with consequences for doctors’ psychological well-being. The aim of this study was to analyze the relationship among emotional stability, psychological well-being, and life satisfaction of medical doctors. We conducted a cross-sectional study on 280 medical doctors from Romania between February 2021 and September 2021, in the period between the third and fourth pandemic waves, who were evaluated by the DECAS, ASSET, and Satisfaction with Life scales. Our results showed that emotional stability is negatively correlated with psychological well-being (r = −0.526, p < 0.000) and positively correlated with life satisfaction (r = 0.319, p < 0.0001). Between psychological well-being and life satisfaction, we found a negative correlation (r = −0.046, p < 0.001). This study shows that there is a correlation among emotional stability, psychological well-being, and life satisfaction, which is why it can be considered that Romanian doctors have generated coping mechanisms during the COVID-19 pandemic.
Introduction
Stress is a widely studied concept and refers to how each of us responds positively or negatively to an internal or external stimulus/condition that often exceeds perceived coping abilities [1]. These stressors can be divided into three categories: circumstantial, occupational, and personal [2]. Occupational stress in the medical world is a global phenomenon faced by modern society, negatively affecting both the physical and the mental health of the physician, followed by consequences for the quality of the medical act to the detriment of the patient [3]. Physicians must constantly face high standards at the workplace, and they frequently face problems such as lack of time due to the increased number of patients, inability to cope with situations due to a lack of skills needed in the specialty they practice, as well as a lack of support from their colleagues [4], and increased number of hours spent in the hospital, especially night shifts with sleep deprivation [5]. Therefore, there are other significant aspects that result in a decrease in the free time intended for the physician's recovery, as well as increased family problems and financial issues. Given the current situation, it has been demonstrated that the pandemic of coronavirus disease 2019 (COVID-19) has raised distress for medical doctors; in addition to all the factors mentioned above, it is important to underline the risk for this professional category to develop different mental and physical disorders, in this pandemic context [6,7]. Furthermore, during the pandemic period, medical doctors presented a higher risk of developing depression and/or anxiety disorders compared to other professions [8]. Moreover, compared with other medical specialties, this risk is higher especially for the doctors who work on the front line treating COVID-19 patients [9]. Other factors that contribute to elevated stress levels in doctors in the pandemic context include a lack of protective equipment [10], overloading during busy periods with many patients [11], the fear of becoming infected, and concern regarding the possibility of infecting family [12], as well as the possibility of friends and relatives avoiding being in the presence of medical staff [13]. Stress levels are used as an indicator in mental health, and more and more studies have shown that employment and labor conditions in the medical field affect the psychological well-being (PWB) of healthcare employees [14,15]. All of these factors increase stress, eventually leading to the development of burnout syndrome [16]. Burnout syndrome has increased in recent years among physicians [17] and is defined as the response to prolonged exposure to high levels of stress at work. It is manifested by emotional exhaustion, episodes of depersonalization, and decreased performance at work [18]. The factors that contribute to the increase in occupational stress consist of personality traits, stressors related to patient wellbeing, level of experience, and attitude at work [19,20]. Occupational stress and involvement at work are negatively correlated, whereby doctors who have a high level of stress tend not to get involved as much as others [21]. Lack of control at work increases the level of occupational stress. Workplace performance is influenced by the doctor's experience and by job stability [22].
Both stress factors and daily challenges influence the doctor's PWB. The most pressing aspects are related to the doctor's responsibility for the patient's wellbeing, the lack of control related to the patient, the high standards that patients and relatives have, and their dissatisfaction with the medical act [23].
PWB is a construct consisting of several dimensions according to the study conducted by Ryff and Keyes (1995): self-acceptance refers to the acceptance of one's own person with advantageous but also disadvantageous personal traits, including acceptance of one's own past; personal growth refers to continuous development by experiencing new things; purpose in life refers to the belief that everyone's life has a purpose which is significant; positive relationships with others; environmental mastery through which everyone has the ability to manage and route things to the desired direction; autonomy, i.e., the determination that each of us has in achieving the proposed goals [24,25]. PWB refers to both continuous personal development and the concept of living well and being well with oneself [26].
Subjective wellbeing refers to the satisfaction and happiness that each of us feels, encompassing a cognitive component and an affective one [26]. The cognitive component is the evaluation of life satisfaction (LS), while the affective component is represented by positive and negative affect [27]. They integrate the levels of individual satisfaction into life roles [28]. Furthermore, PWB is closely related to both job satisfaction and LS. A study conducted in Denmark on a large number of general practitioners showed that one in five doctors faces a high level of stress and low PWB [29]. A recent study on family physicians from China showed that the level of involvement in the workplace is positively correlated with high performance, while PWB is positively correlated with high performance. Fulfilled, happy, and motivated family physicians who identify themselves with the environment where they work and keep proper relationships with their colleagues have increased PWB [30]. Recent research in China showed that the level of PWB is positively correlated with the title of physician and negatively correlated with age and education [31]. PWB is most often associated with personality traits [32].
From the dimensional perspective of personality assessment, the emotional stability (ES) dimension, the fifth dimension of the Big Five Model (FFM = Five Factor Model) is most often associated with subjective wellbeing [33]. People with high ES are characterized as relaxed, emotionally stable, resilient, optimistic, and rational in thinking. On the other hand, people with low ES are characterized as anxious, scared, and easily irritable, with a low tolerance for frustration and an amplified response under stress [34].
Rus et al. highlighted the fact that there is a positive correlation between subjects who obtained low scores in the ES dimension and high levels of stress among medical employees [14]. Low ES was associated with a risk factor for the inability to manage a worklife balance by physicians from all specialties [35]. Furthermore, low ES was positively associated with the emotional exhaustion dimension specific to the burnout syndrome in medical staff from private hospitals from India [36]. Moreover, it is important to mention that, during the COVID-19 pandemic, low levels of ES and extraversion were the main personality dimensions, from the Big Five Model, related to high distress and fear in both the general population and healthcare workers [6,37].
LS is defined as the evaluation of one's own life, as well as the way one feels depending on the objectives or the things that can be obtained in the future [38]. It can also be defined as one's judgment according to one's own balance, expectations, or standards [39,40]. Regarding the factors that influence LS, it has been shown that work-life imbalance, multiple tasks at work, and a lack of support from colleagues negatively affect LS [41]. Factors that positively influence LS are represented by adequate working hours, proper physical health, the existence of the necessary resources for patient care, and working for more than 4 years at the same job [42]. It has also been shown that personality traits have an important role in LS [43]. It has been demonstrated that ES is positively correlated with LS [44].
ES has been described as a factor of resilience to psychological distress in medical workers, during the COVID-19 pandemic [8,45,46]. In addition to ES, another adaptive coping mechanism with stress is represented by PWB [47]. What is important to find out in this context is whether these two psychological dimensions constitute just a resilient factor, as already demonstrated by previous studies, or whether these psychological dimensions are correlated with satisfaction in life, in Romanian medical doctors during the COVID-19 pandemic.
In light of the evidence discussed above, the aim of this study was to establish whether there is a correlation among ES, PWB, and LS in Romanian medical doctors, in the period between the third and fourth pandemic waves of COVID-19. In this context, we hypothesize that the association among ES, PWB, and LS present in Romanian doctors generated different adaptative coping mechanisms, in the period between the third and fourth pandemic waves of COVID-19.
Materials and Methods
This was a cross-sectional study conducted between February 2021 and September 2021 at the "George Emil Palade" University of Medicine, Pharmacy, Science and Technology of Târgu Mures , on physicians who graduated in medical studies and carried out their medical activity in Romania. Due to the fact that the study took place in the period between the third and fourth pandemic waves of COVID-19, it is important to mention that medical doctors included in this study were selected from the second line of COVID-19 treatment. This research was approved by the Ethics Commission of the "George Emil Palade" University of Medicine, Pharmacy, Science, and Technology of Târgu Mures , by decisions no. 1250/28.01.2021 and 1374/20.05.2021. All participants signed the informed consent form before enrolling in the study.
Participants and Procedure
Out of an initial 311 subjects, 280 participants who met the eligibility criteria were included in this study. The sample of the present study was constituted using a simple sample randomization. Therefore, our study group was considered a representative one according to the total number of Romanian doctors working in Romania. According to the official date of Ministry of Health, about 63,000 medical doctors are active in Romania [48]. In rapport with the simple sample calculation method, this study would need to include 245 medical doctors for a representative sample population (representing 80% of the number of doctors from the second line of treating COVID-19, 95% CI). Therefore, in our sample we included 280 medical doctors, and this sample can be considered representative of Romanian medical doctors from the second line of treating COVID-19. Furthermore, 30 subjects were excluded due to the fact that they did not pass the internal validations scales of the DECAS Personality Inventory, while one subject did not carry out activities in Romania. The subjects completed the following scales: DECAS Personality Inventory, ASSET (A Shortened Stress Evaluation Tool), and the Satisfaction with Life Scale. In addition to the applied scales, the following parameters were included in the analysis: age, sex, level of experience. Inclusion criteria were as follows: (1) doctors who carried out medical activity in Romania; (2) doctors who graduated from the Faculty of General Medicine in Romania. Exclusion criteria were as follows: (1) doctors who did not carry out medical activity in Romania; (2) doctors who did not pass the internal validation scales of the DECAS Personality Inventory; (3) doctors who worked on the front line in the fight against the pandemic.
Prior to enrolling in the study, all participants signed the informed consent form. The questionnaires were disseminated online through social media to medical groups from Romania.
Measures
To evaluate the subjects, we administered three scales validated on the Romanian population: the DECAS Personality Inventory (DECAS), A Shortened Stress Evaluation Tool (ASSET), and the Satisfaction with Life Scale (SWLS).
The DECAS Personality Inventory (DECAS) is a personality assessment scale based on the FFM, developed by Sava et al. [49]. It is a scale consisting of 97 statements for the assessment of each personality dimension: openness, extraversion, conscientiousness, agreeableness, and emotional stability. The openness dimension, the least studied dimension in the literature, is assessed through 18 items and a reserve one that targets the following facets: fantasy, aesthetics, feelings, actions, intellectual curiosity, and values. The extraversion dimension is the most obvious dimension and, along with the emotional stability dimension, is found in the descriptions of all reference models of personality assessment, consisting of the following facets: warmth, sociability, assertiveness, activism, sensation seeking, and positive emotions. The conscientiousness dimension includes the following six facets: competence, order, sense of duty, desire to achieve, presumption, and deliberation. The agreeableness dimension has the greatest impact on interpersonal relationships, consisting of the following facets: trust, direct behavior, altruism, goodwill, modesty, and gentleness. The emotional stability dimension includes the following facets: anxiety, anger, depression, self-awareness, impossibility, and vulnerability. In addition, the DECAS personality inventory includes three validation scales built with the purpose of evaluating the sincerity of the answers of the subjects: social desirability (SD), random answers (RD), and approval (AP). The SD validation scale is a factor that measures the tendency of the subjects to put themselves in a favorable light through the answers offered in the questionnaire items. A score of more than 65 (T-scores) obtained by the subject on this scale invalidates the results. The RD validation scale represent a factor which evaluates the subject's tendency to give random answers, whereby a score higher than 70 points (T-scores) on this scale leads to the invalidation of the personality inventory protocol. The AP validation scale is a sensitive factor to the subject's tendency to respond more with "true" or "false", and a score of more than 65 points (T-scores) or a score of less than 35 points (T-scores) invalidates the protocol. Regarding the inventory psychometric properties, internal consistency was calculated following the assessment of a batch of 1524 people with alpha Cronbach coefficient values assessed on the Romanian population ranging from 0.70 for the conscientiousness dimension to 0.75 for the emotional stability dimension. The Cronbach's alpha internal consistency coefficient was 0.69 for SD and 0.71 for AP [49].
A Shortened Stress Evaluation Tool (ASSET) was developed by Cooper and Cartwright, which can be easily used to identify potential stress exposure for employees in various fields [50,51]. The tool measures the following variables as stressors: professional relationships, work-life balance, overload, workplace safety, environmental control, access to resources and communication, and payments and benefits. The second section defines the perception of the involvement level both as an employee of the respective institution and as the involvement level of the institution toward the employee. The third section investigates stress effects on physical health and mental wellbeing, and the fourth section focuses on job aspects related to job satisfaction or physical job conditions [50,51]. The instrument has a total of 63 items scored on a six-point Likert scale and 37 items for biographic data (current job, family, education, lifestyle, and interests). The 63 items are distributed in several subscales aimed at professional relations, work-life balance, overload, workplace safety, control, resources and communication, payments and benefits, work aspects, the perceived commitment of the organization toward its employees, the commitment of members toward their organization, PWB, and physical health. For some dimensions of the scale, for example, PWB, the interpretation of the results is made in the opposite way. The score of this subscale is interpreted as follows: <3-very good level of PWB, <4-good level of PWB, 4-7-medium level of PWB, >7 low level of PWB, >8 very low level of PWB. Therefore, lower scores obtained by the subject for PWB can be interpreted as reduced distress levels for the subject and a good PWB. In terms of internal consistency, the alpha Cronbach coefficient measured on the Romanian population showed an average of 0.73 across all scales, with only two subscales below 0.60 [50].
The Satisfaction with Life Scale (SWLS) was developed by Diener et al. and is a measuring instrument designed to assess subjective wellbeing, from the perspective of its cognitive component. It consists of five questions scored on a seven-point Likert scale. The score of the scale is interpreted as follows: 31-35-extremely satisfied, 26-30-satisfied, 21-25-slightly satisfied, 20-neutral, 15-19-slightly dissatisfied, 10-14-dissatisfied, 5-9-extremely dissatisfied. The alpha Cronbach coefficient assessed on the Romanian population was 0.82, proving its good internal consistency [52].
Statistical Analysis
Statistical analysis was performed using the GraphPad Prism 7 licensed software. The significance level for the p-value was set to 0.05, with a confidence interval CI = 95%. Statistical analysis included elements of descriptive statistics (mean, median, standard deviation) and elements of inferential statistics. To determine whether there was a statistically significant difference between the median values of ES, PWB, and LS in resident doctors and senior doctors, we applied the Mann-Whitney test for unpaired data. To determine the distribution of data series, we applied the Shapiro-Wilk test. The Spearman test, a nonparametric test, was applied to measure the strength and direction of the association among the studied variables (ES, PWB, and LS).
Results
Out of an initial number of 311 subjects, 280 participants who met the eligibility criteria were included in this study, of whom 233 (83.21%) were female and 47 (16.79%) were male. The mean age of the group was 28.81 ± 4.79 years. Regarding experience, 33 (11.78%) were senior doctors and 247 (88.21%) were junior doctors. The distribution in the sample included the follows categories: medical specialties 191 (68.22%), surgical specialties 39 (13.93%), and paraclinical specialties 50 (17.85%). Demographic characteristics are summarized in Table 1. Regarding the implications of ES, our results showed a significant negative correlation between ES and PWB (r = −0.526, p < 0.0001). Furthermore, there was a significant positive correlation between ES and LS (r = 0.319, p < 0.0001). Between LS and PWB, we found a significant negative correlation (r = −0.046, p < 0.001). The correlations among the three variables are found in Table 3.
Discussion
The present study investigated the correlations among ES, one of the dimensions of the Big Five Model, PWB, and LS in medical doctors in the period between the third and fourth pandemic waves of COVID-19.
Given that stress levels were high during the pandemic, our study shows that physicians had a moderate level of PWB, and they were satisfied with life. These results are consistent with previous studies [53,54]. These aspects may be due to the fact that the study was conducted in the period between the third and fourth pandemic waves, at which point the doctors had become familiar with the pandemic, and things had become clearer [55]. Longitudinal studies have shown that resilience has increased and the general population has found a surprising ability to adapt [56,57]. Coping mechanisms such as active attitudes, along with making plans, acceptance, and reinterpretation of reality are positively associated with LS [58]. Other factors that have helped to reduce stress are protective measures, psychological counselors [59], team support, stress monitoring, taking breaks regularly [60], knowledge of the disease [61], and things becoming easier [62].
ES is an independent predictor of LS [41,63]. In our study, the ES dimension was positively correlated with LS. In the literature, there are other similar positive correlations between ES and LS, regardless of the scale or questionnaire applied for personality evaluation [64,65]. This finding is identical to that of Tyssen et al., according to whom doctors with low ES responded excessively to the stressful conditions imposed by their profession with implications for daily activities [66]. During COVID-19, levels of stress were higher, associated with avoidance of the use of coping mechanisms [67], but people with high ES could overcome these issues by following doctors' recommendations [68]. Moreover, low levels of anxiety facilitate adaptability in all existential roles [69].
We found that, between LS and PWB, there was a negative correlation (lower levels on the scale indicating reduced distress levels for the subject and a good PWB). This is confirmed by several studies that showed a relationship between LS and stress or other constructs such as PWB [70,71]. A possibility to increase the level of PWB is by using approach-oriented coping strategies, because they are connected with a higher level of PWB [72]. At the same time, LS, understood as the achievement of goals, leads to beneficial cognition and is negatively correlated with avoidance coping strategies [73].
Our results indicated that the ES dimension was negatively correlated with the PWB of the doctor (lower levels on the scale indicating reduced distress levels for the subject and a good PWB). These observations are also supported by the results of the study by Soh et al., which stated that emotional stability is an important predictor of PWB [74,75]. A relaxed doctor, who controls the situation, with good emotional control and stress resistance, will have a better PWB, which is also reflected by involvement in the professional role [76].
Psychological resilience implies maintaining a consistent level of happiness and PWB in the face of stressors. This means developing strategies in work over the years in order to conserve a good mental health. This can be translated into practices and behaviors that the physicians consider being good to protect their PWB [77]. A low level of PWB manifested by depression and anxiety has direct effects on choosing avoidance coping strategies instead of applying problem-focused strategies [78].
The use of an adaptative coping mechanism/resilience during COVID-19 is influenced by both EA and PWB [79]. A comparative study of resident and senior doctors showed that both categories used coping mechanisms during the pandemic. These mechanisms varied with age, whereby resident doctors were more technology-oriented and practiced more mindfulness than senior doctors [80]. Resilience is associated with maturity, responsibility, optimism, perseverance, and cooperation [81]. Physicians usually present increased resilience through their education, which is necessary to cope with the daily challenges of their chosen profession, especially in the COVID-19 pandemic [82]. Stress can be overwhelming regardless of the level of experience throughout the medical profession [83]. It has been shown that subjective wellbeing and satisfaction with life have an important impact on improving physicians' resilience to stress [84]. In this regard, the level of resilience could be increased by an improvement of ES, PWB, and LS [85]. From this point of view, resilience may be the crucial aspect to focus on when elaborating programs to support mental health [86]. Although ES is a personality trait, personality is a construct that is relatively stable over time with small changes over short periods [87]. It has recently been shown that there is a significant difference in the stability of personality traits between adolescence and adulthood [88]. Accordingly, there is the possibility that practicing therapy to learn techniques can lead to an increase in emotional stability over time [89]. Hypnotherapy combined with behavioral cognitive therapy (CBT) has been shown to significantly improve emotional stability [90]. Another approach is to restore the balance between mind and body by practicing mindfulness [91,92]. Healthcare workers experience increased levels of daily stress. During the pandemic, these concerns have been at a higher level due to overload at work, the fear of infecting both themselves and their relatives, and high levels of uncertainty about the future of the pandemic [93]. Doctors have developed coping mechanisms through social distancing, wearing a mask, collaborating with colleagues to manage patients, and recurrent training and pandemic information received from the institution where they practice [94]. Acceptance and engagement therapy (ACT) is an acceptance-based behavioral intervention that promises to reduce the psychological impact of the pandemic. The ACT increases both behavioral awareness and openness to experience. Through ACT, the doctor takes on the role of observer of their own thoughts [95]. ACT is used to improve the functioning of the workplace, to reduce the stress caused by daily activities, and to improve relationships with others [96].
Limitations
Our study had some limitations that deserve attention in the future. The first limitation is that we used self-administered questionnaires that could have contributed to inaccurate results due to the fact that only the DECAS Personality Inventory has an internal validation scale that can detect distorted responses. It is recommended that future studies be conducted in this direction to figure out which of the variables (ES, PWB, and LS) are interrelated. Furthermore, in the future, the level of resilience/coping mechanisms can be assessed in terms of a correlation with ES, PWB, and LS.
Conclusions
ES and PWB were found to be correlated with LS; thus, it can be considered that Romanian doctors generated coping mechanisms during the COVID-19 pandemic. In addition, the level of emotional stability and psychological well-being of the doctors was moderate, and they perceived an increased level of life satisfaction in the period between the third and fourth pandemic waves, confirming that coping mechanisms were generated to deal with the pandemic. Future research may investigate these coping mechanisms.
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2022-03-08T16:56:19.255Z
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2022-03-01T00:00:00.000
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Improvement of Tubular Permanent Magnet Machine Performance Using Dual-Segment Halbach Array
In this paper, a modification of the dual-segment permanent magnet (PM) Halbach array is investigated to improve the performance of the tubular linear machine, in terms of flux density and output power. Instead of a classical Halbach array with only radial and axial PMs, the proposed model involves the insertion of mig-magnets, which have a magnetized angle shifted from the reference magnetized angles of axial and radial PMs. This structure leads to the elimination of flux leakage and the concentration of flux linkage in middle of the coil; therefore, the output power is increased by 13.2%.
Introduction
Tubular machines are commonly applied in many industrial applications, such as cryocoolers, linear compressors, or refrigerators [1][2][3]. Recent research activities for energy harvesting in vehicle suspension systems [4][5][6] or using ocean wave energy [7][8][9] have proposed a new trend for tubular machines. From a design viewpoint, at a particular volume, increasing the output power is very difficult. L. Zou et al. [10] provide the original design, which is composed of one magnet layer using a Halbach arrangement instead of only axial magnet or radial magnet arrays. In the following step, by using the same Halbach array structure, X. Tang et al. [11] modified the original model with a double layer of permanent magnet; this method eliminates the flux leakage, while concentrating flux density in the middle of the coil. This leads to an eightfold improvement in the output power and a 3.8-fold improvement in the power density compared to the values of the original design.
Another technique was studied by Y. Shen, in which mid-magnets whose magnetization angle is shifted from the reference angle of axial and radial magnets are inserted [12]. There are two possible structures-odd-segment and even-segment-and these structures define the number of magnet segments over a pole pitch as an odd or even number. The FEM and analytical results for a 12-slot/10-pole PM brushless machine illustrate that the 3-segment Halbach array exhibits significantly higher fundamental airgap flux density than that of the magnet cylinder, having a traditional Halbach array with two segments. Under the same theory, W. Zhao designed a tubular linear generator for energy harvesting from body motion [13] using an even-segment Halbach array. The eight-segment dual Halbach array is employed in the optimized generator because its magnetic flux density is greater than that of the four-segment array. However, the effects of the odd-segment model were not presented.
In this paper, an electromagnetic shock absorber for vehicle energy harvesting using a segment-magnet Halbach array is proposed. Unlike most conventional research [6,7], in which a mechanical shock absorber is replaced with an electrical one, the proposed device is a combination 2 of 10 of both parts. Electrical components including a magnetic core, winding, and permanent magnet (PM) are attached to the inner and outer frame of the mechanical shock absorber. To validate with the corresponding experiments, a single PM layer, with a coreless model, is designed, fabricated and tested. To avoid the noise caused by the mechanism, experiments are carried out under lower vibration speeds than those of the previous study [14,15]. In addition, the effects of both the evenand odd-segment PM on the tubular generator are investigated and compared with the conventional one. In previous publications, these effects have mostly been investigated for rotating machine. Finite element analysis (FEA) results show thata under the same operating conditions, a tubular generator using segment-magnet yields has higher power than that of a generator with the original Halbach array structure by 13.2%. In the further study, the optimized dimensions of the PM segments, the magnetized angle, and feasibility will be taken into account.
Design Specifications
This section deals with the design specifications of the tubular machine using the classical Halbach array based on the actual dimensions of a commercial shock absorber in an SUV-Korando car. Analyses using FEM are validated with corresponding experiments. Figure 1 shows the cross-section of the half model and its geometry over one pole pitch, as was presented in Reference [14,15]. The overlapping length of the magnet array and coil windings is about 200 mm but, fortunately, this value is adjustable by ±40 mm. That is also the condition for the outer diameter, which can be flexibly chosen at values between 80 mm and 160 mm, while the inner diameter has to be at least 40 mm, the same as the diameter of the damping part or the mechanical part [14,15].
Electrical components including a magnetic core, winding, and permanent magnet (PM) are attached to the inner and outer frame of the mechanical shock absorber. To validate with the corresponding experiments, a single PM layer, with a coreless model, is designed, fabricated and tested. To avoid the noise caused by the mechanism, experiments are carried out under lower vibration speeds than those of the previous study [14,15]. In addition, the effects of both the even-and odd-segment PM on the tubular generator are investigated and compared with the conventional one. In previous publications, these effects have mostly been investigated for rotating machine. Finite element analysis (FEA) results show thata under the same operating conditions, a tubular generator using segmentmagnet yields has higher power than that of a generator with the original Halbach array structure by 13.2%. In the further study, the optimized dimensions of the PM segments, the magnetized angle, and feasibility will be taken into account.
Design Specifications
This section deals with the design specifications of the tubular machine using the classical Halbach array based on the actual dimensions of a commercial shock absorber in an SUV-Korando car. Analyses using FEM are validated with corresponding experiments. Figure 1 shows the cross-section of the half model and its geometry over one pole pitch, as was presented in Reference [14,15]. The overlapping length of the magnet array and coil windings is about 200 mm but, fortunately, this value is adjustable by ± 40 mm. That is also the condition for the outer diameter, which can be flexibly chosen at values between 80 mm and 160 mm, while the inner diameter has to be at least 40 mm, the same as the diameter of the damping part or the mechanical part [14,15].
Due to the relative position between the coils and the excited flux density, and with the aim of simplifying the drive system, an external circuit is designed with two phases, in which phase 1 is in series connection with coil 1 and coil 3, and phase 2 is in series connection with coil 2 and coil 4. It is mandatory to precisely set up the winding directions to achieve maximum power [14,15]. Detailed specifications of the tubular machine are summarized in Table 1. Due to the relative position between the coils and the excited flux density, and with the aim of simplifying the drive system, an external circuit is designed with two phases, in which phase 1 is in series connection with coil 1 and coil 3, and phase 2 is in series connection with coil 2 and coil 4. It is mandatory to precisely set up the winding directions to achieve maximum power [14,15]. Detailed specifications of the tubular machine are summarized in Table 1. The relative position between the coils and the excited flux, and the external circuit, are shown in the Figure 2. According to References [10,14,16], the maximum output power occurs at the maximum vibrating speed v max when the load resistance R L equals the sum of the coil resistance R c ; this is termed the full load condition. The relative position between the coils and the excited flux, and the external circuit, are shown in the Figure 2. According to References [10,14,16], the maximum output power occurs at the maximum vibrating speed vmax when the load resistance RL equals the sum of the coil resistance Rc; this is termed the full load condition. Equation (1) shows that output power is proportional to the square of the radial flux density, the square of the vibrating speed and the volumes of the coil [10,14,15].
where Br is the radial flux density, vz is the vibrating speed, σ is the conductivity, and Vcoil is the coil volume.
The relationship between the peak to peak stroke, the vibrating speed, and the vibrating frequency is calculated as follows [10,14,15]: where vrms is root-mean-square of the vibration speed, and f is the vibration frequency. Equation (1) shows that output power is proportional to the square of the radial flux density, the square of the vibrating speed and the volumes of the coil [10,14,15].
where B r is the radial flux density, v z is the vibrating speed, σ is the conductivity, and V coil is the coil volume.
The relationship between the peak to peak stroke, the vibrating speed, and the vibrating frequency is calculated as follows [10,14,15]: where v rms is root-mean-square of the vibration speed, and f is the vibration frequency. In addition, the instantaneous voltage of one coil centered at equilibrium position z 0 in the regenerative shock absorber is presented as a function of time, position, magnetic flux density B 0 , Energies 2018, 11, 3132 4 of 10 length of the conductor L, sum of the thicknesses H of a radial and an axial PM, suspension velocity and frequency. For the 0 • coil or for coil 1, which has the maximum magnetic flux density, if the vibration amplitude is small, the voltage is calculated by [10]: and a 90 • coil or coil 3 will have a double frequency wave [10]: The additional iron layer is inserted to reduce the inner diameter of the PMs; this layer is composed of stainless steel, while the back iron is made of an electromagnetic material. Both radial PMs and axial PMs are made of DDP-40SH, which has a relative flux density of B r = 1.26 T at 20 • C and a relative permeability of µ r = 1.05. Windings are wound around and supported by a bobbin with a total number of turns per slot of 180.
Validation with Experimental Data
According to Reference [10], when a mid-sized car is moving at a speed of 60 mph on a road class C, the vibrating frequency of the shock absorber is estimated to be approximately 0.25 m/s. Under these conditions, the peak to peak stroke length and vibrating frequency are about 11.25 mm and 10 Hz, respectively. These conditions of the stroke length, vibrating speed, and linear frequency are considered as a standard for the models studied in this paper. When applying the standard conditions, the maximum and average power can be theoretically calculated as 76.09 W and 37.8 W, respectively.
To prepare for the experiments, a prototype of a single layer coreless model was fabricated and installed, as in the illustrations provided in Figures 3 and 4. Unfortunately, if the linear frequency of the generator is 10 Hz, the required rotating speed of the sub-motor is 3000 rpm, which exceeds the limitation of this motor; therefore, measurements were performed at lower vibrating speeds than in the previous study [14,15]. In addition, the instantaneous voltage of one coil centered at equilibrium position z0 in the regenerative shock absorber is presented as a function of time, position, magnetic flux density B0, length of the conductor L, sum of the thicknesses H of a radial and an axial PM, suspension velocity and frequency. For the 0° coil or for coil 1, which has the maximum magnetic flux density, if the vibration amplitude is small, the voltage is calculated by [10]: and a 90° coil or coil 3 will have a double frequency wave [10]: The additional iron layer is inserted to reduce the inner diameter of the PMs; this layer is composed of stainless steel, while the back iron is made of an electromagnetic material. Both radial PMs and axial PMs are made of DDP-40SH, which has a relative flux density of Br = 1.26 T at 20 °C and a relative permeability of µr = 1.05. Windings are wound around and supported by a bobbin with a total number of turns per slot of 180.
Validation with Experimental Data
According to Reference [10], when a mid-sized car is moving at a speed of 60 mph on a road class C, the vibrating frequency of the shock absorber is estimated to be approximately 0.25 m/s. Under these conditions, the peak to peak stroke length and vibrating frequency are about 11.25 mm and 10 Hz, respectively. These conditions of the stroke length, vibrating speed, and linear frequency are considered as a standard for the models studied in this paper. When applying the standard conditions, the maximum and average power can be theoretically calculated as 76.09 W and 37.8 W, respectively.
To prepare for the experiments, a prototype of a single layer coreless model was fabricated and installed, as in the illustrations provided in Figures 3 and 4. Unfortunately, if the linear frequency of the generator is 10 Hz, the required rotating speed of the sub-motor is 3000 rpm, which exceeds the limitation of this motor; therefore, measurements were performed at lower vibrating speeds than in the previous study [14,15]. With the assumption that the peak to peak stroke length is fixed at 11.25 mm, Figure 6 presents the back EMF waveforms achieved for the load resistance when the vibrating speed is 0.125 m/s, which equals 50% of the standard. The average deviation is approximately 6.7%. In Figure 6b, it can be clearly seen that the frequency of load 2 is double that of load 1; this phenomenon can be predicted using Equations (3) and (4). With the assumption that the peak to peak stroke length is fixed at 11.25 mm, Figure 6 presents the back EMF waveforms achieved for the load resistance when the vibrating speed is 0.125 m/s, which equals 50% of the standard. The average deviation is approximately 6.7%. In Figure 6b, it can be clearly seen that the frequency of load 2 is double that of load 1; this phenomenon can be predicted using Equations (3) and (4). With the assumption that the peak to peak stroke length is fixed at 11.25 mm, Figure 6 presents the back EMF waveforms achieved for the load resistance when the vibrating speed is 0.125 m/s, which equals 50% of the standard. The average deviation is approximately 6.7%. In Figure 6b, it can be clearly seen that the frequency of load 2 is double that of load 1; this phenomenon can be predicted using Equations (3) and (4). With the assumption that the peak to peak stroke length is fixed at 11.25 mm, Figure 6 presents the back EMF waveforms achieved for the load resistance when the vibrating speed is 0.125 m/s, which equals 50% of the standard. The average deviation is approximately 6.7%. In Figure 6b, it can be clearly seen that the frequency of load 2 is double that of load 1; this phenomenon can be predicted using Equations (3) and (4). .
The measured result at a vibrating speed of 0.15 m/s for the average power is approximately 11.46 W, which is nearly the same as the expectation. Figure 8 illustrates the power variation according to various load resistances when the vibrating speed is 0.125 m/s and the peak to peak stroke length is 11.25 mm. Both the analysis and the experiment results present similar trends, with an average deviation of about 13.4%. When the load resistance equals the sum of the coil resistance, the average output power is at its maximum. There are several possible explanations for the differences between the simulation and the experiment in Figures 6-8. As illustrated in Figure 3, owing to the limitations during manufacturing, the permanent magnets were separated into smaller segments of a solid toroidal shape. Moreover, when the prototype is being measured, errors can occur due to the noise caused by the mechanical system. However, the accuracy is much better than the previous design for the coreless model.
The measured result at a vibrating speed of 0.15 m/s for the average power is approximately 11.46 W, which is nearly the same as the expectation. Figure 8 illustrates the power variation according to various load resistances when the vibrating speed is 0.125 m/s and the peak to peak stroke length is 11.25 mm. Both the analysis and the experiment results present similar trends, with an average deviation of about 13.4%. When the load resistance equals the sum of the coil resistance, the average output power is at its maximum. There are several possible explanations for the differences between the simulation and the experiment in Figures 6-8. As illustrated in Figure 3, owing to the limitations during manufacturing, the permanent magnets were separated into smaller segments of a solid toroidal shape. Moreover, when the prototype is being measured, errors can occur due to the noise caused by the mechanical system. However, the accuracy is much better than the previous design for the coreless model. .
The measured result at a vibrating speed of 0.15 m/s for the average power is approximately 11.46 W, which is nearly the same as the expectation. Figure 8 illustrates the power variation according to various load resistances when the vibrating speed is 0.125 m/s and the peak to peak stroke length is 11.25 mm. Both the analysis and the experiment results present similar trends, with an average deviation of about 13.4%. When the load resistance equals the sum of the coil resistance, the average output power is at its maximum. There are several possible explanations for the differences between the simulation and the experiment in Figures 6-8. As illustrated in Figure 3, owing to the limitations during manufacturing, the permanent magnets were separated into smaller segments of a solid toroidal shape. Moreover, when the prototype is being measured, errors can occur due to the noise caused by the mechanical system. However, the accuracy is much better than the previous design for the coreless model.
Tubular Machine with Dual-Segment Halbach Array
In this section, a dual-segment Halbach array is applied to the same tubular generator with the aim of improving the output power. The design specifications with the number of magnets, the sizes, and the magnetized direction are shown in Figure 9 and Table 2.
Tubular Machine with Dual-Segment Halbach Array
In this section, a dual-segment Halbach array is applied to the same tubular generator with the aim of improving the output power. The design specifications with the number of magnets, the sizes, and the magnetized direction are shown in Figure 9 and Table 2. Based on the results in Reference [12], instead of applying only axial and radial PMs over one pole pitch in a classical Halbach array, a number of mid-magnets with different magnetized angles are inserted. There are two applicable approaches: even-segment, with an even number of magnets over one pole pitch, and odd-segment, with an odd number of magnets over one pole pitch. In the original design, the pole pitch is = 27 mm, so the number of PM segments is selected to be 4 in the even-segment case and 5 in the odd-segment case. If there is a higher number of segments, the width of each segment becomes smaller. In the case of the odd-segment, the axial PM with 0° of the magnetized angle is removed [8]. For a fair comparison, the other dimensions, such as the length and diameter of the generator, the number of turns per slot, and the materials, are the same for the 3 models. In addition, in the preliminary investigation, the magnetized angle is equally divided. Figure 10 shows the flux distribution on the 3 different models under a no-load condition, with a vibrating speed of 0.25 m/s, a peak to peak stroke length of 11.25 mm and a linear speed of 10 Hz. According to Equation (1), the output power is proportional to the radial flux density in the middle of the coil. In the cases of the 4-segment and 5-segment magnet, the radial flux density increased by 6.8% and 5.2%, respectively; therefore, the output power can be expected to increase by around 15%. Due to the absence of the axial PM (0° of the magnetized angle), the flux linkage in the odd-segment PM model is slightly higher than that of the even-segment PM model. Figure 11 presents a comparison of the three models in terms of the radial flux density in the middle of the coil and the output power. The other characteristics are summarized in Table 3. Based on the results in Reference [12], instead of applying only axial and radial PMs over one pole pitch in a classical Halbach array, a number of mid-magnets with different magnetized angles are inserted. There are two applicable approaches: even-segment, with an even number of magnets over one pole pitch, and odd-segment, with an odd number of magnets over one pole pitch. In the original design, the pole pitch is τ p = 27 mm, so the number of PM segments is selected to be 4 in the even-segment case and 5 in the odd-segment case. If there is a higher number of segments, the width of each segment becomes smaller. In the case of the odd-segment, the axial PM with 0 • of the magnetized angle is removed [8]. For a fair comparison, the other dimensions, such as the length and diameter of the generator, the number of turns per slot, and the materials, are the same for the 3 models. In addition, in the preliminary investigation, the magnetized angle is equally divided. Figure 10 shows the flux distribution on the 3 different models under a no-load condition, with a vibrating speed of 0.25 m/s, a peak to peak stroke length of 11.25 mm and a linear speed of 10 Hz. According to Equation (1), the output power is proportional to the radial flux density in the middle of the coil. In the cases of the 4-segment and 5-segment magnet, the radial flux density increased by 6.8% and 5.2%, respectively; therefore, the output power can be expected to increase by around 15%. Due to the absence of the axial PM (0 • of the magnetized angle), the flux linkage in the odd-segment PM model is slightly higher than that of the even-segment PM model. Figure 11 presents a comparison of the three models in terms of the radial flux density in the middle of the coil and the output power. The other characteristics are summarized in Table 3.
Conclusions
In this paper, a tubular permanent magnet machine composed of a novel magnet arrangement is proposed and compared with the conventional model in terms of the radial flux density in the middle of the coil and output power. A prototype of a tubular machine composed of the classical
Conclusions
In this paper, a tubular permanent magnet machine composed of a novel magnet arrangement is proposed and compared with the conventional model in terms of the radial flux density in the middle of the coil and output power. A prototype of a tubular machine composed of the classical
Conclusions
In this paper, a tubular permanent magnet machine composed of a novel magnet arrangement is proposed and compared with the conventional model in terms of the radial flux density in the middle of the coil and output power. A prototype of a tubular machine composed of the classical Halbach array was fabricated and validated using the FEM analyzed results, which show an average deviation of 13.5% in terms of the output power.
Unlike devices that use the classical Halbach array, in the proposed model mid-magnets are inserted, which have a magnetized direction that is shifted from that of the original axial and radial PMs. Based on the FEM analyses, in the cases of 4-segment and 5-segment Halbach arrays, the RMS values of the radial flux density increased by nearly 6.8%, which led to an increase in the average output power of 13.2%.
In a further study, the relative dimensions and the magnetized angle between the magnet segments will be optimized.
|
2019-04-16T13:28:54.968Z
|
2018-09-27T00:00:00.000
|
{
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"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/1996-1073/11/11/3132/pdf",
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9837096
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pes2o/s2orc
|
v3-fos-license
|
Changes in abdominal obesity in Chilean university students stratified by body mass index
Background Studies based on Body Mass Index (BMI) and waist circumference (WC) are generally used to examine the prevalence and tendency of overweight and obesity. These studies help determine the socioeconomic development of a country and improve public health policies. Therefore, the goal of this research was to determine the trend of change in abdominal obesity of Chilean university students according to the Body Mass Index (BMI) measured in intervals of three and six years. Methods For this study, a total of 1598 students of both sexes ranging in age from 18 to 26 from a Chilean university were evaluated. Students were assessed commencing in 2007 (372 males and 315 females), 2010 (250 males and 330 females), and ending in 2013 (153 males and 178 females). During the three transversal assessments, weight, height, and waist circumference were evaluated. BMI was calculated for both sexes. Results No significant differences were found in age and BMI during the three years evaluated (2007, 2010, and 2013). In 2013, waist circumference (WC) increased significantly (p < 0.001 for both sexes). Moreover, in 2013, in all the percentiles evaluated, high values of WC were compared in relation to previous years. Furthermore, in 2013, in all four BMI categories (underweight, normal, overweight, and obese), the university students showed significant increases in WC (Females: p = 0.004; Males: p = 0.035) whereas in 2007 and 2010, the values remained relatively stable. Conclusions BMI remained constant during 2007, 2010, and 2013. However, the university students of both sexes showed greater risk of abdominal obesity as a result of increased WC in 2013.
Background
Student trends have become important work for human biological researchers to determine the socioeconomic development of a country for improving public health policies [1]. A secular trend is defined as the differences between individuals or groups of the same age and sex associated with the year or decade of birth [2]. It has become an important biological indicator in terms of health. Furthermore, these types of studies possess a fundamental characteristic that allows one to infer the increase of the prevalence of diseases associated with obesity, hypertension, glucose intolerance, and dyslipidemia, not only the early type found in childhood and adolescence but also during adulthood.
A number of studies have established various criteria to determine secular trends in children, adolescents, and adults [3][4][5]. Currently, researchers have directed their attention to studying the prevalence and tendency of overweight and obesity. These studies are based on Body Mass Index (BMI) and waist circumference (WC) [6], generally used for studying diverse populations worldwide [7].
In general, during the last decades, it has been widely acknowledged that height has tended to stabilize primarily in the developed countries. However, weight continues to increase resulting in forms of obesity [8] and increasing with greater frequency worldwide [7,9]. In fact, various countries in South America are in the process of nutritional transition. Therefore, it is expected that these societies experience important changes in their demographics, in their diets, and in their respective lifestyles.
In essence, Chile is not far behind in this trend. In the past few years, a number of studies have shown an increased prevalence for overweight, obesity, and metabolic risk in young university students [10][11][12][13]. This excessive increase is related to the rapid socio-economic advances seen in the last decades [14,15]. Therefore, the assumption is that young university students studying probably demonstrate significant changes in body fat in short periods of time. Therefore, the objective of this study was to determine the trend in changes in abdominal obesity in Chilean university students in relation to the BMI in measured intervals of three and six years.
Study design, sample and subjects
The research design was cross-sectional. In this study, young Chilean university students from the University of Talca (UTAL), Lircay Campus UTAL (Talca, Chile) answered three transversal questionnaires in 2007, 2010, and 2013. UTAL is a Chilean state university. It is located northeast of the city of Talca. It is 270 km south of Santiago (the capital of Chile). The economy of Talca is based primarily on agriculture and cattle while the wine industry plays a significant role in the local economy.
For the three years of this study, non-probabilistic sampling (quotas) was used. The data collected from the first survey included responses from 687 university students (372 males and 315 females); for the second survey, 580 (250 males and 330 females); and for the third survey, 331 (153 males and 178 females) for a total of 1598 young university students. Furthermore, during all three years, subjects dropped out of the study. The attrition rate ranged from 12.7-to-15.6 % for males and 18.3 % for females. Age ranged from 18 to 26 years. Students below and above the established age range, those not providing informed consent, or students not attending the day of the evaluation were excluded from the research. Students were recruited from their respective faculties and invited to participate voluntarily.
Collection of the anthropometric information was approved by the Ethics Committee of the University of Talca (UTAL). All of the subjects provided informed consent before anthropometric information was collected from them.
Anthropometric measurements
All anthropometric data was collected at the beginning of each academic year of the study (March-April). The technical personnel were trained prior to collecting data in order to become familiar with the standardized evaluation measurement protocol for collecting anthropometric data.
Each research year, 10 % of the sample was evaluated twice. The technical measurement error (TME) was less than 2 % for the three evaluation years. In effect, the protocol described by the International Society for the Advancement of Kinanthropometry (ISAK) [16] was used as the basis for the study. Weight (kg) was measured with a Tanita digital (United Kingdom, Ltd.) scale with a precision of 100 g and a scale of 0 to 50 kg. Height (cm) was measured with an aluminum Seca estadiometer (Seca Gmbh & Co. KG, Hamburg, Germany) graduated in millimeters with a scale of 0 to 250 cm. The circumference of the waist (cm) was measured at the mid-point between the lower ribs and the top of the iliac crest with a metal belt Seca measuring tape in millimeters with a precision of 0.1 cm.
Statistical analysis
The Kolmogorov-Smirnov (K-S) test was run on all the variables. It was conducted separately for each sex and evaluation year in order to determine the normal distribution. The data was analyzed using descriptive statistics in order to find the frequencies and percentages for the variable categories. Means and standard deviation (SD) were determined for the continuous variables. In addition, percentile distribution was determined: p5, p15, p50, p85, and p95.
The differences between the three evaluation years and the differences between the four BMI categories were verified by one-way ANOVA. Post hoc Tukey test was used to identify points of difference. Differences between percentiles were verified through fraction 100 log (percentile from 2007 Table 1 below shows the characteristics and evaluations of the students enrolled in the university during the 3 year project. Table 2 shows the means and ± SD for age, weight, height, and WC for both sexes of the university students. were relatively similar but less than those of 2013. This pattern is reflected in the increase in the number of cases of overweight and obesity observed during 2013. Based on the four categories of BMI, Fig. 1 shows the mean and ± SD values for WC. The students of both sexes classified as underweight BMI (less than 18.5 kg/ m 2 ) showed mean values similar to the WC during the three evaluation years. However, in the other three categories (normal, overweight, and obese), the young university students showed significant increases in WC during 2013 when compared to 2007 and 2010 (p < 0.05) where the values remained relatively stable(p > 0.05
Discussion
The results of this study indicate that BMI did not change over time. However, the WC increased significantly in both sexes especially during the last evaluation. The findings reflect the presence of accelerated secular changes in body fat estimated by WC. These findings are consistent with other studies carried out internationally [18][19][20]. These studies confirmed the increased trends in WC in both sexes extended over time. Since the WC is an anthropometric measure that reflects the central body fat, it is important to point out that the greatest increase in body fat distribution occurred from 2010 to 2013.
These findings are not only worrisome for the sample of university students studied in Talca but also those university students living in the surrounding Maule region of Chile. Therefore, it is important to take preventative measures to control excess weight and obesity in young Chilean university students [10]. Also this research may be used to raise awareness in other countries since recent studies continue to show elevated rates of overweight and obesity in university students around the world [10,13]. Evidently, this trend can be interpreted as greater levels of abdominal body fat creating a potentially greater risk for young university students to develop related illnesses such as type 2 diabetes, hypertension, dyslipidemia, cardiovascular illnesses, cancer, and death caused by obesity [18,21,22]. In general, a large portion of our sample of both males and females in the 2013 evaluation year of this study were found to be above the normal standard values. Young university students included in this study in percentiles 85 and 95 had higher values in the WC. These findings suggest that during very short intervals of time, the body shape of these young university students changed at an accelerated rate.
In essence, our results demonstrate that a large portion of the university population is at significant risk for developing chronic non-communicable diseases. This is due to the greater WC for the same BMI particularly during the 2013 evaluation measurement. In fact, this behavior could be associated with multiple factors such as poor eating habits, an increase in sedentary behaviors [23]. In general, these factors could create the predisposition for the disproportionate increase in WC among the young university students in this sample studied. However, economic development, increased urbanization, and excessive consumption of fats [24] in Chile during the past few years cannot be ruled as contributing factors in these changes observed.
In 1988, the Chilean government created the National Council for Health Promotion and introduced public policies in an attempt to combat the challenges of the epidemiology of obesity [25]. Other endeavors were also undertaken like the proposal for a Global Strategy to Combat Obesity introduced at the 2006 EGO-Chile [26]. Moreover in 2012, approval and publication of the national nutritional labelling law for food were obtained [27]. Despite these government's efforts, abdominal fat, at least among the young university students studied, continues to increase on a grand scale.
Identifying the limitations in this study will help improve the design for future research projects. First of all, the sample cohorts were selected through nonprobabilistic sampling (accidental). This does not allow generalization of the results to other university populations in Chile. Therefore, these findings should be interpreted with caution. In the second place, it was not possible to measure more variables related to body composition, physical activity, and eating habits of the sample. Therefore, in future research, these variables need to be taken into account since controlling for these variables could contribute to identifying the actual factors that contribute to the excessive increase in obesity in young university students. Furthermore, we suggest extending the research to other regions in Chile with the goal of including other universities with similar characteristics.
On the other hand, the process of data collection took place appropriately by maintaining a standardized protocol with an evaluation team with extensive experience in anthropometric measurement. Furthermore, the sample from this project was representative since it included students from the majority of the professional programs. This allowed a greater evaluation of changes in body fat amongst diverse University of Talca Chilean students. Moreover, during the three measurements taken, the same intervals of time were maintained between the evaluations. Furthermore, a strict order and sequencing in the methodology was adhered to ensuring an adequate methodological design.
Conclusion
In conclusion, the results showed that the BMI remained constant during 2007, 2010, and 2013. However, the young university students of both sexes may have a tendency for a greater risk of abdominal obesity. These results suggest a potential adverse cardio metabolic risk in the university populations of Talca. Thus, it is urgent that obesity prevention programs be instituted within the universities.
|
2016-05-04T20:20:58.661Z
|
2015-12-01T00:00:00.000
|
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81967724
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pes2o/s2orc
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v3-fos-license
|
The soil-transmitted helminths in Sri Lanka : a review of the recent literature
The major soil-transmitted helminth (STH) infections caused by Ascaris lumbricoides, Trichuris trichiura and Necator americanus have been recognized as endemic in Sri Lanka for over a century. Although prevalence rates have declined drastically over this period because of mass deworming programmes and improved housing, these infections are still found in high risk communities with poor access to sanitation. The available scientific literature published on STH infections in Sri Lanka from around the year 2000 onwards is reviewed here in three broad areas: prevalence of STH infections and factors affecting transmission, impact of control activities on prevalence and drug resistance, and the impact of STH infections on the health of infected individuals. In conclusion, an overview of the current control strategy adopted by the Ministry of Health in Sri Lanka is presented.
Introduction
The soil-transmitted helminths are among the most common parasites of humans. 1 The aetiological agents include the common roundworm (Ascaris lumbricoides), the whipworm (Trichuris trichiura), the hookworms (Necator americanus and Ancylostoma duodenale), and the threadworm (Strongyloides stercoralis), all of which are primarily parasites of humans.They are known as soil-transmitted helminths (STH) because their continued transmission is dependent on contamination of soil with the microscopic eggs or larvae of these helminths, as a result of open defaecation.
Ascariasis, trichuriasis and hookworm infections (the major STH infections) have been identified by the World Health Organization as common and important causes of morbidity among children.It is estimated that worldwide in 2010, approximately 820 million persons were infected with A. lumbricoides, 460 million with T. trichiura, and 440 million with A. duodenale or N. Americanus. 2 Infections are known to result in a range of clinical manifestations including malnutrition, intestinal and biliary obstruction, dysentery and anaemia.The highest prevalence occurs in areas where sanitation is inadequate and water supplies are unsafe.The strategy recommended by WHO to control morbidity from STH involves the periodic administration of anthelmintic medicines to populations at risk of disease, which includes pre-school age children, school age children and women of reproductive age. 3 New infections of A. lumbricoides and T. trichiura are acquired by ingestion of eggs containing infective larvae, via direct ingestion of contaminated soil, or fruits and vegetables contaminated with infected soil.Infection with the hookworms occurs when the free-living larvae, which are found on the surface of contaminated soil, penetrate bare skin.
As a developing country with a warm, wet, tropical climate, Sri Lanka is also endemic for all three types of STH (excluding A duodenale).Hookworm infections were first identified in this country over one hundred years ago, in the late 19 th century. 4The public health impact of all three STH infections was well recognized [5][6][7][8][9][10][11] and extensive de-worming programmes were conducted over much of the 20 th century.As a result of these programmes, combined with gradual improvements in housing and sanitation, as well as general health literacy, prevalence rates have drifted down steadily, albeit slowly.STH infections have, however remained endemic in Sri Lanka, and there is a significant body of work that has been published over the years, summarized by de Silva in 2005. 12is review summarizes the available scientific literature published on STH infections in Sri Lanka, from around the year 2000 onwards.The review is organized into three broad areas: prevalence of STH infections and factors affecting transmission; impact of control activities on prevalence and drug resistance; and the impact of STH infections on the health of infected individuals.In conclusion, an overview of the current control strategy adopted by the Ministry of Health in Sri Lanka is presented.
Prevalence of STH infections and factors affecting transmission
A nationwide survey of the health 9 -10 year old school children, carried out in 2003, included examination for STH infections. 13A total of 2,173 children from all nine provinces were examined using a single Kato-Katz smear of faecal samples.The cumulative prevalence (percentage of children with any STH infection) was estimated to be 6.9% among those examined.Whipworm was the most prevalent infection (4.0%), followed by roundworm (2.8%) and hookworm (1.2%).Cumulative prevalence was highest in the Eastern (12.3%),Northern (11.1%) and Western provinces (10.0%).
Other studies which have examined prevalence in more restricted populations around the same time include a survey of 743 schoolchildren attending Grades 1 -5 in four schools in the Moneragala District in 1997. 14Fresh stool samples were examined using the Kato-Katz methods.The prevalence of ascariasis, trichuriasis and hookworm infections were 2.0%, 0.7% and 5% respectively.In 2016, a survey of children attending 9 schools in the Kaduwela area which had been severely affected by floods, found that none of the 156 faecal samples examined by normal saline smears were positive for STH eggs. 15detailed study conducted in the estate sector, which remains a high-risk community for STH infections in Sri Lanka, has examined in depth, factors that affect the prevalence of STH The first paper examines the relationships between the prevalence and intensity of human infection with Ascaris and the availability of sanitary facilities, socio-economic status and personal health habits.16 During the period July -December 2000, 176 subjects, who lived on a low-country tea plantation, were investigated using Kato-Katz smears.Half (50.0%) of the subjects were found to be excreting Ascaris eggs.Almost all (96.6%) of the subjects lived in terraces of one-room houses built by the plantation owners, and only 30.7% had access to a latrine.Most (90.3%) obtained their drinking water from common taps, and 48.8% boiled their drinking water.The subjects who only drank water that had been boiled and those who washed their hands before meals were relatively unlikely to be infected.The authors concluded that even in congested living conditions with poor sanitary facilities, good hygiene and the boiling of all drinking water can reduce the risks of Ascaris infection.
The second paper examines the effect of seasonal variations in climatic conditions on transmission of Ascaris infections in two selected low-country plantations, during the period March 2000 -June 2001. 17Faecal samples from 477 persons aged between 2 and 74 years were tested using the Kato-Katz method and the prevalence and intensity of infection determined.Monthly follow-ups were undertaken with similar stool examinations and treatment given with a single dose of mebendazole, if found positive.Infection and re-infection rates were calculated each month.Rainfall and temperature were recorded each day.Total rainfall, number of wet-days and mean temperature was calculated for each month.The prevalence of Ascaris infection was 53.4% and 51.0% at Maliboda and Ayr estates respectively.Highest infection and re-infection rates at Maliboda (37.7%, 37.2%) occurred in June and at Ayr (13.3%, 25.9%) in October 2000 respectively.During the study period, the mean rainfall was 28.1 cm (range 7.4-63.9cm) and mean temperature 27.6 degrees C (range 22.1 degrees -34.4 degrees C).Significant correlations were found between the re-infection rate and rainfall, temperature and the number of wet-days.Similar correlations were observed with the infection rate and temperature and the number of wet-days.Ascaris infections were found to correlate significantly only with the number of wet-days in a month.The authors concluded that the number of wet-days appeared to be a better indicator of Ascaris infections than total rainfall or mean temperature.
The third paper examines the climatic, socio-economic and behavioural factors influencing N americanus infection in the same study population. 18The baseline prevalence of hookworm infection was 28.5%, while the intensity of infection ranged from 0-4,828.5 eggs/g faeces, with a mean of 128.4 eggs/g.The monthly incidence of hookworm infection was found to show three peaks at Maliboda (September 2000 (21.3%),January 2001 (20.8%) and May 2001 (17.5%)), while two peaks were seen at Ayr (September 2000 (25.0%) and February 2001 (29.2%)).The authors found that the incidence of hookworm infection showed statistically significant correlation with mean temperature.Bathing and washing with water from rockpools formed by waterfalls, the use of wells, and a lack of toilets were also found to increase the risk of hookworm infection significantly.Some years later, data from these three studies were re-analysed to evaluate patterns of coinfection and changes in egg deposition. 19This analysis found positive associations between T. trichiura and both N. americanus and A. lumbricoides, but no association between N. americanus and Ascaris.It was shown that N. americanus and Ascaris infections had lower egg depositions when they were in single infections than when they were co-infecting.There was no clear evidence, however, of a similar effect of co-infection in Trichuris egg deposition.The authors concluded that associations in prevalence and egg deposition in STH species may vary, possibly indicating that effects of co-infection are species dependent.
In a separate, cross sectional study conducted in 2013, 258 children aged 1 -6 years, living on Uduwela tea plantation in the Kandy District were examined to determine the prevalence of ascariasis and factors associated with it. 20Data regarding socio-demographic and hygienic habits were collected from heads of households via an interviewer administered structured questionnaire.Wet mount preparation, formaldehyde-ether sedimentation and Kato-Katz techniques were used to examine stool samples for Ascaris eggs, and prevalence was found to be 37.8%.On multivariate logistic regression analysis, factors significantly associated with Ascaris infections included living in attached houses, shared toilet facilities, de-worming period more than three months, maternal education level and living in the "top" government administrative division in the study area.As in the other studies from the estate sector, poor sanitation facilities and poor health education were found to be important factors associated with Ascaris infections.
Impact of control programmes on STH prevalence
Mass deworming programmes were widely pursued by public health authorities in Sri Lanka in the first half of the 20 th century, even though the anthelmintics were not very effective. 12ore recently however, anthelmintics have not been used on a mass scale, except in relation to certain defined, target populations.
For example, a biannual mass deworming programme with single dose mebendazole (500mg) targeting school aged children was implemented in the plantation sector from 1994 to 2005, after it was shown that the prevalence of STH infection among these children was over 90%. 21In 2009, five years after the deworming programme had been abandoned due to lack of funds, faecal samples from 1,890 children attending 114 estate sector schools in five districts (Kandy, Nuwara Eliya, Badulla, Ratnapura and Kegalle) were examined using single Kato-Katz smears. 22Although the prevalence was nowhere near as high as in the 1990s, the overall combined prevalence was still high (29.0%),while 24.4% had ascariasis (found in all five districts), 5.9% had trichuriasis (also seen in all five districts), and 4.7% had hookworm infections (not found in Nuwara Eliya and Badulla districts).This study demonstrated that even after ten years of mass chemotherapy, prevalence can bounce back after cessation of preventive chemotherapy, if the initial force of transmission is strong and other long-term control measures are not concomitantly implemented.
The above results present a stark contrast with findings from the rest of the country.During the period 2002 -2006, the mass drug administration (MDA) programmes conducted by the Ministry of Health to eliminate lymphatic filariasis (LF) as a public health problem from Sri Lanka, involved the annual administration of diethylcarbamazine citrate (DEC) and albendazole to all those living in the nine LF-endemic districts along the Western and Southern coastal belt.Since albendazole is very effective against STH infections too, several studies examined the effect of the LF MDA programme on STH infections.
In the first of these, children attending six selected schools in the Ragama Medical Officer of Health area were examined just before and after the MDA programme in July 2002. 23Of the 265 children examined in the baseline survey, only 12 had any STH infection (prevalence 4.5%).In the follow-up survey only 2/252 children (2.0%) were infected.Like overall prevalence, the mean egg counts (as estimated by the examination of single Kato-Katz smears) also declined from baseline to follow up but the differences were not statistically significant in either event.
The second survey was conducted in 2006 to assess the impact of LF MDA in the Western Province. 24Faecal samples from 448 children who attended 17 schools selected for the national survey conducted in 2003 (just after the 1 st round of LF MDA) were examined using single Kato-Katz smears.Roundworm prevalence was found to be marginally lower in 2006 (4.0%) than in 2003 (4.7%), as was hookworm (0.2% vs 0.4%), whereas whipworm prevalence was higher (13.8% vs 9.4%).However, these differences, as well as that between the geometric mean egg counts, were not statistically significant.Compliance with MDA in 2006, however, as reported by the schoolchildren examined, was only 59%.The findings suggested that four annual rounds of MDA with DEC and albendazole had virtually no effect on STH infections among school children in the Western Province.
A third survey, conducted in 2012, explored the practicality of integrating surveillance for STH, (assessed by Kato-Katz) with transmission assessment surveys for LF in two evaluation units (EUs) in the Gampaha district. 25Each transmission assessment survey tested children (N = 1,462 inland EU; 1,642 coastal EU) sampled from 30 primary schools.Low filarial antigenemia rates (0% and 0.1% for the inland and coastal EUs) suggested that LF transmission was very low in the Gampaha district.The STH rates were also low: 0.8% (inland) and 2.8% (coastal).Most STH detected were low or moderate intensity T. trichiura infections.The added cost of including STH testing was estimated at approximately $5,000 per EU, and the authors concluded that the results suggest that it is feasible to integrate school-based surveillance for STH and LF.
Drug resistance
Large-scale treatment with the benzimidazoles (albendazole and mebendazole) has been adopted globally as a major control strategy against STH infections.Prolonged and repeated treatment with the same anthelmintics has led to the emergence of widespread benzimidazole resistance in veterinary parasites, caused by a single nucleotide polymorphism at codon 200,167 or 198 in the β-tubulin gene. 26Concern that prolonged use of these anthelmintics may select for resistant parasites has promoted efforts to develop genotyping assays to screen for βtubulin polymorphisms in N. americanus.Single nucleotide polymorphism assays were developed based on the Smart amplification method (SmartAmp2) to target the above codons in the β-tubulin isotype 1 gene and applied to 110 N americanus larval samples from the Ratnapura district in Sri Lanka. 26None of the larval samples showed significant levels of polymorphisms either at positions 167 or 200, but a polymorphism was identified at codon position 198A/C in some samples, the first time that this mutant type has been detected in N americanus.The results suggest that although full-blown benzimidazole resistance was not present in the samples tested, the mutations associated with its emergence are detectable.
Impact of STH infections on health
As summarized previously, the early literature from the last century bears ample testimony to the morbidity and even mortality caused by heavy STH infections: severe anaemia, reduction in physical fitness, acute intestinal obstruction, rectal prolapse, etc. 12 As prevalence rates and the intensity of infections have declined, these manifestations have become increasingly rare and in the current context, STH infections are often asymptomatic.As a result, the studies SLJID • www.http://sljol.info/index.php/SLJID• Vol. 8, No. 2, October 2018 published over the last two decades on the association between STH infection and ill health have focussed on more subtle manifestations such as physical growth, anaemia and cognitive development in children.
During the national survey of the health status of school children conducted in 2003, children with STH infections were found to have a significantly higher prevalence of anaemia than those who were uninfected. 13There was however no significant association between STH infection and stunting of growth as assessed by height for age z-scores and body mass index.
A prospective placebo-controlled randomized study was conducted in 2009 -2010 to assess the impact of de-worming and iron supplementation on the cognitive abilities and educational achievement of school-age children in the plantation sector. 27The treatment group (n = 615) received de-worming (mebendazole 500 mg single dose) and weekly iron supplementation (tablets containing 200 mg of ferrous sulphate equivalent to 60 mg of elemental iron) for six months, while the control group (n = 575) received placebo for both the anthelmintic and iron.The prevalence of soil-transmitted helminth (STH) infection was reduced in the treatment group, with significant differences between treatment and control groups in the levels of Ascaris and Trichuris.The results also suggested that the anthelmintic treatment was effective against roundworm and whipworm infections, but not hookworm infection.In contrast to the prevalence of roundworm which dropped from 20.8% at baseline to 14.3% at follow-up 6 months later, and whipworm, which also dropped from 7.5% to 4.9%, hookworm infection rose from 5.5% to 8.0% in the treatment group.This is consistent with the evidence from other efficacy trials that mebendazole is not as effective in treating hookworm as it is in treating roundworm or whipworm infections. 28Final analyses found no impact on haemoglobin levels, nor any significant impact on concentration levels or on educational test scores.The authors concluded that decline in STH prevalence alone, in the absence of improved haemoglobin status, produced no evidence of impact on concentration levels or educational test scores.
A study conducted in 2013, on 489 children aged 1-12 years, living on Uduwela tea estate in the Kandy District, examined the association between ascariasis and physical growth of the children. 29The prevalence of ascariasis was 38.4% and most children (51%) had light intensity infections, 30% had infections of moderate intensity, and 19% had heavy infections.The prevalence of undernutrition was 61.7%; 45% per cent were underweight, while 24.1% and 21.5% of children were stunted and wasted respectively.No significant association was found between Ascaris infections status and undernutrition, but heavy intensity infections were associated with decreased values of weight-for-height z-scores (WHZ).
The commensal flora of the gut is known to play key roles in human health, including nutrient metabolism, protection against pathogens and regulation of both innate and adaptive immune responses.Given that the soil-transmitted helminths and the gut microbial flora share the same environment within the human host, it is thought possible that parasite-microbiota interactions may impact on the health of helminth-infected individuals.A study conducted on 76 subjects from 9 villages in four districts of Sri Lanka explored qualitative and quantitative differences between the microbial community profiles of those infected with one or more STH, and uninfected subjects and volunteers who had been subjected to regular prophylactic anthelmintic treatment. 30High-throughput sequencing of the bacterial 16S rRNA gene, followed by bioinformatics and biostatistical analyses of sequence data revealed no significant differences in alpha diversity (Shannon) and richness between groups.However, beta diversity was significantly increased in infected and treated subjects when individually compared to uninfected volunteers.Among others, bacteria of the families Verrucomicrobiaceae and SLJID • www.http://sljol.info/index.php/SLJID• Vol. 8, No. 2, October 2018 Enterobacteriaceae showed a trend towards increased abundance in infected persons, whereas the Leuconostocaceae and Bacteroidaceae showed a relative increase in the uninfected and treated respectively.
Current control strategy in Sri Lanka
In 2012, the Family Health Bureau of the Ministry of Health issued a general circular letter (Number 02-172/2012) with guidelines for de-worming children and pregnant women in the community setting during the period 2013-2016. 31These guidelines divided the country into areas of high risk (Uva, Sabaragamuwa and Central Provinces) or moderate risk (all other provinces), based on the national survey conducted in 2003 13 , together with the 2009 survey in the plantation sector. 22The guidelines recommend that all children in high risk areas should be treated twice a year with a single dose of mebendazole (500 mg), while children in moderate risk areas should be treated once a year.
The guidelines issued by the Ministry of Health address community-based de-worming of both children and pregnant women.The latter group is included because hookworm infections are known to result in iron deficiency anaemia, which has particularly adverse health consequences for pregnant women, and because the age prevalence of hookworm infections, unlike that of ascariasis and trichuriasis, continues to increase until adulthood.Hence the recommendation, first issued by the Ministry of Health in 1994, is that all pregnant women should be given a single dose of mebendazole 500mg after completion of the first trimester of pregnancy. 12 2017, a second national survey of STH infections was conducted, which showed that overall prevalence has declined in all parts of the country, including the high risk communities in the estate sector and urban low income settlements. 32Based on these findings, the Family Health Bureau is currently in the process of revising the de-worming guidelines issued by the Ministry of Health.
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2018-10-31T00:00:00.000
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Stakeholder perspectives on the value of car parking
Car parking is a routine yet highly complex part of daily life for both drivers and those affected by parking. This paper aims to unravel how key stakeholders value parking, by looking beyond the traditional possibilities associated with supply and demand to help better inform decision makers with their parking related dilemmas, by drawing on a series of in-depth interviews. First, interviews were conducted with eight academics who maintain a research interest in parking, to validate key stakeholders and their parking dilemmas as identified from literature. Second, interviews with 20 representatives spanning an assortment of key stakeholder groups affected by parking were undertaken, to determine their perspectives on the value of parking. The findings indicate that a considerably broader reach of stakeholders are affected by parking than the existing literature suggests, and the process of means by which stakeholders value parking is more sophisticated than previously thought. This new finding dispels traditional beliefs relating to how stakeholders value parking; the article outlines the extent to which such beliefs are mistaken, and provides the foundation for further work to understand the extent of these replacement values.
Introduction
According to a recent report by the RAC Foundation, 'the average car spends about 80% of the time parked at home, is parked elsewhere for about 16% of the time, and is thus only actually in use (i.e. moving) for the remaining 3-4% of the time' (Bates & Leibling, 2012, p. iv). As a consequence, accommodating parking either on or off street in larger towns and cities is a significant problem (Glazer & Niskanen, 1992). In spite of its clear importance, parking remains relatively underexplored by the research community (Verhoef, Nijkamp, & Rietveld, 1995). In particular, car parking can be fraught with misconceptions so it is important to educate and involve stakeholders when making parking policy decisions (Shoup, 1995). The extent to which parking can impact on parking stakeholders essentially derives from how they value parking and what influences them to do so.
The aim of this paper is to investigate how a range of stakeholder groups affected by car parking value parking and what influences them to value parking in the way they do. The objectives are threefold. First, to validate the findings from literature regarding who the key car parking stakeholders are. Second, to discover what parking issues are of concern to them. Third, to establish how car parking is valued by the stakeholders and gain an insight into how these car parking issues influence their perceptions of value. The paper comprises a literature review, a method involving preliminary and principal interview stages, findings from each stage, a discussion and a conclusion.
Literature review
This section aims to identify who the parking stakeholders are and to explore the different meanings behind the word value and subsequent connotations for stakeholders, as presented by literature. An analysis of the main parking issues which may affect the stakeholders is also conducted to open up the possibilities of how stakeholder concerns might influence how they value parking. The section begins by reviewing how parking stakeholders and value are construed in literature and moves on to investigate a range of parking issues likely to concern the stakeholders as current in literature.
Identifying parking stakeholders in literature
The conventional approach when seeking to define the term stakeholder might be to take the strategic organisational perspective of, 'those individuals or groups that depend on an organisation to fulfil their own goals and on whom, in turn, the organisation depends' (Johnson, Whittington, & Scholes, 2011, p. 206). However, literature is less generous in defining parking stakeholders (resulting in a gap), so the term stakeholder must be applied. Nevertheless, literature does offer a number of potential stakeholders who are affected by parking.
In précis, despite a comprehensive review of the parking literature, the smattering of stakeholders found seems somehow incomplete and condensed into pockets comprising individual users, with a particular focus on managing their car parking behaviour. The literature's show of restraint in this area seems unexpected, as a more testing challenge might be to find anyone who is not affected by car parking in some way.
Governmental
Parking policy can help to achieve six desirable urban goals (Mcshane & Meyer, 1982, p. 133): (1) healthy economic climate, and a business community able to support local employment needs; (2) most efficient use of existing transportation, land, and other public resources; (3) ease of mobility/accessibility; (4) equity of resource distribution and preferential allocation of some resources; (5) environmental goals, especially reduced air pollution and the related goal of minimised energy consumption; (6) enhanced amenity and cultural attractiveness; preservation of a city's unique character.
In spite of a potential for making tantalising promises, managing parking effectively seems fraught with challenges for those tasked with making policy decisions, as some of these goals may be construed by some to conflict. For instance, a strategy based on localism forms part of the current UK government's vision for managing sustainable transport and tasks local authorities with the dual objectives of creating growth and cutting carbon (Department for Transport, 2011). Furthermore, the separation of residential and commercial areas by planning officials often results in daily needs not being met within walking distance, consequently intensifying car dependency (Button, 2010). Residential areas neighbouring commercial areas can experience pressure from overspill by commuters (Rye & Ison, 2005), obliging local governments to find solutions such as the implementation of controlled parking zones (CPZs) which may not always meet with residential approval (Rye, Cowan, & Ison, 2006). Even in the case of UK park-and-ride (P&R) schemes (large car parks located on urban peripheries with connecting bus services to access the town centre), originally intended to reduce environmental negativities in urban centres, there is concern about attracting drivers who would otherwise have completed the entire journey by bus (Meek, Ison, & Enoch, 2010). Therefore, it seems that governments have responsibility for making decisions regarding car parking policy in an environment where transport levels of demand have exceeded supply, and where the demand is characteristically both qualitative and differentiated, and where the supply acts as a service and not as a product (Dios Ortúzar & Willumsen, 2007). In other words, car parking supply cannot be stocked up to meet differentiated demand.
Land use
In many cases, the desire to satisfy demand has resulted in an oversupply, typically through the use of minimum parking requirements. These decisions have resulted in more land being used for parking for each new development built, thus leading to several environmental negativities, including urban sprawl (Forinash et al., 2003). A consequence of high levels of parking in central business districts (CBDs) is that it reduces population density, attracts more car use while suppressing public transport use, thus negatively impacting on town and city sustainability (Kenworthy & Laube, 1996). In addition, as commuter car parking in areas subject to minimum parking requirements is often supplied to commuters for free, there is little incentive for individual users to seek alternative modes (Shoup, 1995). Also, where car parking levels are high, it is possible that parking requirements may impinge on public space. Public space is described as follows: 'How good a city is at facilitating exchange determines its healtheconomic, social, cultural and environmental.
Public space forms a vital conduit in this exchange process, providing platforms for everyday interaction and information flowsthe basis and content for the public life of cities' (Tims & Mean, 2005, p. 5). In addition, there is the issue of shared space. Car users' dominance in areas of shared spaces triggers the segregation of car users from nonusers (such as pedestrians) who become less comfortable and confident in engaging with their environment (Kaparias, Bell, Miri, Chan, & Mount, 2012). Therefore, the impact car parking can have on land use seems to be detrimental and widespread, with perhaps the highest costs affecting not only the environment but social spheres also.
Exploring car parking stakeholder value in literature
Directly eliciting from car parking literature how stakeholders value parking is challenging as it is not easily detectible. Potentially it can be found where the predominating focus is rooted in quantifiable economics, most specifically in response to controlling car parking behaviour of individual users. The literature seems preoccupied with exploring offsetting decisions made by individual users when presented with a series of choices based on location, time and price attributes, as evident in the work of Glazer and Niskanen (1992), Anderson and de Palma (2004), Shoup (2006), Arnott and Inci (2006), Calthrop and Proost (2006) and Kelly and Clinch (2009). Economics is applied to a range of parking control policies which are directed at exploiting supply-demand relationships. Because of this, the range of stakeholders reached out to could be limited by making assumptions of how individual users value car parking. This is because the word value can suggest both a quality and a monetary figure (Thomson, Austin, Devine-Wright, & Mills, 2003), in which case those affected by parking may value car parking outside economics. Individuals also express their interpretation of value differently (Zeithaml, 1988) and so in précis, value appears to exist in individual abstract perspectives, thus reflected in stakeholder car parking choices, decisions and behaviour.
Value losses and gains
Established ties between parking and economics, as referred to previously, suggest that parking can be valued via the concept of an individual user offsetting relationship using positive and negative attributes (convenience/price), congruent with provider supply and demand elasticities. This notion may be likened to prospect theory which uses losses and gains to determine value worth (Kahneman & Tversky, 1984) and is potentially reflected in car parking policy decisions, such as with minimum parking requirements where the desire to achieve gains for individual users has appeared to exceed the losses experienced elsewhere.
Literature review summary
In summary, the literature presents a modicum of parking stakeholders, focusing predominantly on user-supplier relationships that are often in accord with economic theories of supply and demand. Furthermore, car parking issues seem challenging for decision makers because although car parking facilities can contribute to satisfying multiple urban aspirations, the resulting policies can result in exacerbating parking problems. Also, wider stakeholder and value concepts, outside economics, are not generally reflected in the parking literature despite their potential for application to car parking. This paper seeks to more comprehensively understand who the stakeholders are that are affected by car parking, what parking issues are of concern to them and what impact these issues have on how they value parking.
Method
This study is appropriate for a qualitative approach as its purpose is to explore and interpret phenomena that are not currently available anywhere else (Silverman, 2011). To meet the aim of the study, semi-structured in-depth exploratory interviews were conducted: preliminary (expert) interviews, and principal (sector leader) interviews.
Preliminary interviews
The preliminary interviews with academics, all with a published interest in parking, were selected initially from the literature review and then via a 'snowballing' technique whereby participants suggests further experts whose contribution they deem to be of benefit, in line with inductive theory building analysis (Miles & Huberman, 1994). For the interviewees selected, see Table 1. They are referred to from now on by their representative letter, such as A, B or C, etc.
Eight such interviews were conducted (seven via telephone, one via Skype) with 19 questions focusing on three topic areas: car parking stakeholder identification, parking issues and value. The questions asked had three aims. First, to identify who the academics consider are the stakeholders affected by car parking. (To reliably achieve this aim the academics are presented with a table of stakeholders, as suggested by the literature, at the end of the interview, presenting the opportunity for further comment.) Second, to establish what the academics consider are the key parking issues that are of concern to the stakeholders. And third, to gauge the academics' opinion on how they think the stakeholders might value parking. Satisfying these three aims helped to focus the questions for the principal set of interviews with representatives of the stakeholders themselves.
Principal interviews
The principal set of interviews was conducted with those considered to be sector leaders of parking stakeholder groups as classified and agreed by the academics. Nineteen telephone interviews plus one via email were conducted comprising 20 questions pertaining to three topics based on analysis of the academics' responses: car parking stakeholders, issues and value. The overriding aim was not only to reveal how the stakeholders value parking but to understand the environment in which their parking issues affect their value of parking. The sub-aims were threefold: first, to further endorse stakeholder
Analysis strategy
After transcription, the analysis strategy was similar for both sets of interviews. To start, NVivo TM data management software is employed to assist with handling the volume of textual data generated. Following data input, an inductive technique following a methodical process of building patterns and applying codes was used. Initial descriptive codes were allocated to segments of transcript where common themes began to emerge, first from the individual responses to an individual question, second from the collective responses to an individual question and third, from the collective responses to the collective questions. Analysis coding enables the data to be synthesised while maintaining interaction (Miles & Huberman, 1994) and facilitates thorough investigation.
Preliminary interview findings 4.1. Stakeholder identification and classification
In the preliminary series of interviews the academics were asked to identify and then classify who they consider to be the key stakeholders affected by parking. They were also afforded the opportunity to comment on an existing classified set of stakeholders as elicited from the parking literature. This resulted in minor adjustments (underlined, see Table 2). 'The academics' responses approve four primary groups of parking stakeholders which serve to identify the sector leader interviews to be undertaken for the principal interview stage, namely non-consumers, consumers, suppliers and governmental. Each of these primary groups comprises sub-groups which describe their roles. Of particular interest here, is that the 'local business sector' sub-group straddles the 'consumers' and 'suppliers' primary group categories, whereby retailers, employers and financiers to developers are classified as being consumers, while developers, architects and professional associations are more likely to be suppliers. Overall, a total of five sets of four interviews were conducted with sector leaders of the parking stakeholder groups (see Table 3). Table 4 presents what academics perceive to be the key car parking issues of concern to the stakeholders and how they believe that these are currently being addressed. The academics described various car parking characteristics (see Table 4, 1.0) which they considered were common causes of most parking issues troubling car parking stakeholders.
Parking issues according to academics
The second order categories (1.2-1.6) were felt by the academics to be of similar significance; however, 'Land used for parking limits other opportunity uses' (1.1) was of highest interest to the academics located in America. H referred to dilemmas and consequences: 'Cities in America provide way too much parking. By providing more parking they think they are competing with the suburbs and it actually has the exact opposite effect. We find that in cities with the highest amount of parking, there is less population and less jobs per square mile.' G was also frustrated by parking's emotional characteristic: 'Expert knowledge is given no credence compared to emotional knowledge.' He felt that this resulted in 'terrible parking policies'. 'Potential parking issue solutions' (2.0) has three second-order category subsets which the academics identified as commonly implemented policy practice. Dissatisfaction with all three subsets was conveyed across the academics with G venting particular disapproval for 'Free or low cost to the user' (2.3): 'If you're giving away something for free, and there are many who do not need it, it leads to a lot of abuse.'
How stakeholders value parking according to academics
The different ways that stakeholders may value parking according to the academics are presented in Table 5. Table 5 presents the second-order categories in no significant order except for 'Objective based' (2.1), as the majority of academics felt that how stakeholders might value parking was almost entirely dependent on their primary objective; C says 'Value is about objectives'. Possibly, the remaining second-order categories may be construed to be stakeholder objectives but the academics spoke about them in terms of values. For instance regarding 'Lifestyle facilitator' (2.7), E believes that, 'Parking allows you to have accessibility to whatever it is you're doing. It's the most important factor in the way people value parking.' And B agrees: 'Parking is something that makes their [users'] lifestyle possible.'
How stakeholders value parking
The sector leaders speaking from the perspective of representing the stakeholder groups define their value of parking in eight different ways organised into a matrix, Table 6. How the groups value parking is expressed in either a positive or a negative way, or both, such as in the cases of the local business sector, parking industry and governmental groups, which interchange between the two depending on the context of the value.
In the first instance it is visible from Table 6 that the non-consumer group entertains no positive ways to value car parking. Instead it has a negative perspective on two ways that parking can impact on the environment. In contrast, the consumer group is singularly positive demonstrating values focusing on the environment, access and economics.
The remaining three groups appear divided on at least one environmental value. The local business sector assumes a similar division about an additional environmental value but holds a positive value of the commercial aspect of parking. The parking industry and governmental groups are equally weighted but with one point of difference. The parking industry group exclusively holds a positive value of the combined convenience, safety and price and the governmental group is positive in reference to an efficient transport system. No single group holds all eight values and it seems that occupying a central place in the matrix enables the local business sector to see both sides of a value, which may be indicative of their position within the macro environment. Each of the eight values is explored in turn.
Efficient use of land
The question, Do you consider that using land for parking is an efficient use of land?, is responded to by all interviewees. Three of the non-consumer group place a negative value on using land for car parking (NC3 is the exception, 'you need to strike a balance') and NC1's response is representative, 'It's a highly inefficient use of space.' The non-consumer group responses concern wider implications than the unrefined issue of 'efficient use of land', such as a lack of priority for cycle parking (NC4) and a car-dominant mindset of policy makers (NC2). The non-consumer group seems conscious of the impact parking has on land use only in terms of a loss and therefore manifests a strong negative value of parking.
In contrast, the consumer group maintains a positive view as it believes that, in some circumstances, using land for parking carries two key gains, facilitating access and helping to sustain economic activity. C1: 'I feel that the car brings enormous benefits to sections of the population … [the elderly] are engaged in community life, they are also far more self-sufficient than they might be if they were relying on lifts or relying on public transport.' C2 expresses a similar sentiment with regard to the disabled community who are often challenged by alternatives. C3 refers to economic matters: 'For local economies parking is the lifeblood. It is essential to the community and to the economy.' C4 agrees: 'If land used for parking contributes to economic activity then there is nothing wrong with that.' The rest of the groups are divided. On the one hand there is negativity; LBS1 feels that local authorities should 'hand the town centre back over to pedestrians', by rationalising parking and PI4, 'If you look at land costs, almost everywhere in the city it would be more economically beneficial to use the land for something else other than parking.' Yet, on the other there is positivity; G1: 'Broadly, yes. In as far as its necessary to get people to and from places.' PI1, 2, 3 and G2 use the phrase, 'It depends', and then describe a balancing act between competing land uses, commercial viability and environmental impacts.
Impact on public space
This is a priority for the non-consumer group and stimulates a negative value of car parking as the group perceives that parking can place public space at risk of experiencing a loss. NC1 gives a typical response from the non-consumer perspective: 'People bring vitality, life and economy to a space whereas cars bring dead space to public areas … parking on the footpath is a critical issue as it's advocating public space to vehicles … it takes away space for people to walk in and enforces the ownership of space by vehicles.' NC2 raises the issue of broader losses potentially caused by parking's impact on public space such as a loss to quality of life and a negative economic health value.
The issue is not referred to by the other groups except by the local business sector which is split over the matter. On the one hand there is empathy for the non-consumer group's view: LBS1, 'It shouldn't be allowed to override or displace other attributes of society, or the natural and built environment.' Yet, on the other, there is potential for commercial gain for elements of the local business sector if they are in a position to satisfy demand, such as in the case of developers: LBS1, 'Integral parking is perceived as quite a benefit to prospective tenants.'
Facilitates access
The consumer group gives a sense that parking is intrinsic to everyday life as it enables lifestyles by providing accessibility, which the group values as a positive gain. C1: 'Parking is important because it allows all of us to do what is considered to be very normal activities that make up life in the United Kingdom. That's engaging in work, sporting activities, social activities, and so on.' C3 agrees and C2 emphasises the importance of parking facilities to particular groups such as rural communities who are otherwise restricted by a lack of alternatives. C4 reiterates the perceived positive gains: 'There are many social advantages to car park provision in the same ways that there are social advantages to owning a car.' Comparable responses emerge from the parking industry. PI1: 'Society is dependent on the motor car. Therefore if we want to be a car owning, car-borne society we have to manage parking.' PI4 also sees the positive gains from access and PI3 is consistent yet mindful of the challenges involved: 'Parking is an essential part of any journey. You have to have somewhere to park; the question is what is the most efficient way of doing that.' The governmental group also positively values parking's ability to facilitate access. At a regional level, G1: 'It isn't so much to do with parking it is actually to do with access. So let's equate parking with access more readily, let's not make it all about the car.' Parking facilitating access and the consequential potential business and community gains are underlined at the national level by G4: 'Parking plays an important role for both business and leisure; without it we would not be able to access even our most essential amenities.'
Sustains economic activity
Sustains economic activity is ascribed by the consumer group as a way in which to find positive gains and therefore value from parking and in particular, free parking.
For instance, C2's response is representative of the consumer group: 'Out-of-town shopping centres have free parking and so customers go out-of-town. What is happening today is that there are fewer shops on the high street, one of the reasons for this I'm sure of, is that parking is so expensive.' Valuing parking through its potential ability to sustain economic activity is not referred to by the other sector leaders except for G1, at a regional level, who is sceptical about the motivation behind pro-free parking opinion: 'This link between business viability and free parking is a growing one and I have a feeling that it's almost used in some cases as an excuse by smaller shops. My shop is failing because you've made parking more difficult, is something I hear even when I show them that I've actually made parking easier and more people are getting there as a result, they still insist on taking the irrational sort of view.'
A commercial product
The local business sector group alludes to a positive value of parking by treating it as a commercial product: LBS2, 'I value it as a commercial product.' The rest of the group discuss their perceptions of the local business sector environment and how parking can be indirectly used to attract commercial gain. LBS1, whose employer provides consultancy services to various entities including developers, believes that 'Parking is still seen as a benefit, or an advantage, or as a value for developments.' Likewise, LBS4 is representative of an organisation with a significant retail portfolio: 'Parking is important to me and my business as it provides a major role in our properties.' These statements are supported by a sector leader from the governmental group G1: 'In strict commercial terms parking space can be an incredibly commercially viable asset', indicating that the local business sector can realise a positive commercial gain from parking, despite it not being their priority business. Most surprising perhaps, is that the word commercial does not feature in any of the parking industry's transcriptions.
Revenue stream
Positive gain from parking as a revenue stream is inferred from both the parking industry and the government group's transcripts. From the parking industry perspective, PI1 says: 'Parking is a revenue generator'; and PI2 and PI4 expand by detailing how the positive gains can be felt by the user; PI4: 'Income needs to go back to the motorist in the form of better quality, facilities, better management, and through the work of Park Mark by the British Parking Association (BPA).' P2: 'If you put the right amount of resources in parking has a value to the customer but if parking is given no value, then it has no value.' From the governmental perspective, G4 (national level) states: 'Parking is an important funding stream for local authorities, even more so given the wider financial constraints in which we find ourselves.' Equally, the positive value of a revenue stream is emphasised by G3 (local level): 'Parking revenue is very important for a local authority … The revenue is reinvested into public transport … So it's important in terms of our wider transport priorities in the city.' This is further supported by some speculative remarks made by the other stakeholder group respondents, aimed predominantly at the governmental group. PI4's comment is characteristic: 'Parking does give them an income and increasingly these days it is an important income particularly in times of austerity.' Yet PI3's observation is representative of an underlying unease or suspicion about the issue: 'They strenuously deny that they are using parking as a way of generating income … it clearly does provide a contribution towards their finances.' 5.1.7. Convenience, safety and price Convenience, safety and price are value perceptions held by the parking industry regarding how it perceives that the individual users it serves receive positive gain from parking. PI2: 'Convenience is always going to be the first choice, and when having a choice, choose the safe one, rather than the cheapest one. So convenience, safety, price is my view.' Two of these values, 'convenience' and 'price', are understood by PI3 who describes a troubling example of how an outpatient might value parking: 'It may seem [unfair] to charge somebody who is going for cancer treatment to park their car at a hospital, however it may be a better thing than not charging and that person not finding a space because they've been taken up by a lazy commuter who can't walk a bit further to another car park and pay for parking.' This is supported by PI3 who is fearful of the impacts of an increasing trend to make hospital parking free of charge leaving patients struggling to find available spaces: 'What was called the tax on the sick has now become an attack on the sick.' Price is raised by the majority of the consumer group and their comments imply that they generally accept paying for parking. C1's remark is typical: 'I think charging for parking is entirely fair, simply in terms of paying for a service which somebody has had to provide.' The other groups take a similar view; NC3, LBS4 and G1 describe priced parking as 'necessary'; NC2 as 'fair'; G2 as 'logical'; NC1 says, 'I take a user payer approach to this'; G4, 'It is important that we have a system that is fair to the motorist, but which also recognises the cost of regulating and maintaining parking facilities.' Priced parking seems to concern all the stakeholder groups, yet only the parking industry seems to understand its potential as a positive gain or value to the consumer, such as to an outpatient. Instead, the consumer group, along with the other groups, perceives cost as an expected part of a parking activity and is less attentive to its broader potential. With regard to 'convenience' and 'safety', the consumer group seems to prefer to discuss access to enable a lifestyle (see 5.1.3), adding a depth to the value. Safety is absent in all the different groups' transcripts, except for C2 who links the safety of parking facilities with price, concluding that safety is a minor concern outside the parking industry.
Part of an efficient transport system
This is of positive value to the majority of the governmental group. G3 (local level) gives a typical response to the question, How do you value parking?: 'Parking is an integral part of our transport policy for the city and for the city centre. Our policy in the city is very clear, we will continue to accommodate the car and we will promote public transport.' G4 (national level) develops the point further: 'I want a system of parking which supports local businesses and allows local residents and visitors to access our towns and cities conveniently. But it must also help to ease congestion, thereby reducing the time taken to navigate urban areas cutting unnecessary emissions.' The perceived positive gains resulting from a vision of an efficient transport system demonstrate a depth of understanding by the governmental group of the range of issues that parking can impact on. This is not articulated by the remainder of the sector leader interviewees as discussion is dominated by specific issues, such as managing the high street (NC1), parking on private land (PI2) or adequate provision (PI3), despite asking all the groups an identical set of questions.
Stakeholder perspectives on how they each value car parking
The ways in which the stakeholders describe how they value parking are influenced by a range of parking issues which the sector leaders drew attention to during the interviews. These are classified into three key groups of government, land use and a focus on the consumer (as presented in Table 7).
Government
Four of the five stakeholder groups express negative concerns about issues related to the government, as given in Table 8.
The devolution of UK parking powers from national to local levels is of particular concern and is frequently held responsible by the stakeholder groups for leaving some local authorities feeling isolated and deficient in essential knowledge and expertise, which they believe is necessary to enable them to resolve their local issues. As the instigators of devolution, the UK government (national level) seems determined to persevere and encourage a local approach. Through its lack of references the parking industry gives a sense of operating independently from government, possibly due to a lack of national guidelines and standards.
Land use
Land use is of most significant concern to the non-consumer and governmental groups (see Table 9).
The non-consumer group perceives that the government consistently delivers cardominant policies in terms of land use, thus disseminating a distorted view of what optimal land uses might otherwise be. For the non-consumers, a consequence of witnessing a car-dominant landscape is a general tolerance of parking intruding into public space in the absence of any clear alternatives or retribution. Public space is found to be something of particular value to the non-consumer group (see 5.1.2) and as such nonconsumers feel that government should demonstrate a responsibility for its prioritisation. The governmental levels show awareness of competing land uses but they also experience a broader range of pressures, such as austerity and accessibility, which they perceive as something they must be accountable for and are challenged to prioritise and offset.
Focus on the consumer
The parking industry stakeholder group seems to express its value of parking through focusing on consumer issues, such as: facilitates access (see 5.1.3); convenience, safety and price (see 5.1.7). Table 10 presents some of the issues the stakeholders feel are of concern to the consumer which seem to influence how the parking industry values parking.
The industry is responsive to consumer parking dilemmas and so endeavours to provide facilities accordingly by delivering competitive standards. It speaks of reforming consumer perceptions of paying for parking as a 'grudge purchase' by supplying consumers with facilities they might instead value, hence convenience, safety and price (see 5.1.7). Parking consumers are of no concern to the non-consumer group and of secondary value to the local business sector and governmental groups, and as such are scarcely mentioned, but they are of significant interest to the parking industry.
Discussion and conclusion
The use of in-depth exploratory style interviews conducted to identify who the stakeholders are that are affected by parking, what parking issues are of concern to them, how they value parking and how the issues that concern them impact on their value of parking has to some extent confirmed and added to the literature review findings.
Stakeholder identification
The academics interviewed sanctioned and contributed to the stakeholders identified from the literature and the classification of their groups (see Table 2), thus comprehensively authenticating and advancing the range of car parking stakeholders that literature currently offers. This establishes a platform for further investigation into stakeholders which extend beyond those within the consumer group. It is hoped that in the future, an equal number of stakeholder groups will be afforded the same attention.
Parking issues which motivate how stakeholders value parking
Parking policy's capacity to contribute to the six desirable urban goals (see 2.2.1) as specified by the literature appears to address many of the key parking issues held by the stakeholders which form the motivation behind how they value parking. What this paper has achieved is that it has exposed which stakeholder group values which urban goal and whether they perceive that value to be either a positive or negative value of parking.
For instance, the second goal mentions the efficient use of land. This was perceived by the academics to be of priority interest to the stakeholders and was indeed discussed by all of the stakeholder groups in terms of how they value parking. However, only the non-consumer group shared with the academics the wholly negative view of using land for parking (mostly in relation to the fifth urban goal), leaving the consumer group in opposition through their perceptions of only the positives (mostly in relation to the first and third urban goals), and the rest of the groups divided between positive and negative views. Governmental Representatives from local, regional and central UK government G1, 2, 3, 4 For the non-consumer group, a clear issue was the impact that car parking can have on public space; they seem to value public space for qualities similar to those presented in the literature, as given by Tims and Mean (2005, p. 5).They perceived this impact as a significant loss or disbenefit to society and thus a negative value, endorsed by the sixth desirable urban goal, and to some extent the fourth. Car parking's impact on public space was of no concern to the rest of the stakeholder groups (or to the academics), except for the local business sector, which was again split between the views of the non-consumer group and the first urban goal of a healthy economic climate.
Access was a key issue raised by the majority of stakeholders, with three of the five groups understanding only the benefits that car parking can bring to enabling a lifestyle for consumers of car parking. Access forms the third urban goal and was also perceived by the academics to be a way in which only the consumer group of stakeholders would value parking (see Table 5, 2.7). Additionally, at least three of the ways that the Table 4. Academics' perception of car parking issues which are of concern to stakeholders and commonly implemented policy solutions.
First-order category
Second-order category Description Parking is often provided to the user for free or at a low cost stakeholder groups positively value car parking are economically underpinned (see Table 6): sustains economic activity (see 5.1.4), a commercial product (see 5.1.5) and revenue stream (see 5.1.6). Despite none of these being raised as a parking issue by the academics, they were touched on in the academics' perception of how those in the consumer/supplier stakeholder group (all stakeholders except for the non-consumer and governmental groups) might value car parking (see Table 5, 2.2revenue stream) and are perhaps best reflected in only the first of the desirable urban goals. The second desirable urban goal, in addition to mentioning land, also considers the efficient use of existing transportation. This seems to be a contributor to a key positive way in which only the governmental stakeholder group values car parking (see 5.1.8). The academics come close by referring to 'policy facilitator' (see Table 5, 2.3) as a way they perceive local governmental stakeholders might value car parking, but neglect to address it specifically as an issue, instead considering it more tenuously as 'parking is one component' (see Table 4, 1.4).
A missing element from the desirable urban goals is that of convenience, safety and price (see 5.1.7), as motivators behind a key way in which the parking industry stakeholder group values car parking. Instead, both convenience and price are extensively addressed in the literature through such works as Glazer and Niskanen (1992), Anderson (2004), Shoup (2006), Arnott and Inci (2006), Calthrop and Proost (2006) and Kelly and Clinch (2009); the issue of safety is not sufficiently attended to. This paper develops the findings from literature by presenting clarification in the positive and negative ways that particular stakeholder groups value parking, as motivated by their issues with parking. What specifically influences the positive and negative values is focused on next.
Influencers of how stakeholders value car parking
The literature review revealed two prevalent concerns, governmental (see 2.2.1) and land use (see 2.2.2). These encompass a multitude of parking issues which concern the car parking stakeholders interviewed. They are significant as they seem to be key influencers of how some stakeholder groups value car parking. 'If we reduce the amount of parking, we can increase the amount of public space that is available for people to walk and spend time in … how you impact on public space should be reflected in a pricing structure, but people don't feel they should have to pay for parking, they feel annoyed about it and that public space should be managed for parking.' (NC1) 'It is an efficient use of land … but it has to compete with other demands for the use of land.' (G2 national level) Frustrations experienced by users have been responded to by the parking industry yet passing the costs on to the consumer leads to consumer perspective of parking as a 'grudge purchase' 'Individuals have to pay real cash to park, they have to get money out of their purse put it in the machine and the whole process focuses attention on to actually paying money. In addition, people feel that the roads belong to them, they pay our taxes for providing and maintaining them so why should they have to pay any extra to park.' (PI3) 'There is no such thing as a free parking space. Someone's paying for it so why should that not be the user. ' (PI2) For instance, the preliminary interview findings disclosed that car parking experiences included various parking characteristics (see Table 4) which can intensify more widespread complications involving issues of supply and demand (Dios Ortúzar & Willumsen, 2007). Included in 'Characteristics of parking issues' (see Table 4, 1.0) as given by the academics, are six second-order categories, many of which include dilemmas experienced by the governmental stakeholder group, such as parking is complicated (1.2) and challenging decision making (1.6). Indeed, almost all the stakeholder groups expressed concern that the government might be struggling to manage car parking to meet with their satisfaction (see Table 7). Consequently, it is the stakeholder groups' perceptions of how the government manages parking that influences the stakeholders to value parking in the ways in which they do.
With regard to land use, car parking's impact on public space was a key concern for the non-consumer group and contributed to its negative value of parking (see 5.1.2). Indeed, the literature seems to agree by also presenting negative environmental impacts of car parking (Forinash et al., 2003;Kenworthy & Laube, 1996). The findings from the preliminary interviews pointed to land use as a dominant issue for the academics although their concern was more focused on land opportunity uses and was neglectful of perceiving land use as connected with public space.
The parking industry group's value of car parking was found to be influenced by their focus on consumers (see 5.2.3 'Focus on the consumer'). This group seems preoccupied with pacifying their perceptions of consumer frustration, such as over paying for parking, by delivering what the parking industry deem to be good value themselves, that is, convenience, safety and price (see 5.1.7 'Convenience, safety and price)'. The closest representation of this in literature is perhaps best presented in 2.3 'Exploring car parking stakeholder value in literature', which explores consumer behaviour in relation to convenience and price. Despite being of significance to the parking industry, safety as an influencer of how the parking industry values parking seems not to be reflected in the car parking literature.
Summary
This paper aimed to unravel how key stakeholders value parking and has productively achieved this aim through a series of clear and comprehensible tables. The tables concisely present who the key stakeholders affected by car parking are, the issues that lie beneath how they value car parking, the key ways in which they value parking and what influences their value of parking. Despite the literature's acknowledgement of a range of stakeholders, the findings have extended that range and exposed that car parking stakeholders value parking in multiple ways which stretch beyond conventional economic controls. Those making decisions in car parking, particularly at a policy level, may benefit from the findings in application.
|
2018-12-02T05:20:24.635Z
|
2014-01-01T00:00:00.000
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250337710
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pes2o/s2orc
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v3-fos-license
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Outcomes of laboratory-confirmed SARS-CoV-2 infection during resurgence driven by Omicron lineages BA.4 and BA.5 compared with previous waves in the Western Cape Province, South Africa
Highlights • Severe hospitalization or risk of death was similar for BA.4/BA.5 and BA.1 infections.• Previous infection and vaccination strongly protected against severe COVID-19.• Growing population immunity against COVID-19 resulted in reduced severe disease.• Booster vaccinations are important to reduce the public health impact of COVID-19.
We, therefore, compared outcomes of laboratory-confirmed SARS-CoV-2 infections during the April-June 2022 resurgence (proxy for BA.4/ BA.5 infection) with outcomes during each of the four previous waves in South Africa, each of which was caused by a different variant or sublineage, using data on patients with laboratory-confirmed SARS-CoV-2 infection aged ≥20 years using public sector services in the Western Cape Province, South Africa.
Methods
We conducted a cohort study using de-identified data from the Western Cape Provincial Health Data Centre (WCPHDC) of public sector patients aged ≥20 years with a laboratory-confirmed COVID-19 diagnosis (positive SARS-CoV-2 polymerase chain reaction (PCR) or antigen test). The Western Cape has nearly 7 million inhabitants, of whom approximately 75% use public sector health services ( Western Cape Department of Health, 2020 ). The WCPHDC and approach for this study have previously been described in detail ( Boulle et al . , 2019 ;Davies et al . , 2022 ;Hussey et al . , 2022 ; Western Cape Department of Health in collaboration with the National Institute for Communicable Diseases, 2020 ). Briefly, for this analysis, waves of infection were defined as starting and ending when the 7-day moving average of public sector COVID-19 hospital admissions exceeded and dropped below 5 and 12 per million population, respectively. We included cases diagnosed from 7 days before the wave start date to 7 days before the wave end date to account for the lag between infection/first symptoms and hospitalization. We thus included data on cases diagnosed from May 1-May 21, 2022, for the BA.4/BA.5 wave, with followup through to June 11, 2022. This corresponds to the period when BA.4/BA.5 dominated in the province, accounting for 90% of sequenced cases in the Western Cape (4 95/54 8; the remainder were BA.2 [n = 51] with one BA.1 and one recombinant) as shown in Figure 1 ( Network for Genomic Surveillance in South Africa, 2022 ).
We used Cox regression adjusted for age, sex, geographic location, comorbidities, service pressure (number of weekly admissions in the health district) at the time of diagnosis, previously diagnosed infection ( ≥1 laboratory-confirmed SARS-CoV-2 diagnosis ≥90 days previously), and SARS-CoV-2 vaccination to assess differences in the following COVID-19 outcomes between waves driven by different variants: (i) death and (ii) death or severe hospitalization (admission to intensive care or mechanical ventilation or oral/intravenous steroid prescription). We only included outcomes within 21 days of COVID-19 diagnosis for comparable ascertainment across all waves. All deaths within 21 days of a COVID-19 diagnosis were included unless a clear non-COVID-19 cause of death was recorded. For patients with recorded South African national identity numbers, data are linked to the South African vital registry to identify deaths not recorded in the WCPHDC. Vaccination data were obtained by linking the South African national identifier to the Electronic Vaccine Data System, which records all vaccines administered in the country. The only vaccines available in South Africa to date are BNT162b2 and Ad26.COV2.S. For the regression models, vaccination status was categorized into five groups: (i) "≥3 doses" (three or more homologous or heterologous doses of any vaccine), (ii) "two doses" (two doses of any vaccine), (iii) "single dose Ad26.COV2.S" (single dose of Ad26.COV2.S), (iv) "single dose BNT162b2" (single dose of BNTB162b2), or (v) "unvaccinated". Participants were considered to be in a particular vaccine group if they had received their last dose ≥7 days before COVID-19 diagnosis for "≥3 doses", ≥14 days before for "two doses", and ≥28 days before for the single dose categories.
The study was approved by the University of Cape Town and Stellenbosch University Health Research Ethics Committees and Western Cape Government: Health. Individual informed con-
Discussion
Using the period of diagnosis as a proxy for being infected with different Omicron sublineages in the Western Cape, we found no difference in the risk of severe COVID-19 hospitalization or death during the BA.4/BA.5 period compared to the BA.1 period, both of which had better outcomes than previous waves. Strong protection against severe COVID-19 conferred by previous infection and vaccination was retained in the BA.4/BA.5 wave, with three homologous doses of Ad26.COV2.S or BNT162b2 or a heterologous combination of these provides 83% protection (95% CI 60; 93%) against severe COVID-19 hospitalization or death among laboratory-confirmed cases.
A study in animals recently suggested that BA.4/BA.5 may be more pathogenic than BA.2 ( Kimura et al . , 2022 ). Although we did not compare BA.4/BA.5 with BA.2 directly, as BA.2 did not cause a distinguishable surge in infections in the Western Cape, disease severity of BA.2 and BA.1 are similar ( Lewnard et al . , 2022 ), and we found no evidence of worse clinical outcomes with BA.4/BA.5 compared to BA.1. Nonetheless, our findings need to be interpreted in the context of South African SARS-CoV-2 epidemiology with progressively increasing seroprevalence due to previous infection (mostly undiagnosed) and/or vaccination ( Bingham et al . , 2022 ;Madhi et al . , 2022 ;Sun et al . , 2022 ). For example, among blood donors, after the BA.1 wave, the estimated national prevalence of anti-nucleocapsid antibodies was 87% (indicating previous infection), with a further 10% having anti-spike antibodies only (suggesting vaccination without previous infection) ( Bingham et al . , 2022 ). Since anti-nucleocapsid antibodies have lower sensitivity for identifying previous infections and may wane, it is possible that previous exposure to SARS-CoV-2 infections and/or vaccination may even exceed 97%. Indeed, our finding that the aHR shifted toward a lower risk of severe outcomes during BA.4/BA.5 vs BA.1 in models not accounting for vaccination and previously diagnosed infection suggests that the observed continued ecologic decoupling of COVID-19 cases and severe outcomes, is at least partly due to growing protection against severe disease from both previous infection and vaccination. The observed clinical outcomes of infection with BA.4/BA.5 may therefore be different in settings with N/A, not applicable. a Date of diagnoses for cases included in each wave. We included cases diagnosed from 7 days before the "wave start" to the date of "wave end" (deemed to occur when 7-day moving average of daily new public sector admissions exceeded 5 million [start] and dropped below 12 million [end] respectively). b Vaccination is summarized as vaccine type and number of doses provided diagnosis was ≥28 days after first dose, ≥14 days after second dose, and ≥7 days after third dose; c Admission to an intensive care unit, mechanical ventilation, or prescription of oral or intravenous steroids; not reported for wave one as steroids not widely used until after June 16, 2020. different previous variant infection and vaccination exposure. With the progression of the SARS-CoV-2 pandemic globally, it is increasingly difficult to determine the clinical severity of any variant in a completely naïve individual. However, for health service planning, this is less relevant than the real-world effect in populations with varying degrees of immune protection ( Mefsin et al . , 2022 ). For example, although we showed a similar risk of severe hospitalization or death in the BA.4/BA.5 and BA.1 waves when adjusted for vaccination and previously diagnosed infection, the actual burden of admissions and deaths was much lower in the BA.4/BA.5 waves, with the peak 7-day moving average of admissions and deaths being 222 and 36 in the BA.1 wave vs 66 and nine in the BA.4/BA.5 wave. The ability to use routine data to rapidly assess the relative severity of waves caused by different lineages and variants adjusted for comorbidities, vaccination and previous infection has been especially valuable for local health service planning .
To our knowledge, this is one of the first comparisons of the clinical severity of BA.4/BA.5 infections with previous variants with relatively complete adjustment for comorbidities and vaccination among all diagnosed cases. Nonetheless, this type of data and analysis has several limitations, which have been described in detail previously . These include using the time of infection as a proxy for the variant causing infection rather than actual genomic sequencing or PCR test proxies which would be more accurate, could allow assessing the biological effect associated with specific mutations and would overcome challenges with comparing disease severity across waves due to differences in testing practices, treatment availability, and health service pressures. Notably, testing in the BA.4/BA.5 wave was at the lowest levels since the start of the pandemic with less testing of patients with milder disease; hence we may have over-estimated disease severity in this wave. For example, the peak weekly testing rate in the BA.4/BA.5 wave in the Western Cape was only 1/3 of that during the BA.1 wave (256 vs 756 tests per week per 10 0,0 0 0 population). Although we would have liked to assess the effects of time since vaccination and homologous vs heterologous vaccine doses, it was not possible to do this analysis due to small numbers of participants with each of the different vaccine combinations and durations since the last dose ( Lyke et al . , 2022 ). The rou- Table 2 Associations between different infection periods and severe COVID-19 outcomes adjusted for patient characteristics, sub-district, vaccination, and previously diagnosed infection using Cox regression. tine healthcare data used did not allow us to distinguish between severe hospitalizations and deaths where the diagnosis of COVID-19 may have been incidental or contributory rather than causal. We also had incomplete ascertainment of key covariates, especially previously diagnosed infection, due to substantial missed diagnoses (only 19% of our BA.4/BA.5 cases had previously diagnosed infection, whereas seroprevalence studies suggest at least 87% of the population had previous infection before the BA.4/BA.5 wave) ( Bingham et al . , 2022 ) and only including infections that were diagnosed more than 90 days apart. Similarly, due to the small numbers of patients with previously diagnosed infection and severe disease in the BA.4/BA.5 wave (n = 6), we were unable to assess whether there were differences in the extent of protection conferred by previous infection with different variants. Even in those with previously diagnosed infection it is possible that they had ad-ditional unascertained infections in other waves that may have impacted on their protection against severe disease due to BA.4/BA.5. Further, we had no data on vaccinations received outside of the province or without submitting a South African identity number and no data on undiagnosed comorbidities as we can only adjust for those algorithmically identified in the WCPHDC.
In conclusion, we found similar disease severity among diagnosed COVID-19 cases in the BA.4/BA.5 and BA.1 periods, both of which were associated with less severe outcomes than waves caused by previous SARS-CoV-2 variants. This finding is in the context of growing immunity against SARS-CoV-2 with strong protection against severe outcomes conferred by previous infection and vaccination, especially > 3 doses. Three homologous doses of Ad26.COV2.S or BNT162b2 or a heterologous combination provided 83% protection (95% CI 60; 93%) against severe COVID-19 hos-pitalization or death among laboratory-confirmed cases. Ensuring that individuals at high risk of severe COVID-19 outcomes have at least three vaccine doses remains a key strategy to limit the public health impact of further COVID-19 waves. Further research is needed to understand the specific differences in viral phenotype caused by the mutations in BA.4 and BA.5, as these mutations may occur in future variants and subvariants. In addition, it would be useful to quantify the protection provided by different types of immunity, such as natural infection with different variants, hybrid immunity (natural infection with vaccination), and heterologous versus homologous vaccination and waning of immunity.
Declaration of competing interest
All authors have no competing interests to declare.
Funding
We acknowledge funding for the Western Cape Provincial Health Data Centre (WCPHDC) from the Western Cape Department of Health, the US National Institutes for Health (R01 HD080465, U01 AI069924), the Bill and Melinda Gates Foundation (1164272, 1191327), the United States Agency for International Development (72067418CA0 0 023), the European Union (101045989) The funders had no role in the study design, data collection, data analysis, data interpretation, or writing of this report. The opinions, findings, and conclusions expressed in this manuscript reflect those of the authors alone. For the purposes of open access, the author has applied CC-BY public copyright to any author-accepted version arising from this submission.
Ethical approval
The study was approved by the University of Cape Town and Stellenbosch University Health Research Ethics Committees and Western Cape Government: Health. Individual informed consent requirement was waived for this secondary analysis of deidentified data.
|
2022-07-01T13:06:50.678Z
|
2022-11-01T00:00:00.000
|
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235825741
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pes2o/s2orc
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v3-fos-license
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Enhanced Detection of Mycobacterium bovis-Specific T Cells in Experimentally-Infected Cattle
Bovine tuberculosis (bTB), caused by infection with Mycobacterium bovis, continues to be a major economic burden associated with production losses and a public health concern due to its zoonotic nature. As with other intracellular pathogens, cell-mediated immunity plays an important role in the control of infection. Characterization of such responses is important for understanding the immune status of the host, and to identify mechanisms of protective immunity or immunopathology. This type of information can be important in the development of vaccination strategies, diagnostic assays, and in predicting protection or disease progression. However, the frequency of circulating M. bovis-specific T cells are often low, making the analysis of such responses difficult. As previously demonstrated in a different cattle infection model, antigenic expansion allows us to increase the frequency of antigen-specific T cells. Moreover, the concurrent assessment of cytokine production and proliferation provides a deeper understanding of the functional nature of these cells. The work presented here, analyzes the T cell response following experimental M. bovis infection in cattle via in vitro antigenic expansion and re-stimulation to characterize antigen-specific CD4, CD8, and γδ T cells and their functional phenotype, shedding light on the variable functional ability of these cells. Data gathered from these studies can help us better understand the cellular response to M. bovis infection and develop improved vaccines and diagnostic tools.
INTRODUCTION
Bovine tuberculosis (bTB), is a chronic bacterial infection caused primarily by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (1). This group of genetically-related mycobacteria also includes M. tuberculosis, M. cannetii, M. africanum, M. pinnipeii, M. microti, M. caprae, and M. mungi, which are known to infect and result in similar disease pathology in multiple hosts (2). Worldwide, bTB is a major cause of economic hardship. In 1995, it was estimated that bTB causes >50 million cattle infections resulting in $3 billion of losses annually (3,4). The number of infected cattle and associated economic loss are likely higher today and its zoonotic nature poses a legitimate public health risk.
As with other intracellular pathogens, protective immune responses against tuberculosis (TB) are associated with interferon gamma (IFN-γ) production derived from T helper 1 (T H 1) CD4 T cells (5)(6)(7)(8)(9). Delayed-type hypersensitivity reactions and IFN-γ release assays (IGRA) are commonly used to assess Mycobacterial reactivity or infection in various species [reviewed in Schiller et al. (10), Walzl et al. (11)]. However, these responses may not necessarily correlate with protection. In cattle, vaccination against bTB results in the induction of IFN-γ responses that can be measured ex vivo following overnight antigen stimulation, yet neither the presence nor the levels of IFN-γ induced translate into levels of protection afforded by vaccination (4,11,12). Immune protection from future infections is mediated by the induction and maintenance of memory responses. Memory T cells are a heterogenous population of cells including T effector memory (T EM ), T central memory (T CM ), and T resident memory (T RM ) cells, which display distinct functional, effector, and migratory phenotypes (13). T EM tend to have a fast response to antigen, retain their effector function (i.e., cytokine production), and are relatively short-lived. In contrast, T CM respond slower to antigen, show increased proliferative capabilities, can generate T effector and T EM cells, and are longlived. Previously, our laboratory demonstrated that following M. bovis infection in cattle, both T EM and T CM CD4 T cells are generated (14). In addition, we demonstrated that following M. bovis infection, bovine T CM are highly proliferative to antigen stimulation, and that T CM cells can revert in phenotype to generate T EM and T effector phenotypes (14).
While IFN-γ may not serve as a correlate of protection, it nevertheless plays a central role in the response to TB. Long-term culture systems, that measure IFN-γ from T CM cells, appear to be better predictors of vaccine efficacy as compared to ex vivo IFNγ production (15)(16)(17). These data would suggest that perhaps the T cell source of IFN-γ is a better predictor of protection rather than the overall levels of IFN-γ. Since proliferation is another characteristic feature of memory responses, typically associated with T CM , we wondered if the concurrent assessment of proliferation and IFN-γ production would allow us to better understand the source of IFN-γ and the overall functional phenotype of memory responses following M. bovis infection.
In the work presented here, we utilize an in vitro recall response assay, whereby antigen-specific cells are expanded, and proliferation and IFN-γ production are assessed concurrently. Additionally, we assess the potential of these antigen-specific cells to produce IFN-γ via restimulation, thereby enhancing their detection.
Animals and Mycobacterium bovis Aerosol Challenge
Holstein steers (∼6 months of age) were obtained from a tuberculosis-free source and housed at the National Animal Disease Center, agricultural biosafety level 3 (AgBSL3) animal facility. Animals were allowed to acclimate for 2 weeks prior to challenge. All animal studies were conducted with approval from the Institutional Animal Care and Use Committee (AICUC) at the National Animal Disease Center in Ames, Iowa.
Mycobacterium bovis strain 10-7428, a field strain of low passage (<3), which has been shown to be virulent in a calf aerosol model (18). The inoculum was prepared using standard techniques (19) M. bovis aerosol infection in cattle has been previously described (18,20,21). Briefly, eight (8) steers were infected with a single dose of virulent M. bovis strain 10-7428 by nebulization of inoculum into a mask (Equine AeroMask R , Trudell Medical International, London, ON, Canada) covering the nostrils and mouth. Six (6) age-matched steers were used as non-infected controls. All experimental animal procedures were conducted in accordance with recommendations in the Care and Use of Laboratory Animals of the National Institutes of Health and the Guide for the Care and Use of Agricultural Animals in Research and Teaching (22,23). All animal-related procedures were also approved by the USDA-National Animal Disease Center Animal Care and Use Committee.
Isolation of Peripheral Blood Mononuclear Cells
Whole blood was collected via venipuncture of the jugular vein into EDTA tubes. Blood was processed for isolation of PBMC as described earlier (24), with some modifications. Briefly, 10 ml of blood were diluted 1:2 in sterile, culture grade, Dubelcco's phosphate-buffered saline (DPBS) (Gibco, Thermo Fisher, Waltham, MA) and centrifuged at 1,200 × g for 30 min at room temperature (RT). The buffy coats were harvested and overlayed onto 5 ml of 1.077 Ficoll (Sigma-Aldrich, St. Louis, MO), and centrifuged again at 1,200 × g for 30 min at RT. PBMC were then harvested and washed once in sterile PBS at 300 × g for 10 min. Cells were then counted on a hemocytometer using trypan blue staining to determine number and viability. Cells were then resuspended to the desired concentration using complete RPMI 1640 (Gibco Life Tech, Thermo Fisher) media, as described previously (24).
PBMC Labeling, in vitro Antigen Stimulation, and Restimulation
In order to assess antigen-specific responses, PBMC were first labeled using the CellTrace R violet proliferation kit (Invitrogen, Thermo Fisher), according to manufacturer's recommendations. Following labeling, 1 × 10 6 cells were plated onto 96-well, flat bottom plates and left unstimulated, or stimulated with PPDb (5 µg/well), or Concanavalin A (ConA, 0.5µg/well, Sigma-Aldrich), in a total volume of 200 µl. Cells were then incubated at 37 • C with 5% CO 2 for 7 days. In order to assess intracellular cytokine production, cultured PBMC were treated with either a 1× solution of eBioscience TM Protein transport inhibitor (500× Brefeldin A, Thermo Fisher) or with a 1× solution of eBioscience TM Cell Stimulation cocktail plus Protein transport inhibitor [500× phorbol 12-myristate 13acetate (PMA), ionomycin, Brefeldin A] (Thermo Fisher) and incubated overnight for ∼16 h prior to harvest on day 7 (24).
Surface and Intracellular Staining
For surface and intracellular cytokine staining, PBMC were harvested on day 7 of culture and washed with DPBS via centrifugation at 300 × g for 5 min at RT. Staining was performed as described previously (24). Briefly, cells were incubated with a fixable viability dye (eBioscience TM , Thermo Fisher) and then stained for surface markers using FITC-labeled anti-bovine CD4 (CC8, Bio-Rad, Hercules, CA), APC-labeled anti-bovine CD8 (CC63, Bio-Rad), and anti-bovine γδ (IgG2b, TCR1-N24, Washington State University, Pullman, WA; BUV-labeled anti-IgG2b, BD Bioscience, San Jose, CA) antibodies. Cells were then fixed and permeabilized using BD Cytofix/Cytoperm TM kit (BD Bioscience) according to manufacturer's recommendation and stained with an anti-bovine, PE-labeled IFN-γ antibody (CC302, Bio-Rad). Cells were resuspended in FACS buffer and then analyzed using a BD FACSymphony TM A5 flow cytometer (BD Bioscience). Data was analyzed using FlowJo R software (Tree Star, Inc., San Diego, CA).
Statistical Analysis
Statistical analyses were performed using GraphPad Prism 8 (GraphPad software, San Diego, CA). Pair-wise comparisons of means were performed using t-tests, and multiple comparisons mixed-effects analysis were performed using Sidak's multiple comparisons test. P ≤ 0.05 were considered statistically-significant.
T Cell Population Subsets
Analysis of T cell population subsets from cultured PMBC from control and M. bovis-infected animals at various time points post-challenge was performed; gating scheme for the analysis is shown in Supplementary Figure 1. Frequencies of CD4, CD8, and γδ T cells did not differ significantly between control and infected animals at all time points analyzed (Figures 1A-C).
Overall, frequencies of all three subsets were relatively stable through the course of infection, with γδ T cells comprising the majority of the circulating pool of T cells ( Figure 1C).
M. bovis Infection
In order to assess antigen-specific responses, PBMC from control and M. bovis-infected animals were stimulated in vitro with PPDb and the frequency of proliferating CD4, CD8, and γδ T cells were determined. Representative histograms for assessment of proliferation can be seen in Supplementary Figure 2. Following antigen stimulation, we observed a significant increase in the frequency of proliferating CD4 T cells from M. bovis-infected animals as compared to controls at 4, 8-, and 48-weeks postinfection (Figure 2A). CD8 T cells from infected animals at 4and 8-weeks post-infection showed an increase in the frequency of proliferating cells, as compared to control animals, however, this change was not statistically significant ( Figure 2B). Similarly, proliferation was observed in γδ T cells from M. bovis-infected animals, however, the frequency of proliferating cells was only statistically different from control animals at 4 weeks postinfection ( Figure 2C). Altogether, these data indicate that the majority of the proliferative response to PPDb antigens occurred within the CD4 T cell compartment of PBMCs from M. bovisinfected animals.
T Cell IFN-γ Responses Following M. bovis Infection
The frequency of IFN-γ-producing cells following in vitro stimulation of PBMC from control and infected animals was also assessed. Similar to the proliferation data, IFN-γ production was predominantly observed within the CD4 T cell compartment of infected animals, at all time points analyzed (Figure 3). However, despite observing an increased frequency of IFNγ-producing CD4 T cells from M. bovis-infected animals as compared to controls, these differences were not statistically significant (p = 0.09, 0.08, and 0.30, respectively, for each time point) ( Figure 3A). At 4-and 8-weeks post-infection, we observed an increase in the frequency of IFN-γ-producing CD8 T cells as compared to controls, however, this increase was not statistically significant ( Figure 3B). The IFN-γ contribution from γδ T cells was relatively minimal, and no differences were seen between infected and control animals ( Figure 3C). Congruent with the proliferation findings from above, the majority of IFN-γ produced in response to PPDb antigens is derived from CD4 T cells following M. bovis infection.
Concurrent Assessment of Proliferation and IFN-γ Responses to Mycobacterial Antigens
Assessing proliferation and cytokine production concurrently, allows for further characterization of the functional potential of antigen-specific cells. By analyzing cells in this fashion, four distinct functional subsets were identified: cells that proliferate and produce IFN-γ (double function), cells that only produce IFN-γ, cells that only proliferate, and cells that neither proliferate nor produce IFN-γ (double negative) in response to antigen (Supplementary Figure 3). These functional subsets were identifiable for CD4 (Figure 4), CD8 (Supplementary Figure 4), and γδ (Supplementary Figure 5) T cells. However, not unexpectedly, these subsets were more discernable within the CD4 T cell compartment, as this is the T cell population with the greatest frequency of cells responding to antigen stimulation. Functionally, antigen-specific CD4 T cells are primarily capable of either proliferating or both proliferating and producing IFN-γ. At 4, 8-, and 48-weeks post-infection, CD4 T cells that Despite the reduced frequency of responding CD8 T cells, a similar pattern of functional phenotype was observed for this subset (Supplementary Figure 4). Proliferative responses made up the majority of the functional potential of CD8 T cells, followed by double producers, and a smaller percentage of IFN-γ-only producing cells (Supplementary Figures 4, 6, middle panel). Interestingly, γδ T cells showed primarily a proliferative response (Supplementary Figure 5), making up over 90% of the antigen-specific response at all time points analyzed (Supplementary Figure 6, bottom panel). Altogether, these data showed that when functional phenotypes are assessed concurrently, proliferation appears to predominate for CD4, CD8, and γδ T cells. Additionally, CD4-, CD8-, and γδ-derived IFN-γ arises from cells that have also proliferated in response to antigen, with a much smaller contribution arising from nonproliferating cells.
Enhancing Cytokine Production Following Mycobacterial Antigen Stimulation
Above findings indicated that proliferation comprises the majority of the antigen-specific response, and that only a subset of proliferating cells produced IFN-γ in response to PPDb stimulation. We wondered if the remainder of proliferating cells had the potential to make cytokines, when re-stimulation was provided in vitro. In order to determine if these cells had the potential to produce IFN-γ, 16 h prior to harvest on day 7, cells were re-stimulated with PMA/Ionomycin, a pan-T cell stimulator, in the presence of brefeldin A, as described previously (24). Indeed, following restimulation with PMA/Ionomycin, we observed a lower percentage of cells that only proliferated (Supplementary Figure 3 Altogether, these data demonstrate that M. bovis-specific T cells that proliferate to antigen stimulation are all capable of producing IFN-γ, and that this response can be enhanced by providing restimulation.
CONCLUSIONS
Understanding the functional role of T cells following M. bovis infection in cattle will provide insights for the development of vaccine and diagnostic interventions. Proliferation and cytokine production are two major functional characteristics of activated, antigen-specific T cells. Production of IFN-γ from T cells is intimately tied with T H 1 responses, which are necessary for the clearance of intracellular pathogens, including mycobacteria. However, IFN-γ levels do not always correlate with protection (4,11). In the work presented here, we sought to further dissect the functional potential of M. bovis-specific T cells by concurrently assessing two commonly-measured functional phenotypes: proliferation and IFN-γ production. In doing so, we were able to characterize three distinct functional subsets for all three T cell populations: cells with the potential to proliferate and produce IFN-γ (dual function), cells that only produce IFN-γ, and cells that only proliferate in response to antigen stimulation. Furthermore, we demonstrate that T cells with dual function and cells that proliferate-only make up the majority of the M. bovis-specific T cell response. We also demonstrate that when restimulated, antigen-specific cells that proliferate-only, are capable of producing IFN-γ.
The frequency of CD4, CD8, and γδ T cells remains relatively stable throughout the course of infection, or at least through the time points analyzed in this study (4,8, and 48 weeks post-infection). In addition, we found that M. bovis-specific proliferative responses can be seen in all three T cell populations, yet significant increases in the frequency of proliferating cells were only observed for CD4 T cells at all three time points. Similarly, IFN-γ responses were primarily seen within CD4 T cells, and to a lesser extent with CD8 T cells. Interestingly, despite this increase in the frequency of IFN-γ-producing CD4 T cells in M. bovis-infected animals, these changes were not statistically significant when compared to CD4 T cells from control animals. We attribute this to the variability in responses observed for IFN-γ in outbred populations, such as cattle. It should be noted that moderate variability was also observed for proliferative responses. Cattle represent an outbred population, and while experimental infections provide some level of control, variability in animal responses are expected. Overall, however, we show that proliferation and IFN-γ responses following M. bovis infection are primarily found within the CD4 T cell compartment, with CD8 and γδ T cells providing minor contribution to these responses, consistent with previous observations from our laboratory (14).
Concurrent measurement of proliferation and IFN-γ provides another level of insight into the functional potential of antigenspecific T cells. As a result, we were able to identify three distinct antigen-specific T cell populations with functional potential. We observed that following M. bovis challenge, there are two proliferating populations, one that produces IFN-γ and one that only proliferates. These two populations could represent distinct effector or memory subsets. We have previously demonstrated that following M. bovis infection, cattle do develop T central memory (T CM ) and T effector memory (T EM ) responses (14). Furthermore, we have demonstrated that antigen stimulation not only results in proliferation of T CM , but also in a switch from T CM to T EM , which are capable of producing IFN-γ (14). By measuring the functional phenotypes concurrently, we are likely seeing those distinct subsets. The work presented here did not include surface markers to corroborate memory phenotypes and only characterized proliferation and IFN-γ production. Despite this, it should be noted that by measuring proliferation and cytokine production concurrently, we were able to identify two functionally-distinct populations of cells responding to antigen stimulation. This approach may allow further characterization into other memory and/or effector subsets, which cannot be identified using surface markers as they have either not yet been identified or are not available for cattle.
Concomitant immunity, is a mechanism of immunity whereby a persistent, low-grade infection results in protection from subsequent re-infection with the same pathogen. This type of protection has been shown in other infectious models such as Leishmania (25). Unlike the classical idea of "T cell memory, " concomitant immunity is primarily driven by a unique subset of CD4 effector cells that are derived from T CM cells, are nonproliferative, are high IFN-γ producers, and are long-lived but only under conditions of persistent antigen availability [reviewed in Reyed and Rafati (26)]. In the mouse, this subset of T effector cells can be characterized by expression of the surface marker Ly6C (26), but this marker has not been characterized in other species. Therefore, tracking of this specific cell type becomes difficult in other species. This phenomenon, and its potential role in mediating protection in tuberculosis, have been recently described in a non-human primate model (27). Further support for concomitant immunity is found in human cohort studies as well as epidemiological data suggesting that prior infection with M. tuberculosis provides protection against subsequent infections (28,29). The role of concomitant immunity has not yet been explored for bTB. While we may be unable to characterize Ly6Cexpressing T effector memory subset based on surface expression markers, it may be possible to characterize these cells based on their non-proliferative and high-IFN-γ expression profile, which this assay would facilitate. Additional surface and intracellular markers could be added in order to expand the profiling of memory subsets using this assay. We propose that by refining the concurrent analysis of functional phenotypes, we may be able to explore some of these questions in cattle, thus providing further insights into the cellular immune response to M. bovis in its natural host.
The presence of antigen-specific cells that proliferate but do not produce IFN-γ in response to antigen stimulation led us to ask the question about the functional potential of these cells. To address this, we stimulated these cells with PMA/Ionomycin and measured cytokine production. Interestingly, as seen with T cells from cattle vaccinated against brucellosis (24), this subset of proliferating T cells is capable of producing IFN-γ. These data beg the question as to why the distinction in functional phenotype. As mentioned earlier this could be related to memory subtypes (T CM vs. T EM ), or perhaps this distinction is related to the nature of antigen stimulation. We assume that these antigenspecific T cells constitute a heterogenous populations of cells with a wide T cell repertoire and antigenic specificity. One hypothesis would be that the quantity and quality of the antigen may be responsible for driving function. Indeed, it has been previously shown that thresholds of T cell receptor signaling and duration of signaling determine T cell fate including functions such as cytokine production and proliferation [reviewed in Zikherman and Au-Yeung (30)]. We cannot discard the possibility that the antigen in this assay reaches varying degrees of stimulation for different T cell receptors. Further analysis utilizing defined antigens (i.e., single peptides or peptide cocktails) for stimulation using this assay, would allow us to determine if antigenspecificity and/or availability drives the functional distinction observed here.
Restimulation with PMA/ionomycin also allowed for the enhanced detection of antigen-specific, IFN-γ-producing CD4 cells (i.e., cells proliferating and producing IFN-γ). The restimulation step demonstrated that these proliferating cells are capable of producing IFN-γ when added stimulation is provided. However, we did not assess whether these cells could produce any other cytokines. Polyfunctional T cells (i.e., cells with the ability to produce multiple cytokines) have been shown to correlate with protection in various infectious models (31)(32)(33). However, the role of polyfunctional T cells in TB remains poorly understood, with conflicting data for their role in protection vs. active disease (34,35). Work from our laboratory has shown that in cattle polyfunctional (IFNγ/TNF-α/IL-2) CD4 T CM cells are associated with protective responses following BCG vaccination, while IFN-γ/TNF-α T CM cells are associated with higher bacterial burdens (36). The data presented here suggests that a large proportion of M. bovis-specific CD4 T cells only proliferate upon antigen stimulation, but are capable of producing cytokines in response to restimulation. It may be possible that a significant portion of antigen-specific cells are missed when cytokine analysis is performed in isolation. Assessment of cytokine polyfunctionality using restimulation, as described here, may provide a way to enhance our ability to detect these T cell subsets with polyfunctional phenotypes.
Altogether, the work presented here utilized an in vitro assay relying on enrichment of M. bovis-specific T cells via antigen stimulation to characterize functional phenotypes of proliferation and/or IFN-γ-production. The data demonstrate that the majority of antigen-responsive CD4 T cells proliferate in response to antigen, followed by cells capable of proliferating and producing cytokine. This type of approach, concurrent assessment of two major functions of activated T cells, along with further phenotypic analysis, are likely to increase our fundamental understanding of T cell responses involved in bTB.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.
ETHICS STATEMENT
The animal study was reviewed and approved by National Animal Disease Center Institutional Animal Care and Use Committee (NADC IACUC).
AUTHOR CONTRIBUTIONS
PB, CK, and MP: experiment design, sample collection, experiments, and manuscript editing. PB: data analysis and manuscript preparation. All authors contributed to the article and approved the submitted version.
FUNDING
Financial support for these studies were provided by the United States Department of Agriculture, Agricultural Research Service (ARS) and Animal and Plant Health Inspection Service (APHIS).
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2021-07-15T13:40:31.040Z
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2021-07-14T00:00:00.000
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Matrix-valued orthogonal polynomials related to the quantum analogue of (SU(2)×SU(2),diag)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(\mathrm{SU}(2) \times \mathrm{SU}(2), \mathrm{diag})$$\end{document}
Matrix-valued spherical functions related to the quantum symmetric pair for the quantum analogue of (SU(2)×SU(2),diag)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(\mathrm{SU}(2) \times \mathrm{SU}(2), \mathrm{diag})$$\end{document} are introduced and studied in detail. The quantum symmetric pair is given in terms of a quantised universal enveloping algebra with a coideal subalgebra. The matrix-valued spherical functions give rise to matrix-valued orthogonal polynomials, which are matrix-valued analogues of a subfamily of Askey–Wilson polynomials. For these matrix-valued orthogonal polynomials, a number of properties are derived using this quantum group interpretation: the orthogonality relations from the Schur orthogonality relations, the three-term recurrence relation and the structure of the weight matrix in terms of Chebyshev polynomials from tensor product decompositions, and the matrix-valued Askey–Wilson type q-difference operators from the action of the Casimir elements. A more analytic study of the weight gives an explicit LDU-decomposition in terms of continuous q-ultraspherical polynomials. The LDU-decomposition gives the possibility to find explicit expressions of the matrix entries of the matrix-valued orthogonal polynomials in terms of continuous q-ultraspherical polynomials and q-Racah polynomials.
Introduction
Shortly after the introduction of quantum groups, it was realised that many special functions of basic hypergeometric type [15] have a natural relation to quantum groups, see e.g. [9,Chap. 6], [20,31] for references. In particular, many orthogonal polynomials in the q-analogue of the Askey scheme, see e.g. [26], have found an interpretation on compact quantum groups analogous to the interpretation of orthogonal polynomials of hypergeometric type from the Askey scheme on compact Lie groups and related structures, see e.g. [46,47].
In case of the harmonic analysis on classical Gelfand pairs, one studies spherical functions and related Fourier transforms, see [43]. For our purposes, a Gelfand pair consists of a Lie group G and a compact subgroup K so that the trivial representation of K in the decomposition of any irreducible representation of G restricted to K occurs with multiplicity at most one. The spherical functions are functions on G which are left-and right-K -invariant. The zonal spherical functions are realised as matrix elements of irreducible G-representations with respect to a fixed K -vector. For special cases, the zonal spherical functions can be identified with explicit special functions of hypergeometric type, see [43,Chap. 9], [12,§IV]. The zonal spherical functions are eigenfunctions to an algebra of differential operators, which includes the differential operator arising from the Casimir operator in case G is a reductive group. For special cases with G compact, we obtain orthogonality relations and differential operators for the spherical functions, which can be identified with orthogonal polynomials from the Askey scheme. For the special case G = SU(2) × SU(2) with K ∼ = SU(2) embedded as the diagonal subgroup, the zonal spherical functions are the characters of SU (2), which are identified with the Chebyshev polynomials U n of the second kind by the Weyl character formula. The Gelfand pair situation has been generalised to the setting of quantum groups, mainly in the compact context, see e.g. Andruskiewitch and Natale [3] for the case of finite dimensional Hopf algebra with a Hopf subalgebra, Floris [13], Koornwinder [32], Vainermann [45] for more general compact quantum groups, and, for a non-compact example, Caspers [7].
The notions of matrix-valued and vector-valued spherical functions have already emerged at the beginning of the development of the theory of spherical functions, see e.g. [14] and references given there. However, the focus on the relation with matrix-valued or vector-valued special functions only came later, see e.g. references given in [18,44]. Grünbaum et al. [17] give a group theoretic approach to matrixvalued orthogonal polynomials emphasising the role of the matrix-valued differential operators, which are manipulated in great detail. The paper [17] deals with the case (G, K ) = (SU (3), U(2)). Motivated by [17] and the approach of Koornwinder [29], the group theoretic interpretation of matrix-valued orthogonal polynomials on (G, K ) = (SU (2) × SU (2), SU(2)) is studied from a different point of view, in particular with less manipulation of the matrix-valued differential operators, in [23,24], see also [18,44]. The point of view is to construct the matrix-valued orthogonal polynomials using matrix-valued spherical functions, and next using this group theoretic interpretation to obtain properties of the matrix-valued orthogonal polynomials. This approach for the case (G, K ) = (SU(2) × SU (2), SU(2)) leads to matrix-valued orthogonal polynomials for arbitrary size, which can be considered as analogues of the Chebyshev polynomials of the second kind. A combination of the group theoretic approach and analytic considerations then allows us to understand these matrix-valued orthogonal polynomials completely, i.e. we have explicit orthogonality relations, three-term recurrence relations, matrix-valued differential operators having the matrixvalued orthogonal polynomials as eigenfunctions, expression in terms of Tirao's [41] matrix-valued hypergeometric functions, expression in terms of well-known scalarvalued orthogonal polynomials from the Askey scheme, etc. This has been analytically extended to arbitrary size matrix-valued orthogonal Gegenbauer polynomials [25], see also [39] for related 2 × 2 cases.
The interpretation on quantum groups and related structures leads to many new results for special functions of basic hypergeometric type. In this paper, we use quantum groups in order to obtain matrix-valued orthogonal polynomials as analogues of a subclass of the Askey-Wilson polynomials. In particular, we consider the Chebyshev polynomials of the second kind, recalled in (5.6), as a special case of the Askey-Wilson polynomials [4, (2.18)]. Moreover, we know that the Chebyshev polynomials occur as characters on the quantum SU(2) group, see [48, §A.1]. The approach in this paper is to establish the quantum analogue of the group theoretic approach as presented in [23,24], see also [18,44], for the example of the Gelfand pair G = SU(2) × SU (2) with K ∼ = SU (2). For this approach, we need Letzter's approach [34][35][36] to quantum symmetric spaces using coideal subalgebras. We stick to the conventions as in Kolb [27] and we refer to [28, §1] for a broader perspective on quantum symmetric pairs. So we work with the quantised universal enveloping algebra U q (g) = U q (su (2) ⊕ su(2)), introduced in Sect. 3, equipped with a right coideal subalgebra B, see Sect. 4. Once we have this setting established, the branching rules of the representations of U q (g) restricted to B follow by identifying B with the image of U q (su (2)) (up to an isomorphism) under the comultiplication using the standard Clebsch-Gordan decomposition. In particular, it gives explicit intertwiners. Next we introduce matrix-valued spherical functions in Sect. 4. Using the matrix-valued spherical functions, we introduce the matrix-valued orthogonal polynomials. Then we use a mix of quantum group theoretic and analytic approaches to study these matrix-valued orthogonal polynomials. So we find the orthogonality for the matrix-valued orthogonal polynomials from the Schur orthogonality relations, and the three-term recurrence relation follows from tensor product decompositions of U q (g)-representations, and the matrix-valued q-difference operators for which these matrix-valued orthogonal polynomials are eigenvectors follow from the study of the Casimir elements in U q (g). More analytic properties follow from the LDU-decomposition of the matrix-valued weight function, and this allows to decouple the matrix-valued q-difference operators involved. The decoupling gives the possibility to link the entries of the matrix-valued orthogonal polynomials with (scalar-valued) orthogonal polynomials from the q-analogue of the Askey scheme, in particular the continuous q-ultraspherical polynomials and the q-Racah polynomials. The approach of [17] does not seem to work in the quantum case, because the possibilities to transform q-difference equations are very limited compared to transforming differential equations. We note that in [3, §5] matrix-valued spherical functions are considered for finite dimensional Hopf algebras with respect to a Hopf subalgebra.
The approach to matrix-valued orthogonal polynomials from this quantum group setting also leads to identities in the quantised function algebra. This paper does not include the resulting identities after using infinite dimensional representations of the quantised function algebra. Furthermore, we have not supplied a proof of Lemma 5.4 using infinite dimensional representations and the direct integral decomposition of the Haar functional, but this should be possible as well.
In general, the notion of a quantum symmetric pair seems to be best-suited for the development of harmonic analysis in general and in particular of matrix-valued spherical functions on quantum groups, see e.g. [28,[34][35][36][37][38] and references given there. When considering other quantum symmetric pairs in relation to matrix-valued spherical functions, the branching rule of a representation of the quantised universal enveloping algebra to a coideal subalgebra seems to be difficult. In this paper, it is reduced to the Clebsch-Gordan decomposition, and there is a nice result by Oblomkov and Stokman [38,Proposition 1.15] on a special case of the branching rule for quantum symmetric pair of type AIII, but in general the lack of the branching rule for the quantum symmetric pairs is an obstacle for the study of quantum analogues of matrix-valued spherical functions of e.g. [17,18,38,44].
The matrix-valued orthogonal polynomials resulting from the study in this paper are matrix-valued analogues of the Chebyshev polynomials of the second kind viewed as an example of the Askey-Wilson polynomials. We expect that it is possible to obtain matrix-valued analogues of the continuous q-ultraspherical polynomials viewed as subfamily of the Askey-Wilson polynomials using the approach of [25] using the Askey-Wilson q-derivative instead of the ordinary derivative. We have not explicitly worked out the limit transition q ↑ 1 of the results, but by the set-up it is clear that the formal limit gives back many of the results of [23,24].
The contents of the paper are as follows. In Sect. 2, we fix notation regarding matrix-valued orthogonal polynomials. In Sect. 3, the notation for quantised universal enveloping algebras is recalled. Section 4 states all the main results of this paper. It introduces the quantum symmetric pair explicitly. Using the representations of the quantised universal enveloping algebra and the coideal subalgebra, the matrixvalued polynomials are introduced. We continue to give explicit information on the orthogonality relations, three-term recurrence relations, q-difference operators, the commutant of the weight, the LDU-decomposition of the weight, the decoupling of the q-difference equations and the link to scalar-valued orthogonal polynomials from the q-Askey scheme. The proofs of the statements of Sect. 4 occupy the rest of the paper. In Sect. 5, the main properties derivable from the quantum group set-up are derived, and we discuss in Appendix 1 the precise relation of the branching rule for this quantum symmetric pair and the standard Clebsch-Gordan decomposition. In Sect. 6, we continue the study of the orthogonality relations, in which we make the weight explicit. This requires several identities involving basic hypergeometric series, whose proofs we relegate to Appendix 2. Section 7 studies the consequences of the explicit form of the matrix-valued q-difference operators of Askey-Wilson type to which the matrix-valued orthogonal polynomials are eigenfunctions.
In preparing this paper, we have used computer algebra in order to verify the statements up to certain size of the matrix and up to certain degree of the polynomial in order to eliminate errors and typos. Note, however, that all proofs are direct and do not use computer algebra. A computer algebra package used for this purpose can be found on the homepage of the second author. 1 The convention on notation follows Kolb [27] for quantised universal enveloping algebras and right coideal subalgebras and we follow Gasper and Rahman [15] for the convention on basic hypergeometric series and we assume 0 < q < 1.
Matrix-valued orthogonal polynomials
In this section, we fix notation and give a short background to matrix-valued orthogonal polynomials, which were originally introduced by Krein in the forties, see e.g. references in [5,10]. General references for this section are [5,10,16], and references given there.
Assume that we have a matrix-valued function W : ] almost everywhere. We use the notation A > 0 to denote a strictly positive definite matrix. Moreover, we assume that all moments exist, where integration of a matrix-valued function means that each matrix entry is separately integrated. In particular, the integrals are matrices in M 2 +1 (C). It then follows that for matrix-valued polynomials P, exists. This gives a matrix-valued inner product on the space M 2 +1 (C)[x] of matrixvalued polynomials, satisfying where G n > 0. Moreover, the leading coefficient of P n is non-singular. Any other family of polynomials (Q n ) n∈N so that Q n is a matrix-valued polynomial of degree n and Q n , Q m = 0 for n = m satisfies P n (x) = Q n (x)E n for some non-singular E n ∈ M 2 +1 (C) for all n ∈ N. We call the matrix-valued polynomial P n monic in case the leading coefficient is the identity matrix I . The polynomials P n are called orthonormal in case the squared norm G n = I for all n ∈ N in the orthogonality relations (2.2). The matrix-valued orthogonal polynomials P n always satisfy a matrix-valued threeterm recurrence of the form for matrices A n , B n , C n ∈ M 2 +1 (C) for all n ∈ N. Note that in particular A n is nonsingular for all n. Conversely, assuming P −1 (x) = 0 (by convention) and fixing the constant polynomial P 0 (x) ∈ M 2 +1 (C) we can generate the polynomials P n from the recursion (2.3). In case the polynomials are monic, the coefficient A n = I for all n and P 0 (x) = I as the initial value. In general, the matrices satisfy G n+1 A n = C * n+1 G n , G n B n = B * n G n , so that in the monic case C n = G −1 n−1 G n for n ≥ 1. In case the polynomials are orthonormal, we have C n = A * n−1 and B n Hermitian. Note that the matrix-valued 'sesquilinear form' (2.1) is antilinear in the first entry of the inner product, which leads to a three-term recurrence of the form (2.3) where the multiplication by the constant matrices is from the right, see [10] for a discussion. In be the matrix-valued polynomial defined by P V n (x) = ι * V P n (x)ι V , where P n are the monic matrix-valued orthogonal polynomials for the weight W . Then P V n form a family of monic Vendomorphism-valued orthogonal polynomials, and P n (x) = P V n (x) ⊕ P V ⊥ n (x). The same decomposition can be written down for the orthonormal polynomials.
The projections on invariant subspaces are in the commutant * -algebra {T ∈ M 2 +1 (C) | T W (x) = W (x)T ∀x}. In case the commutant algebra is trivial, the matrix-valued orthogonal polynomials are irreducible. The primitive idempotents correspond to the minimal invariant subspaces, and hence they determine the decomposition of the matrix-valued orthogonal polynomials into irreducible cases.
Remark 2.1
In [42] the authors discuss non-orthogonal decompositions by considering, instead of the commutant algebra, the real vector space It follows that if I R A , then the weight W reduces, non-unitarily, to weights of smaller size. Koelink and Román [22,Example 4.3] showed that A = {W (x) : x ∈ (−1, 1)} so that, in our case, both decompositions coincide.
We denote by E i, j ∈ M 2 +1 (C) the matrix with zeroes except at the (i, j)th entry where it is 1. So for the corresponding standard basis {e k } 2 k=0 we set E i, j e k = δ j,k e i .
We usually use the basis {e k } 2 k=0 in describing the results for the matrix-valued orthogonal polynomials, but occasionally the basis is relabelled {e k } k=− , as is customary for the U q (su (2))-representations of spin . In the latter case, we use superscripts to distinguish from the previous case: E i, j e k = δ j,k e i , i, j, k ∈ {− , . . . , }.
Quantised universal enveloping algebra
We recall the setting for quantised universal enveloping algebras and quantised function algebras, and this section is mainly meant to fix notation. The definitions can be found at various sources on quantum groups, such as the books [9,11,20], and we follow Kolb [27].
Fix for the rest of this paper 0 < q < 1. The quantised universal enveloping algebra can be associated to any root datum, but we only need the simplest cases g = sl(2) and g = sl(2) ⊕ sl (2). The quantised universal enveloping algebra is the unital associative algebra generated by k, k −1 , e, f subject to the relations where we follow the convention as in [27, §3]. For our purposes, it is useful to extend the algebra with the roots of k and k −1 , denoted by k 1/2 , k −1/2 satisfying The extended algebra is denoted by U q (sl(2)), and it is a Hopf algebra with comultiplication , counit ε, antipode S defined on the generators by The Hopf algebra has a * -structure defined on the generators by We denote the corresponding Hopf * -algebra by U q (su (2)).
The identification as Hopf * -algebras with [21,30] The irreducible finite dimensional type 1 representations of the underlying * -algebra have been classified. Here type 1 means that the spectrum of K 1/2 is contained in q 1 2 Z . For each dimension 2 + 1 of spin ∈ 1 2 N, there is a representation in H ∼ = C 2 +1 with orthonormal basis {e − , e − +1 , . . . , e } and on which the action is given by . Finally, recall that the centre Z(U q (su(2))) is generated by the Casimir element ω, (3.4) We use the notation U q (g) to denote the Hopf * -algebra U q (su(2) ⊕ su (2)), which we identify with U q (su (2) for any fixed i and the generators with different index i commute. The tensor product of two Hopf * -algebras is again a Hopf * -algebra, where the maps on a simple tensor X 1 ⊗ X 2 are given by, see e.g. [9,Chap. 4], where we use leg-numbering notation. The irreducible finite dimensional type 1 representations of U q (g) are labelled by ( 1 , 2 ) ∈ 1 2 N × 1 2 N, and the representations t 1 , 2 from U q (g) to End(H 1 , 2 ), H 1 , 2 = H 1 ⊗H 2 , are obtained as the exterior tensor product of the representations of spin 1 and 2 of U q (su (2)). Here type 1 means that the spectrum of K 1/2 i , i = 1, 2, is contained in q 1 2 Z . We have used the notation , ε and S for the comultiplication, counit and antipode for all Hopf algebras, respectively. From the context, it should be clear which comultiplication, counit and antipode is meant. The corresponding dual Hopf * -algebra related to the quantised function algebra is not needed for the description of the results in Sect. 4, and it will be recalled in Sect. 5.1.
spaces have been introduced and studied in detail by Letzter [34][35][36], see also Kolb [27]. In particular, Letzter has shown that Macdonald polynomials occur as spherical functions on quantum symmetric pairs motivated by the works of Koornwinder, Dijkhuizen, Noumi and others. In our case, B ⊂ U q (g), as in Definition 4.1, is the appropriate right coideal subalgebra. Using the explicit branching rules for t ( 1 , 2 ) | B of Theorem 4.3, we introduce matrix-valued spherical functions in Definition 4.4. To these matrix-valued spherical functions, we associate matrix-valued polynomials in (4.8), and we spend the remainder of this section to describe properties of these matrix-valued polynomials. This includes the orthogonality relations, the three-term recurrence relation and the matrix-valued polynomials as eigenfunctions of a commuting set of matrix-valued q-difference equations of Askey-Wilson type. Moreover, we give two explicit descriptions of the matrix-valued weight function W , one in terms of spherical functions for this quantum symmetric pair and one in terms of the LDU-decomposition. The LDU-decomposition gives the possibility to decouple the matrix-valued q-difference operator, and this leads to an explicit expression for the matrix entries of the matrix-valued orthogonal polynomials in terms of scalar-valued orthogonal polynomials from the q-Askey scheme in Theorem 4.17.
For the symmetric pair (G, K ) = (SU(2) × SU(2), SU(2)), K = SU(2) corresponds to the fixed points of the Cartan involution θ flipping the order of the pairs in G. The quantised universal enveloping algebra associated to G is U q (g) as introduced in Sect. 3. As the quantum analogue of K , we take the right coideal subalgebra B ⊂ U q (g), i.e. B ⊂ U q (g) is an algebra satisfying (B) ⊂ B⊗U q (g), as in Definition 4.1. Letzter [34,Sect. 7,(7.2)] has introduced the corresponding left coideal subalgebra, and we follow Kolb [27, §5] in using right coideal subalgebras for quantum symmetric pairs. Note that we have modified the generators slightly in order to have B * 1 = B 2 . Definition 4.1 The right coideal subalgebra B ⊂ U q (g) is the subalgebra generated by K ±1/2 , where K = K 1 K −1 2 , and Remark 4.2 (i) B is a right coideal as follows from the general construction, see [27,Proposition 5.2]. It can be verified directly by checking it for the generators.
is in B⊗U q (g) by a straightforward calculation. Since B 2 = B * 1 , it also follows for B 2 , since K ±1/2 are self-adjoint. The relations, cf. [27,Lemma 5.15], hold in U q (g) as can also be checked directly.
Then we see that k 1/2 → K −1/2 , q f k 1/2 → B 1 , and q −1 k −1/2 e → B 2 under the map ι•(Ψ ⊗Id)• . In particular, the relations (4.1) follow. We conclude that B is isomorphic as a * -algebra to (U q (su(2)) ⊂ U q (g) by the * -isomorphism ι • (Ψ ⊗ Id). (iii) In particular, B ∼ = U q (su(2)) as * -algebras. So the irreducible type 1 representations of B are labelled by the spin ∈ 1 2 N. This can be made explicit by t : B → End(H ) and setting with the notation of (3.3). We use the same notation t for these representations here and in (3.3), since they correspond under the identification of B as U q (su (2)). (iv) Let σ be the * -algebra isomorphism on U q (g) = U q (su(2)) ⊗ U q (su (2)) by flipping the order in the tensor product, or equivalently by flipping the subscripts 1 ↔ 2. Then σ : B → B is an involution B 1 ↔ B 2 , K ↔ K −1 . On the level of representations of U q (g) and B, it follows t 1 , 2 (σ (X )) = P * t 2 , 1 (X )P, X ∈ U q (g), where P :
Theorem 4.3
The finite dimensional representation t 1 , 2 of U q (g) restricted to B decomposes multiplicity-free into irreducible representations t of B: With respect to the orthonormal basis {e p } p=− of H and the orthogonal basis where C 1 , 2 , i, j, p are Clebsch-Gordan coefficients satisfying The proof of Theorem 4.3 is a reduction to the well-known Clebsch-Gordan decomposition for the quantised universal enveloping algebra U q (su (2)), see e.g. [9,20], using Remark 4.2. The proof is presented in Appendix 1. In particular, (β 1 , 2 ) * β 1 , 2 is the identity on H . Note that In general, the decomposition of an irreducible representation restricted to a right coideal subalgebra seems a difficult problem. In this particular case, we can reduce to the Clebsch-Gordan decomposition, and yet another known special case is by Oblomkov We use the reparametrisation of (4.3) by 2 N and let ( 1 , 2 ) ∈ 1 2 N × 1 2 N so that [t 1 , 2 | B : t ] = 1. The spherical function of type associated to ( 1 , 2 ) is defined by (ii) Note that the condition (4.3) is symmetric in 1 and 2 . With the notation of Remark 4.2(iv), we have Φ 2 , 1 (Z ) = J Φ 1 , 2 (σ (Z ))J for Z ∈ U q (g). This follows from β 2 , 1 = Pβ 1 , 2 J , which is a consequence of (8.2).
In case = 0, H 0 ∼ = C, we need 1 = 2 . Then Φ 0 1 , 1 are linear maps U q (g) → C. In particular, Φ 0 0,0 equals the counit ε, and the spherical function ϕ = 1 2 (q −1 + Fig. 1 The spherical functions Φ 1 , 2 when = 2 and interpretation of ϕ · Φ 5/2,5/2 in terms of the matrix-valued spherical functions. The reparametrisation ξ is depicted q)Φ 0 1/2,1/2 is scalar-valued linear map on U q (g). The elements Φ 0 n/2,n/2 can be written as a multiple of U n (ϕ), where U n denotes the Chebyshev polynomial of the second kind of degree n, see Proposition 5.3. This statement can be considered as a special case of Theorem 4.8, but we need the identification with the Chebyshev polynomials in the spherical case = 0 in order to obtain the weight function in Theorem 4.8. Proposition 5.3 will follow from Theorem 4.6. The identification of the spherical functions for = 0 with Chebyshev polynomials corresponds to the classical case, since the spherical functions on G × G/G are the characters on G and the characters on SU(2) are Chebyshev polynomials of the second kind, as the simplest case of the Weyl character formula. It also corresponds to the computation of the characters on the quantum SU(2) group by Woronowicz [48], since the characters are identified with Chebyshev polynomials as well.
Next Theorem 4.6 gives the possibility to associate polynomials in ϕ to spherical functions of Definition 4.4. Theorem 4.6 essentially follows from the tensor product decomposition of representations of U q (g), which in turn follows from tensor product decomposition for U q (su(2)), and some explicit knowledge of Clebsch-Gordan coefficients.
Theorem 4.6 Fix ∈ 1 2 N and let ( 1 , 2 ) ∈ 1 2 N × 1 2 N satisfy (4.3), then for constants A i, j we have In order to interpret the result of Theorem 4.6, we evaluate both sides at an arbitrary X ∈ U q (g). The right-hand side is a linear combination of linear maps from H to itself after evaluating at X . For the left-hand side, we use the pairing of Hopf algebras so that multiplication and comultiplication are dual to each other and the left-hand side has to be interpreted as which is a linear combination of linear maps from H to itself, using (X ) = (X ) X (1) ⊗ X (2) . The convention in Theorem 4.6 is that A i, j is zero in case ( 1 + i, 2 + j) does not satisfy (4.3). The proof of Theorem 4.6 can be found in Sect. 5.2.
Since B is a right coideal subalgebra, we see that the left-hand side of Theorem 4.6 has the same transformation behaviour as (4.5). Indeed, for X ∈ B and Y ∈ U q (g) we have where we have used that X (1) ∈ B by the right coideal property and (4.5) for ϕ = Φ 0 1/2,1/2 in the second equality, and the counit axiom (X ) ε(X (1) )X (2) = X in the fourth equality and then (4.5) for Φ 1 , 2 and the fact that ϕ(Y (1) ) is a scalar. Similarly, the invariance property from the right can be proved. Theorem 4.6 leads to polynomials in ϕ by iterating the result and using that A 1/2,1/2 is non-zero.
Corollary 4.7
There exist 2 + 1 polynomials r ,k n,m , 0 ≤ k ≤ 2 , of degree at most n so that The aim of the paper is to show that the polynomials r ,k n,m give rise to matrix-valued orthogonal polynomials. Put where the matrix-valued polynomials P n are taken with respect to the relabelled standard basis e p = e p− , p ∈ {0, 1, . . . , 2 } so that P n = 2 i, j=0 r ,i n, j ⊗ E i, j . From Corollary 4.12 or Theorem 4.17, we see that the polynomial r ,i n, j has real coefficients. The case = 0 corresponds to a three-term recurrence relation for (scalar-valued) orthogonal polynomials, and then the polynomials coincide with the Chebyshev polynomials U n viewed as a subclass of Askey-Wilson polynomials [4, (2.18)], see Proposition 5.3.
We show that the matrix-valued polynomials (P n ) ∞ n=0 are orthogonal with respect to an explicit matrix-valued weight function W , see Theorem 4.8, arising from the Schur orthogonality relations. The expansion of the entries of weight function in terms of Chebyshev polynomials is given by quantum group theoretic considerations except for the calculation of the coefficients in this expansion. The matrix-valued orthogonal polynomials satisfy a matrix-valued three-term recurrence relation as follows from Theorem 4.6, which in turn is a consequence of the decomposition of tensor product representations of U q (g). However, in order to determine the matrix coefficients in the matrix-valued three-term recurrence we use analytic methods. The existence of two Casimir elements in U q (g) leads to the matrix-valued orthogonal polynomials being eigenfunctions of two commuting matrix-valued q-difference operators, see [23] for the group case. This extends Letzter [35] to the matrix-valued set-up for this particular case. The q-difference operators are the key to determining the entries of the matrix-valued orthogonal polynomials explicitly in terms of scalar-valued orthogonal polynomials from the q-Askey scheme [26], namely the continuous q-ultraspherical polynomials and the q-Racah polynomials. In this deduction, the LDU-decomposition of the matrix-valued weight function W is essential, since the conjugation with L allows us to decouple the matrix-valued q-difference operator.
In the remainder of Sect. 4, we state these results explicitly, and we present the proofs in the remaining sections. First we give the main statements which essentially follow from the quantum group theoretic set-up, except for explicit calculations, and these are Theorems 4.8, 4.11, 4.13. The remaining Theorems 4.15, 4.17 are obtained using scalar orthogonal polynomials from the q-analogue of the Askey scheme [26] and transformation and summation formulas for basic hypergeometric series [15].
We start by stating that the matrix-valued polynomials (P n ) ∞ n=0 introduced in (4.8) are orthogonal with the conventions of Sect. 2. The orthogonality relations of Theorem 4.8 are due to the Schur orthogonality relations. The expansion of the entries of the weight function in terms of Chebyshev polynomials follows from the fact that the entries are spherical functions, i.e. correspond to the case = 0 so that they are polynomial in ϕ. The non-zero entries follow by considering tensor product decompositions, but the explicit values for the coefficients α t (m, n) in Theorem 4.8 require summation and transformation formulae for basic hypergeometric series.
Theorem 4.8
The polynomials (P n ) ∞ n=0 of (4.8) form a family of matrix-valued orthogonal polynomials so that P n is of degree n with non-singular leading coefficient. The orthogonality for the matrix-valued polynomials (P n ) n≥0 is given by where the squared norm matrix G m is diagonal: .
Moreover, for 0 ≤ m ≤ n ≤ 2 the weight matrix is given by The proof of Theorem 4.8 proceeds in steps. First we study explicitly the case = 0, motivated by the works of Koornwinder [30], Letzter [34][35][36] and others. Secondly, we show that taking traces of a matrix-valued spherical function of type associated to ( 1 , 2 ) times the adjoint of a spherical function of type associated to ( 1 , 2 ) gives, up to an action by an invertible group-like element of U q (g), a polynomial in the generator for the case = 0. Then the explicit expression of the Haar functional on this polynomial algebra, stated in Lemma 5.4, gives the matrix-valued orthogonality relations. Finally, the explicit expression for the weight is obtained by analysing the explicit expression of W in terms of the matrix entries of the intertwiners β 1 , 2 in case 1 + 2 = . These matrix entries are Clebsch-Gordan coefficients.
The leading coefficient of P n can be calculated explicitly from the proof of Theorem 4.8:
Corollary 4.9
The leading coefficient of P n is a non-singular diagonal matrix: The weight W is not irreducible, see Sect. 2, but splits into two irreducible block matrices. The symmetry J of the weight function of Theorem 4.8 is essentially a consequence of Remark 4.5(ii), but we need the explicit expression of the weight in order to prove that the commutant algebra is not bigger, see also [22, §4].
Proposition 4.10 The commutant algebra
is spanned by I and J , where J : e p → e 2 − p , p ∈ {0, . . . , 2 }, is a self-adjoint involution. Then J P n (x)J = P n (x) and J G n J = G n . Moreover, the weight W decomposes into two irreducible block matrices W + and W − , where W + , respectively W − , acts in the +1-eigenspace, respectively −1-eigenspace, of J . So for P + + P − = I , where P + , P − are the orthogonal self-adjoint projections P + = 1 The special cases for = 1 2 and = 1 are given at the end of this section. In particular, we identify all scalar-valued orthogonal polynomials occurring in this framework explicitly in terms of Askey-Wilson polynomials.
Theorem 4.6 can be used to find a three-term recurrence relation for the matrixvalued orthogonal polynomials P n , cf. Sect. 2, so the underlying tensor product decompositions provide the three-term recurrence relation. However, the resulting expressions for the entries of the coefficients of the matrices are rather complicated expressions in terms of Clebsch-Gordan coefficients. For the corresponding matrixvalued monic polynomials Q n (x) = P n (x)lc(P n ) −1 , see Corollary 4.9 for the explicit expression for the leading coefficient, we can derive a simple expression for the matrices in the three-term recurrence relation once we have obtained more explicit expressions for the matrix entries of Q n . This is obtained in Sect. 7 using an explicit link of the matrix entries to scalar orthogonal polynomials in the q-Askey scheme.
Theorem 4.11
The monic matrix-valued orthogonal polynomials (Q n ) n≥0 satisfy the three-term recurrence relation Note that X n → 0, Y n → 1 4 as n → ∞. The three-term recurrence relation for the matrix-valued orthogonal polynomials P n is given in Corollary 4.12, which follows from Theorem 4.11, since we have G n+1 A n = lc(P n+1 ) * lc(P n ), G n B n = lc(P n ) * X n lc(P n ), and G n−1 C n = lc(P n−1 ) * Y n lc(P n ). For future reference, we give the explicit expressions in Corollary 4.12.
Corollary 4.12
The matrix-valued orthogonal polynomials (P n ) n≥0 satisfy the threeterm recurrence relation Note that the case = 0 gives a three-term recurrence relation that can be solved in terms of the Chebyshev polynomials, see Proposition 5.3.
In the group case, the spherical functions are eigenfunctions of K -invariant differential operators on G/K , see e.g. [8,14]. For matrix-valued spherical functions this is also the case, see [40], and this has been exploited in the special cases studied in [17,23,24]. In the quantum group case, the action of the Casimir operator gives rise to a q-difference operator for the corresponding spherical functions, see [35]. The first occurrence of an Askey-Wilson q-difference operator, see [4,15,19], in this context is due to Koornwinder [30]. For the matrix-valued orthogonal polynomials, we have a matrix-valued analogue of the Askey-Wilson q-difference operator, as given in Theorem 4.13. We obtain two of these operators, one arising from the Casimir operator for U q (su (2)) in the first leg of U q (g) and one from the U q (su (2)) Casimir operator of the second leg. This is related to a kind of Cartan decomposition of U q (g), cf. (4.5), which, however, does not exist in general for quantised universal enveloping algebras. We can still resolve this problem using techniques based on [8, §2], see the first part of the proof in Sect. 5. The proof of Theorem 4.13 is completed in Sect. 7.
Theorem 4.13 Define two matrix-valued q-difference operators by
where the multiplication by the matrix-valued functions M i (z) and M i (z −1 ) is from the left and η q is the shift operator defined by . The matrix-valued function M 1 is given by The matrix-valued orthogonal polynomials P n are eigenfunctions for the operators D i with eigenvalue matrices given by Λ n (i) such that D i P n = P n Λ n (i) and Explicitly, where η q and η q −1 are applied entry-wise to the matrix-valued orthogonal polynomials P n .
Theorem 4.13 shows that J D 1 J = D 2 , since J is constant. In particular, D 1 + D 2 commutes with J and reduces to a q-difference operator for the matrix-valued orthogonal polynomials associated with the weight W + or W − , see Proposition 4.10. Similarly, D 1 − D 2 anticommutes with J .
Note that the expression M i (z)P(qz) + M i (z −1 )P(z/q) is symmetric in z ↔ z −1 for any matrix-valued polynomial P, and hence again is a function in x = μ(z). The case = 0 corresponds to only one q-difference operator, which we rewrite as For the Chebyshev polynomials, U n (x) = (q n+2 ; q) −1 n p n (x; q, −q, q 1/2 , −q 1/2 |q) rewritten as Askey-Wilson polynomials [4, (2.18) By [16, §2] it suffices to check Corollary 4.14 for P = P n , Q = P m , so that by Theorems 4.13 and 4.8 we need to check that Λ n (i) * G n δ m,n = G n Λ m (i)δ m,n , which is true since the matrices involved are real and diagonal.
The orthogonality measure is a positive measure in case 0 < q < 1 and β real with |β| < 1. Explicitly, (4.11) Note that in the special case β = q 1+k , k ∈ N, the weight function is polynomial in x = cos θ , and We use the continuous q-ultraspherical polynomials (4.10) for any β ∈ C. In particular, for β = q −k with k ∈ N the sum in (4.10) is restricted to n − k ≤ r ≤ k, and in particular C n (x; q −k ; q) = 0 in case n − k > k. With this convention, we can now describe the LDU-decomposition of the weight matrix, and state the inverse of the unipotent lower triangular matrix L in Theorem 4.15.
Theorem 4.15
The matrix-valued weight W as in Theorem 4.8 has the following LDU-decomposition: The inverse of L is given by Note that T , L and L −1 are matrix-valued polynomials, which is clear from the explicit expression and (4.12). It is remarkable that the LDU-decomposition is for arbitrary size 2 + 1, but that there is no dependence of L on the spin and that the dependence of T on the spin is only in the constants c k ( ).
We prove the first part of Theorem 4.15 in Sect. 6. The proof of Theorem 4.15 is analytic in nature, and a quantum group theoretic proof would be desirable. The statement on the inverse of L(x) is taken from [1], where the inverse of a lower triangular matrix with matrix entries continuous q-ultraspherical polynomials in a more general situation is derived. The inverse L −1 is derived in [1,Example 4.2]. The inverse of L in the limit case q ↑ 1 was derived by Cagliero and Koornwinder [6], and the proof of [1] is of a different nature than the proof presented in [6]. Using the lower triangular matrix L of the LDU-decomposition of Theorem 4.15, we are able to decouple D 1 of Theorem 4.13 after conjugation with L t (x). We get a scalar q-difference equation for each of the matrix entries of L t (x)P n (x), which is solved by continuous q-ultraspherical polynomials up to a constant. Since we have yet another matrix-valued q-difference operator for L t (x)P n (x), namely L t D 2 (L t ) −1 with D 2 as in Theorem 4.13, we get a relation for the constants involved. This relation turns out to be a three-term recurrence relation along columns, which can be identified with the three-term recurrence for q-Racah polynomials. Finally, use (L t (x)) −1 to obtain an explicit expression for the matrix entries of the matrix-valued orthogonal polynomials of Theorem 4.17. Before where n ∈ {0, 1, 2, . . . , N }, N ∈ N, μ(x) = q −x + γ δq x+1 and so that one of the conditions αq = q −N , or βδq = q −N , or γ q = q −N holds.
Note that the left-hand side is a polynomial of degree at most n, whereas the righthand side is of degree n + j − i. In particular, for j > i the leading coefficient of the right-hand side of Theorem 4.17 has to vanish, leading to Corollary 4.18.
Corollary 4.18 With the notation of Theorem 4.17, we have for j
By evaluating Corollary 4.7 at 1 ∈ U q (g), we obtain Corollary 4.19, which is not clear from Theorem 4.17.
Examples
We end this section by specialising the results for low-dimensional cases. The case = 0 reduces to the Chebyshev polynomials U n (x) of the second kind as observed following Theorem 4.13. This is proved in Proposition 5.3, which is required for the proofs of the general statements of Sect. 4. In this case we work with 2 × 2 matrices. By Proposition 4.10, we know that the weight is block-diagonal, so that in this case we have an orthogonal decomposition to scalar-valued orthogonal polynomials. To be explicit, the matrix-valued weight W is given by In this case, see Sect. 2, the polynomials P n diagonalise since the leading coefficient is diagonalised by conjugation with the orthogonal matrix Y , and we write ). Then we can identify p ± n by any of the results given in this section, and we do this using the three-term recurrence relation of Corollary 4.12. After conjugation, the three-term recurrence relation for p + n is given by and the three-term recurrence relation for p − n is obtained by substituting x → −x into the three-term recurrence relation for p + n . The explicit expressions for p + n and p − n are given in terms of continuous q-Jacobi polynomials P which is a q-analogue of [24, §8.2]. Moreover, writing down the conjugation of the q-difference operator D 1 + D 2 of Theorem 4.13 for the case = 1 2 for the conjugated polynomials gives back the Askey-Wilson q-difference for the continuous q-Jacobi polynomials P . Working out the eigenvalue equation for D 1 − D 2 gives a simple q-analogue of the contiguous relations of [24, p. 5708].
Example: = 1
For = 1 we work with 3×3 matrices. By Proposition 4.10, we can block-diagonalise the matrix-valued weight: where W + is a 2×2 matrix-valued weight and W − is a scalar-valued weight. Explicitly, n is a scalar-valued polynomial. Conjugating Corollary 4.12, the three-term recurrence relations for P + n and p − n are The scalar-valued polynomial p − n can be identified with the continuous q-ultraspherical polynomials: The 2 × 2 matrix-valued polynomials P + n are solutions to the matrix-valued qdifference equation D P + n,1 = P + n Λ n . Here D = M(z)η q + M(z −1 )η q −1 is the restriction of the conjugated D 1 + D 2 to the +1-eigenspace of J . The explicit expressions for M(z) and Λ n are These results are q-analogues of some of the results given in [24, §8.3], see also [39]. Note moreover that W − (0) is a multiple of the identity so that the commutant of W − equals the commuting algebra of Tirao and Zurrián [42], see also [22]. Since the commutant is trivial, the weight W − is irreducible, which can also be checked directly.
Quantum group-related properties of spherical functions
In this section, we start with the proofs of the statements of Sect. 4 which can be obtained using the interpretation of matrix-valued spherical functions on U q (g).
Matrix-valued spherical functions on the quantum group
In this subsection, we study some of the properties of the matrix-valued spherical functions which follow from the quantum group theoretic interpretation. In particular, we derive Theorem 4.3 from Remark 4.2. The precise identification with the literature and the standard Clebsch-Gordan coefficients is made in Appendix 1, and we use the intertwiner and the Clebsch-Gordan coefficients as presented there. We also need the matrix elements of the type 1 irreducible finite dimensional representations. Define t m,n : U q (su(2)) → C, t m,n (X ) = t (X )e n , e m , n, m ∈ {− , . . . , }, where we take the inner product in the representation space H for which the basis {e n } n=− is orthonormal. Denoting then α, β, γ , δ generate a Hopf algebra, where the Hopf algebra structure is determined by duality of Hopf algebras. Moreover, it is a Hopf * -algebra with * -structure defined by α * = δ, β * = −qγ , which we denote by A q (SU (2)). Then the Hopf * -algebra A q (SU (2)) is in duality as Hopf * -algebras with U q (su (2)). In particular, the matrix elements t m,n ∈ A q (SU (2)) can be expressed in terms of the generators and span A q (SU (2)). Moreover, the matrix elements t m,n ∈ A q (SU (2)) form a basis for the underlying vector space of A q (SU (2)). The left action of U q (g) on A q (SU (2)) is given by (X · ξ)(Y ) = ξ(Y X) for X, Y ∈ U q (g) and ξ ∈ A q (SU (2)). Similarly, the right action is given by (ξ · X )(Y ) = ξ(XY ) for X, Y ∈ U q (g) and ξ ∈ A q (SU (2)). A calculation gives k 1/2 · t m,n = q −n t m,n and t m,n · k 1/2 = q −m t m,n so that α · k 1/2 = q 1/2 α, β · k 1/2 = q −1/2 β, γ · k 1/2 = q 1/2 γ , δ · k 1/2 = q −1/2 δ. Since k 1/2 and its powers are group-like elements of U q (g), it follows that the left and right action of k 1/2 and its powers are algebra homomorphisms. See e.g. [9,11,20,21], and references given there.
In the same way, we view where the functions are taken with respect to the Hopf algebra tensor product U q (g) = U q (su (2)) ⊗ U q (su (2)). In particular, for λ, μ ∈ 1 2 Z we find the expression t 1 Similarly, the Hopf * -algebra spanned by all the matrix elements t 1 2 and let A be the commutative subalgebra of U q (g) generated by A and A −1 . Recall the spherical function Φ 1 , 2 from Definition 4.4, and recall the transformation property (4.5).
Definition 5.1 The linear map
So the spherical function Φ 1 , 2 is a spherical function of type by (4.5).
(ii) A spherical function Φ of type restricted to A is diagonal with respect to the basis {e p } p=− . Moreover, for each λ ∈ Z, (iii) Assume that Φ is a spherical function of type and that with all linear maps Φ m,n on U q (g) in the linear span of the matrix elements This proves (i).
To obtain (ii), write Φ = m,n=− Φ m,n ⊗ E m,n , and observe Finally, for (iii) note that for and, by the multiplicity free statement in Theorem 4.3, this is the only (up to a constant) possible linear combination of the matrix elements t 1 m 1 ,n 1 ⊗ t 2 m 2 ,n 2 for fixed ( 1 , 2 ) which has this property. Hence, (iii) follows.
The special case Φ 0 1/2,1/2 : U q (g) → C can now be calculated explicitly using the Clebsch-Gordan coefficients C 1/2,1/2,0 m,−m,0 from (8.5). Explicitly, This element is not self-adjoint in A q (SU (2))⊗A q (SU (2)). Recall the right action of . This is analogous to the construction discussed at the beginning of this subsection. Then is self-adjoint for the * -structure of A q (SU (2)) ⊗ A q (SU (2)). Then by construction Proof of Theorem 4.6 As U q (g)-representations, the tensor product decomposition
The recurrence relation for spherical functions of type
where Ψ i, j m,n is in the span of the matrix elements t 1 +i r 1 ,s 1 ⊗ t 1 + j r 2 ,s 2 . Note that (4.7), and a similar calculation for multiplication by an element from B from the other side, shows that ϕΦ 1 , 2 has the required transformation behaviour (4.5) for the action of B from the left and the right. Since the matrix elements t 1 r 1 ,s 1 ⊗ t 1 r 2 ,s 2 form a basis for A q (G), it follows that each Ψ i, j satisfies (4.5) so that by Proposition 5.2(iii) It remains to show that A 1/2,1/2 = 0. In order to do so, we evaluate the identity of Theorem 4.6 at a suitable element of U q (g). For the U q (su (2))-representations in (3.3), it is immediate that t (e k ) = 0 for k > 2 and that t r,s (e 2 ) = 0 except for the case (r, s) = (− , ) and then t − , (e 2 ) = 0. Extending to U q (g), we find that where the right-hand side is non-zero in case m = − 1 + 2 , n = 1 − 2 . So if we evaluate the identity of Theorem 4.6 at E 2 1 +1 , it follows that only the term with (i, j) = (1/2, 1/2) on the right-hand side of Theorem 4.6 is non-zero, and the specific matrix element is given by (5.4) and this is non-zero for the same matrix element (m, n) = (− 1 + 2 , 1 − 2 ) by (5.4).
Note that we can derive the explicit value of A 1/2,1/2 from the proof of Theorem 4.6 by keeping track of the constants involved. However, we do not need the explicit value except in the case = 0.
Proposition 5.3 For n ∈ N, we have
Recall that the Chebyshev polynomials of the second kind are orthogonal polynomials: see e.g. [15,19,26].
Orthogonality relations
In this subsection, we prove Theorem 4.8 from the quantum group theoretic interpretation up to the calculation of certain explicit coefficients in the expansion of the entries of the weight. Recall the Haar functional on A q (SU (2)). It is the unique left and right invariant positive functional h 0 : A q (SU (2)) → C normalised by h 0 (1) = 1, see e.g. [9], [20, §4.3.2], [21,48]. The Schur orthogonality relations state (2)). Then the functional h = h 0 ⊗ h 0 is the Haar functional on A q (G). We can identify the analogue of the algebra of bi-K -invariant polynomials on G as the algebra generated by the self-adjoint element ψ, and give the analogue of the restriction of the invariant integration in Lemma 5.4.
Lemma 5.4 A functional τ : U q (g) → C that satisfies the transformation behaviour τ ((AX A −1 )Y Z) = ε(X )ψ(Y )ε(Z ) for all X, Z ∈ B and all Y ∈ U q (g) is a polynomial in ψ as in (5.2). Moreover, the Haar functional on the * -algebra C[ψ] ⊂ A q (G) is given by
Proof From Proposition 5.2(iii) and (5.3), we see that any functional on U q (g) satisfying the invariance property is a polynomial in ψ, since this space is spanned by Φ 0 for all X, Z ∈ B and all Y ∈ U q (g).
In particular, any such trace is a polynomial in the generator ψ by Lemma 5.4.
Corollary 5.6
Fix ∈ 1 2 N, then for k, p ∈ {0, 1, . . . , 2 }, Proof of Theorem 5.5 Write Ψ = m,n=− Ψ m,n ⊗ E m,n , Φ = m,n=− Φ m,n ⊗ E m,n , with Ψ m,n and Φ m,n linear functionals on U q (g) so that (5.8) Since B is a right coideal, we can assume that Z (1) ∈ B, so, by the transformation property (4.5), Ψ m, (1) ). Using this, and t k,n (Z (1) ) = t n,k (Z * (1) ) by the * -invariance of B and the unitarity of t , and next move this to the Φ-part, and summing over n and the transformation property (4.5) for Φ, we obtain (2) ) * = ε(Z ) by the antipode axiom in a Hopf algebra. For the invariance property from the left, we proceed similarly using that A is a group-like element. So for Y ∈ U q (g) and X ∈ B we have Proceeding as in the previous paragraph using X (1) The result follows if we prove (X ) X * (1) A −2 S(X (2) ) * A = A −1 ε(X ). In order to prove this, we need the observation that S(X * ) = A −2 S(X ) * A 2 for all X ∈ U q (g), which can be verified on the generators and follows since the operators are antilinear homomorphisms, see Remark 5.7. Now we obtain the required identity: Remark 5.7 The required identity S(X * ) = A −2 S(X ) * A 2 for all X ∈ U q (g) can be generalised to arbitrary semisimple g. Indeed, since the square of the antipode S is given by conjugation with an explicit element of the Cartan subalgebra associated to ρ = 1 2 α>0 α, see e.g. [33, Exercise 4.1.1], and since in a Hopf * -algebra S • * is an involution, we find that S( Using the Clebsch-Gordan decomposition, we see that t The statement on the adjoint follows immediately from (5.8) and ψ being selfadjoint, see (5.2).
We now can start the first part of the proof of Theorem 4.8, except for the fact that we have to determine certain constants. This is contained in Lemma 5.8.
Lemma 5.8 We have
The proof of Lemma 5.8 is a calculation using Theorem 4.6, which we postpone to Sect. 6.3.
First part of the proof of Theorem 4.8 Using the notation of Sect. 4, we find from Corollary 4.7 and (4.8) that where we use that the action by A −1 from the right is an algebra homomorphism, since A −1 is group like, and that ψ is self-adjoint. By Proposition 5.2, Lemma 5.4 and (5.7), we have after a straightforward calculation. Plugging in Lemma 5.8 in (5.9) and rewriting prove the result using Corollary 5.6.
Note that we have not yet determined the explicit values of α t (m, n) in Theorem 4.8 and we have not shown that W is a matrix-valued weight function in the sense of Sect. 2. The values of the constants α t (m, n) will be determined in Sect. 6.1 and the positivity of W (x) for x ∈ (−1, 1) will follow from Theorem 4.15.
q-Difference equations
It is well known, see e.g. Koornwinder [30], Letzter [35], Noumi [37], that the centre of the quantised enveloping algebra can be used to determine a commuting family of q-difference operators to which the corresponding spherical functions are eigenfunctions. In this subsection, we derive the matrix-valued q-difference operators corresponding to central elements to which we find matrix-valued eigenfunctions.
The centre of U q (g) is generated by two Casimir elements, see Sect. 3: Because of Proposition 5.2 and (3.4), we find The goal is to compute the radial parts of the Casimir elements acting on arbitrary spherical functions of type in terms of an explicit q-difference operator. In order to derive such a q-difference operator, we find a BAB-decomposition for suitable elements in U q (g) in Proposition 5.10. This special case of a BAB-decomposition is the analogue of the K AK -decomposition, which has not a general quantum algebra analogue. For this purpose, we first establish Lemma 5.9, which can be viewed as a quantum analogue of [8,Lemma 2.2], and gives the BAB-decomposition of F 2 A λ .
Lemma 5.9 Recall
Proof Recall Definition 4.1, so the result follows from
Proposition 5.10
The BAB-decomposition for the Casimir elements 1 and 2 is given by Proof We first concentrate on 2 , and the statement for 1 follows by flipping the order using σ as in Remark 4.2(iv). We need to rewrite E 2 F 2 A λ , which we do in terms of F 2 A λ and next using Lemma 5.9. The details are as follows.
Using Definition 4.1 and the commutation relations, and pulling through F 2 to the left, we get Similarly, and only slightly more involved, we obtain Using (5.11) and (5.12), we eliminate the term with F 1 F 2 , and shifting λ to λ + 1 gives Apply Lemma 5.9 on the first two terms on the right-hand side of (5.13) and note that the remaining terms in (5.13) and in 2 A λ can be dealt with by observing that K 2 = K −1/2 A = AK −1/2 . Taking corresponding terms together proves the BABdecomposition for 2 A λ after a short calculation.
Our next task is to translate Proposition 5.10 into an operator for spherical functions of type , from which we derive eventually, see Theorem 4.13, an Askey-Wilson qdifference type operator for the matrix-valued orthogonal polynomials P n . Let Φ be a spherical function of type , then we immediately obtain from Proposition 5.10 (5.14) The analogous expression for Φ( 2 A λ ) of (5.14) can be obtained using the flip σ , see Remark 4.2(iv). In particular, it suffices to replace all t (X ) in (5.14) by J t (X )J , see Remark 4.2(iv), to get the corresponding expression. By Proposition 5.2(ii), we know that Φ(A λ ) is diagonal. Note that Φ · 1 : U q → End(H ) is also a spherical function of type by the centrality of the Casimir operator 1 λ ) is diagonal, which can also be seen directly from (5.14). We can calculate the matrix entries of Φ( 1 A λ ) using the upper triangular matrices t (B 1 K −1/2 ), t (B 1 ) having only non-zero entries on the superdiagonal, the lower triangular matrices t (K −1/2 B 2 ), t (B 2 ) having only non-zero entries on the subdiagonal and the diagonal matrices t (K ±1/2 ), t ( For Φ a spherical function of type , we view the diagonal restricted to A as a vector-valued functionΦ: So we can regard the Casimir elements as acting on the vector-valued functionΦ, and the action of the Casimir is made explicit in Proposition 5.11.
Proposition 5.11
Let Φ be a spherical function of type , with corresponding vectorvalued functionΦ : A → H representing the diagonal when restricted to A. Then a tridiagonal and N 1 (z) is a diagonal matrix with respect to the basis {e n } n=− of H with coefficients Moreover, for 2 the action is Remark 5.12 The proof of Theorem 4.13 does not explain why the matrix coefficients of η q and η q −1 in Theorem 4.13 are related by z ↔ z −1 . In Proposition 5.11, there is a lack of symmetry between the up and down shift in λ, and only after suitable multiplication withΦ 0 from the left and right the symmetry of Theorem 4.13 pops up. It would be desirable to have an explanation of this symmetry from the quantum group theoretic interpretation. Note that the symmetry can be translated to the requirement The remark following (5.14) on how to switch to the second Casimir operator gives the conjugation between M 1 and M 2 , respectively N 1 and N 2 . Note that in case = 0, Φ andΦ are equal, and we find that Proposition 5.11 gives the operator for Φ : U q (g) → C a spherical function (of type 0), which should be compared to [30,Lemma 5.1], see also [35,37].
Proof Consider (5.14) and calculate the (m, m)-entry. Using the explicit expressions for the elements t (X ) for X ∈ B, see (4.2), (3.1), in (5.14), we find which we can rewrite as stated with the matrices M 1 (q λ ) and N 1 (q λ ). The case for 2 follows from the observed symmetry, or it can be obtained by an analogous computation.
Corollary 5.13 AssumingΦ 0 (A λ ) is invertible, the matrix-valued polynomials P n satisfy the eigenvalue equations
and Λ n (i) are defined in (5.16) and μ(x) = 1 2 (x + x −1 ). It remains to prove the assumption in Corollary 5.13 for sufficiently many λ, and to calculate the coefficients in the eigenvalue equations explicitly. This is done in Sect. 7.
Having established (5.17), we can prove Corollary 4.9 by considering coefficients in the Laurent expansion.
Proof of Corollary 4.9
The left-hand side of (5.17) can be expanded as a Laurent series in q λ by Proposition 5.2(ii) and (5.15). The leading coefficient of degree + n is an antidiagonal matrix since the Clebsch-Gordan coefficient is zero unless i + j = 2 . With a similar expression forΦ 0 (A λ ) on the right-hand side, expanding P n μ(q λ+1 ) = lc(P n )q n(λ+1) 2 −n + lower order terms gives
Symmetries
Even though we can use the explicit expression of the weight W to establish Proposition 4.10, we show the occurrence of J in the commutant from Remark 4.5(ii).
Note that (5.19), after applying the Haar functional on the * -algebra generated by ψ as in Lemma 5.4, also gives J G n J = G n as is immediately clear from the explicit expression for the squared norm matrix G n in Theorem 4.8 derived in Sect. 5.3. It moreover shows that (J P n J ) n∈N is a family of matrix-valued orthogonal polynomials with respect to the weight W . It is a consequence of Corollary 4.9, see Sect. 2, that J P n J = P n , since J lc(P n )J = lc(P n ) by Corollary 4.9.
Note that we have now proved Proposition 4.10 except for the ⊃-inclusion in the first line. This is done after the proof of Theorem 4.8 is completed at the end of Sect. 6.1.
As a consequence of the discussion on symmetries, we can formulate the symmetry forΦ n in Lemma 5.14. Proof This is a consequence of initial observations in Sect. 5.5. For i, j ∈ {0, . . . , 2 }, using (5.15) and σ (A λ ) = A λ we obtain
The weight and orthogonality relations for the matrix-valued polynomials
In this section, we complement the quantum group theoretic proofs of Sect. 5 of some of the statements of Sect. 4 using mainly analytic techniques. In Sect. 6.1, we prove the statement on the expansion of the entries of the weight function in terms of Chebyshev polynomials of Theorem 4.8. In Sect. 6.2, we prove the LDU-decomposition of Theorem 4.15. In Sect. 6.3, we prove Lemma 5.8 using a special case and induction using Theorem 4.6 in the induction step.
Explicit expressions of the weight
In order to prove the explicit expansion of the matrix entries of the weight W in terms of Chebyshev polynomials, we start with the expression of Corollary 5.6 for the matrix entries of the weight W . After pairing with A λ , we expand as a Laurent polynomial in q λ in Proposition 6.1. Then we can use Lemma 6.2, whose proof is presented in Appendix 2.1.
Proof We obtain from Corollary 5.6 using that A λ is a group-like element and S(A λ ) * = A −λ . By Proposition 5.2(ii) where the Clebsch-Gordan coefficients are to be taken as zero in case |i −n| > − 1 2 k, respectively | j − n| > − 1 2 p. Now put s = j − i, then s runs from − 1 2 (k + p) up to 1 2 (k + p), and we have the Laurent expansion
Plugging in (8.3) gives the explicit expression for d s (k, p).
In order to show that d s ( p, k) = d −s ( p, k), we note that p(ψ)(A λ ) = p(μ(q λ )) is symmetric in λ ↔ −λ for any polynomial p and so Corollary 5.6 implies the symmetry.
Note that for a matrix element of P n (ψ) * W (ψ)P m (ψ) a similar expression can be given, cf. the first part of the proof of Theorem 4.8, but this is not required. The symmetry in the Laurent expansion of W (ψ) k, p (A λ ) does not seem to follow directly from known symmetries for the Clebsch-Gordan coefficients, see e.g. [20,Chap. 3].
Now we can proceed with the proof of Theorem 4.8, for which we need Lemma 6.2. Lemma 6.2 For ∈ 1 2 N and for k, p ∈ {0, . . . , 2 } subject to k ≤ p and k + p ≤ 2 we have with the notation of Proposition 6.1 and Theorem 4.8, Lemma 6.2 contains the essential equality for the proof of the explicit expression of the coefficients of the matrix entries of the weight of Theorem 4.8. The proof of Lemma 6.2 can be found in Appendix 2.1, and it is based on a q-analogue of the corresponding statement for the classical case [24,Theorem 5.4]. Previously in 2011, Mizan Rahman (private correspondence) has informed one of us that he has obtained a q-analogue of the underlying summation formula for [24,Theorem 5.4]. It is remarkable that Rahman's q-analogue is different from the one needed here in Lemma 6.2.
Second part of the proof of Theorem 4. 8 We prove the statement on the explicit expression of the matrix entries of the weight in terms of Chebyshev polynomials. By Corollary 5.6, we have for all λ ∈ Z. Since the coefficients α r (k, p) are completely determined by W (ψ) k, p and since W (ψ) k, p = W (ψ) 2 −k,2 −k = W (ψ) p,k * , we can restrict to the case k ≤ p, k + p ≤ 2 . For this case, the result follows from Lemma 6.2, and hence the explicit expression for α r (k, p) in Theorem 4.8 is obtained.
Note that proof of Theorem 4.8 is not yet complete, since we have to show that the weight is strictly positive definite for almost all x ∈ [−1, 1], see Sect. 2. This will follow from the LDU-decomposition for the weight as observed in Corollary 4.16, but in order to prove the LDU-decomposition of Theorem 4.15 we need the explicit expression for the coefficients α t (m, n) of Theorem 4.8.
In Sect. 5.5, we observed that J commutes with W (x) for all x. In order to prove Proposition 4.10, we need to show that the commutant is not larger, and for this we need the explicit expression of α t (m, n) of Theorem 4.8.
Proof of Proposition 4.10 Let Y be in the commutant, and write W (x) = 2 n=0 W k U n (x) for W k ∈ Mat 2 +1 (C) using Theorem 4.8. Then [Y, W k ] = 0 for all k. The proof follows closely the proofs of [24,Proposition 5.5] and [25,Proposition 2.6]. Note that W 2 and W 2 −1 are symmetric and persymmetric (i.e. commute with J ). Moreover, (W 2 ) m,n is non-zero only for m + n = 2 and (W 2 −1 ) m,n is non-zero only for |m +n −2 | = 1. From the explicit expression of the coefficients α t (m, n), we find that all non-zero entries of W 2 and W 2 −1 are different apart from the symmetry and persymmetry. The proof of Proposition 4.10 can then be finished following [24, Proposition 5.5].
LDU-decomposition
In order to prove the LDU-decomposition of Theorem 4.15 for the weight, we need to prove the matrix identity termwise. So we are required to show that for the expression of W (x) in Theorem 4.8 and for the expressions of L(x) and T (x) in Theorem 4.15. Because of symmetry, we can assume without loss of generality that m ≥ n. Then (6.2) is equivalent to Proposition 6.3 after taking into account the coefficients in the LDU-decomposition, so it suffices to prove Proposition 6.3 in order to obtain Theorem 4.15.
Before embarking on the proof of Proposition 6.3, note that each summand on the right-hand side of the expression of Proposition 6.3 is an even, respectively odd, polynomial for m + n even, respectively odd, since the continuous q-ultraspherical polynomials are symmetric and since w(x; q 2k+2 |q 2 ) = 4(1 − x 2 ) k j=1 1 − 2(2x 2 − 1)q 2 j + q 4 j , see (4.12), is an even polynomial with a factor (1 − x 2 ). In the proof of Proposition 6.3 we use Lemma 6.4. Lemma 6.4 Let 0 ≤ k ≤ m ≤ n and t ∈ N. Then the integral is equal to zero for t > m, and for 0 ≤ t ≤ m, the integral above is equal to In Lemma 6.4, we use the notation (4.13) for the q-Racah polynomials. Lemma 6.4 shows that the expansion as in Proposition 6.3 is indeed valid, and it remains to determine the coefficients β k (m, n).
The proof of Lemma 6.4 follows the lines of the proof of [23, Lemma 2.7], see Appendix 2.2. The main ingredients are, cf. the proof in [23], the connection and linearisation coefficients for the continuous q-ultraspherical polynomials dating back to the work of L.J. Rogers (1894-5), see e.g. [2, §10.11], [19, §13.3], [15, (7.6.14), (8.5.1)]. Write the product C m−k (x; q 2k+2 |q 2 )C n−k (x; q 2k+2 |q 2 ) as a sum over r of continuous q-ultraspherical polynomials C r (x; q 2k+2 |q 2 ) using the linearisation formula and write U n+m−2t , which is a continuous q-ultraspherical polynomial for β = q, in terms of C s (x; q 2k+2 |q 2 ) using the connection formula. The orthogonality relations for the continuous q-ultraspherical polynomials then give the integral in terms of a single series. The details are in Appendix 2.2. From this sketch of proof, it is immediately clear that Lemma 6.4 can be generalised to a more general statement. This is the content of Remark 6.5, whose proof is left to the reader. Remark 6.5 For integers 0 ≤ t, k ≤ m ≤ n and parameters α, β, we have Note that the 4 ϕ 3 -series in Remark 6.5 is balanced, but in general is not a q-Racah polynomial.
In the proof of Proposition 6.3, and hence of Theorem 4.15, we need the summation formula involving q-Racah polynomials stated in Lemma 6.6. Its proof is also given in Appendix 2.2.
With these preparations, we can prove Proposition 6.3, and hence the LDUdecomposition of Theorem 4.15.
Proof of Proposition 6.3 Since we have the existence of the expression in Proposition 6.3, it suffices to calculate β k (m, n) having the explicit value of the α t (m, n)'s from Theorem 4.8. Multiply both sides by 1 2π √ 1 − x 2 U m+n−2t (x) and integrate using the orthogonality for the Chebyshev polynomials so that Lemma 6.4 gives The q-Racah polynomials R k (μ(t); 1, 1, q −m−1 , q −n−1 ; q 2 ) satisfy the orthogonality relations Using the orthogonality relations, we find the explicit expression of β k (m, n) in terms of α t (m, n), and using the explicit expression of α t (m, n) of Theorem 4.8 gives This expression is summable by Lemma 6.6. Collecting the coefficients proves the proposition.
Last part of the proof of Theorem 4.8 Now that we have proved Theorem 4.15, Corollary 4.16 is immediate, since the coefficients of the diagonal matrix T (x) are positive on (−1, 1). So the weight is strictly positive definite on (−1, 1), which is the last step to be taken in the proof of Theorem 4.8.
Summation formula for Clebsch-Gordan coefficients
In this subsection, we prove Lemma 5.8, which has been used in the first part of the proof of Theorem 4.8, see Sect. 5.3. The proof of Lemma 5.8 is somewhat involved, since we employ an indirect way using induction and Theorem 4.6.
Proof of Lemma 5.8 Assume for the moment that Assuming (6.3) the lemma follows, using It remains to prove (6.3). We do this in case 1 + 2 = . Put ( 1 , 2 ) = (k/2, − k/2) = ξ(0, k) for k ∈ N and k ≤ 2 . Using the explicit expression for the Clebsch-Gordan coefficients (8.3), we find that in this special case the left-hand side of (6.3) equals where the sum runs over i for − + k/2 + m ≤ i ≤ − k/2 + m. Substitute i → p − + k/2 + m to see that this equals Simplifying the expression, we find that since the 2 ϕ 1 can be summed by the reversed q-Chu-Vandermonde sum [15,(II.7)].
To prove Lemma 5.8 in general, we set hence it is sufficient to calculate f 1 , 2 (−2). We will show by induction on n that f ξ(n,k) (−2) is independent of (n, k), or equivalently that f 1 , 2 (−2) is independent of ( 1 , 2 ). Since we have established the case n = 0, Lemma 5.8 then follows.
In order to perform the induction step, we consider the recursion of Theorem 4.6.
q-Difference operators for the matrix-valued polynomials
We continue the study of q-difference operator for the matrix-valued orthogonal polynomials started in Corollary 5.13. In particular, we show that the assumption on the invertibility ofΦ 0 follows from the LDU-decomposition in Theorem 4.15. Then we make the coefficients in the matrix-valued second-order q-difference operator of Corollary 5.13 explicit in Sect. 7.1. Comparing to the scalar-valued Askey-Wilson q-difference operators, see e.g. [2,15,19,26], we view the q-difference operator as a matrix-valued analogue of the Askey-Wilson q-difference operator. Next, having the matrix-valued orthogonal polynomials as eigenfunctions to the matrix-valued Askey-Wilson q-difference operator, we use this to obtain an explicit expression of the matrix entries of the matrix-valued orthogonal polynomials in terms of scalar-valued orthogonal polynomials from the q-Askey scheme by decoupling of the q-difference operator using the matrix-valued polynomial L in the LDU-decomposition of Theorem 4.15.
From this expression, we can obtain an explicit expression for the coefficients in the matrix-valued three-term recurrence relation for the matrix-valued orthogonal polynomials, hence proving Theorem 4.11 and Corollary 4.12.
Explicit expressions of the q-difference operators
In order to make Corollary 5.13 explicit, we need to study the invertibility of the matrix Φ 0 (A λ ). For this, we first use Theorem 5.5 and its Corollary 5.6, and in particular (6.1).
Proof of Theorem 4.13 Corollary 5.13 gives a second-order q-difference equation for the matrix-valued orthogonal polynomials for an infinite set of λ. So it suffices to check that for i = 1, 2 the matrix-valued functionsM i ,Ñ i of Corollary 5.13 coincide which is an easy check using the explicit expressions of Theorem 4.13. Now (7.2) and (5.16) for n = 0 show that any equation of (7.1) implies the other equation of (7.1). Indeed, assuming the second equation of (7.1) holds, then implying the first equation of (7.1). By Proposition 5.2(ii) and (5.15), the matrix entries ofΦ 0 (A λ ) are Laurent series in q λ . Setting z = q λ , we see that in order to verify (7.1) entry-wise, we need to check equalities for Laurent series in z.
We first consider the second equality of (7.1) for i = 1. In this case, the matrices N 1 and N 1 are band-limited. Hence, the (m, n)th entry of both sides of (7.1) involves either two or one terms, so we need to check 3) The proof of (7.3) involves the explicit expression of the spherical functions in terms of Clebsch-Gordan coefficients using Proposition 5.2(ii). It is given in Appendix 2.3. The statements for the second q-difference equation with i = 2 follows from the symmetries of Proposition 5.11 and Lemma 5.14.
The explicit expressions have been obtained initially by computer algebra, and then later the proof as presented here and in Appendix 2.3 has been obtained.
Explicit expressions for the matrix entries of the matrix-valued orthogonal polynomials
Having established the q-difference equations for the matrix-valued orthogonal polynomials of Theorem 4.13 and having the diagonal part of the LDU-decomposition of the weight in terms of weight functions for the continuous q-ultraspherical polynomials in Theorem 4.15, it is natural to look at the q-difference operators conjugated by the polynomial function L t . It turns out that this completely decouples one of the second-order q-difference operators of Theorem 4.13. This gives the opportunity to link the matrix entries of the matrix-valued orthogonal polynomials to continuous q-ultraspherical polynomials. In order to determine the coefficients, we use the other q-difference operator and the orthogonality relations. Having such an explicit expression, we can determine the three-term recurrence relation for the monic matrixvalued orthogonal polynomials straightforwardly, and hence also for the matrix-valued orthogonal polynomials P n , since we already have determined the leading coefficient in Corollary 4.9. The first step is to conjugate the second-order q-difference operator D 1 of Theorem 4.13 with the matrix L t of the LDU-decomposition of Theorem 4.15 into a diagonal q-difference operator. This conjugation is inspired by the result of [23, Theorem 6.1]. This conjugation takes D 2 in a three-diagonal q-difference operator. For any n ∈ N, let R n (x) = L t (x)Q n (x), where Q n (x) = P n (x) lc(P n ) −1 denote the corresponding monic polynomial. Note that we have determined the leading coefficient lc(P n ) in Corollary 4.9. Then (R n ) n≥0 forms a family of matrix-valued polynomials, but note that the degree of R n is larger than n, and that the leading coefficient of R n is singular. Note that R n satisfy the orthogonality relations Proof We start by observing that the monic matrix-valued orthogonal polynomials Q n are eigenfunctions of the second-order q-difference operators D i of Theorem 4.13 for the eigenvalue lc(P n )Λ n (i)lc(P n ) −1 = Λ n (i), since the matrices are diagonal and thus commute. By conjugation, we find that R n satisfy using the notationL t (z) = L t (μ(z)), etc., with x = μ(z) = 1 2 (z + z −1 ) as before. It remains to calculate K i (z) explicitly. We show in Appendix 2.4 that the expressions for K i are correct by verifying for i = 1, 2.
Lemma 7.2 For n ∈ N and 0 ≤ i, j ≤ 2 , we have where C n (x; β|q) are the continuous q-ultraspherical polynomials (4.10) and β n (i, j) is a constant depending on i, j and n.
Proof Evaluate D 1 R n (x) = R n (x)Λ n (1) in entry (i, j). Since D 1 is decoupled, we get a q-difference equation for the polynomial R n i, j , which, after simplifying, is All polynomial solutions of this q-difference are given by a multiple of the Askey-Wilson polynomials, p n+ j−i (x; q i+1 , −q i+1 , q 1/2 , −q 1/2 |q), see [ Our next objective is to determine the coefficients β n (i, j) of Lemma 7.2. Having exploited that the matrix-valued polynomials R n are eigenfunctions for the decoupled operator D 1 of Theorem 7.1, we can use Lemma 7.2 in (7.4) to calculate the (i, j)th coefficient of (7.4): using that lc(P m ) and the squared norm matrix G m are diagonal matrices, see Corollary 4.9 and Theorem 4.8. The integral in (7.6) can be evaluated by (4.11). In particular, the case m + j = n + i of (7.6) gives the explicit orthogonality relations Theorem 7. 3 We have Proof From Theorem 7.1, we have Evaluate (7.8) in entry (i, j) and use Lemma 7.2 to find a three-term recurrence relation in i of β n (i, j), Multiply by (1 − z 2 )(1 − q 2 ) 2 and evaluate the Laurent expansion at the leading coefficient in z of degree n + j − i + 3. The leading coefficient in z of the continuous q-ultraspherical polynomialC n (αz; β|q) is (β;q) n (q;q) n α n . After a straightforward computation, this leads to the three-term recurrence relation This recursion relation can be rewritten as the three-term recurrence relation for the q-Racah polynomials after rescaling, see [ for some constant γ n ( j) independent of i. Plugging this expression in (7.7) for i = j gives |γ n ( j)| 2 by comparing with the explicit orthogonality relations for the q-Racah polynomials, see [ , and since the explicit expression of L j,i shows that the leading coefficient (of degree j − i) is positive, we see that the leading coefficient (of degree n + j − i) of R i, j in case j ≥ i is positive. Since γ n ( j) is independent of i, we take i = 0, which shows that γ n ( j) is positive.
Proof of Theorem 4.17 Using Theorem 7.3 with the explicit inverse of L(x) as given in Theorem 4.15 gives an explicit expression for the matrix entries of Q n (x) = (L(x) −1 ) t R n (x). Then we obtain the matrix entries of P n (x) = Q n (x)lc(P n ) from this expression and Corollary 4.9, stating that the leading coefficient is a diagonal matrix.
Three-term recursion relation
The matrix-valued orthogonal polynomials satisfy a three-term recurrence relation, see Sect. 2. Moreover, Theorem 4.6 shows that the three-term recurrence relation can in principle be obtained from the tensor product decomposition. However, in that case we obtain the coefficients of the matrices in the three-term recurrence relation in terms of sums of squares of Clebsch-Gordan coefficients, and this leads to a cumbersome result. In order to obtain an explicit expression for the three-term recurrence relation as in Theorem 4.11 and Corollary 4.12, we use the explicit expression obtained in Theorem 4.17 and Lemma 7.4, which is [23, Lemma 5.1]. Lemma 7.4 is only used to determine X n . Lemma 7.4 Let (Q n ) n≥0 be a sequence of monic (matrix-valued) orthogonal polynomials and write Q n (x) = n k=0 Q n k x k , where Q n k ∈ Mat N (C). The sequence (Q n ) n≥0 satisfies the three-term recurrence relation So we start by calculating the one-but-leading term in the monic matrix-valued orthogonal polynomials. (7.9) and this expression shows that deg(Q n (x) i, j ) = n + j − i. So in case j − i ≤ 0, we can only have a contribution to Q n n−1 in case i − j = 0 or i − j = 1. The first case does not give a contribution, since (7.9) is even or odd for n + j − i even or odd. So we only have to calculate the leading coefficient in (7.9) for i − j = 1. With the explicit value of β n (i, j) as in Sect. 7.2 or Theorem 4.17 and Corollary 4.19, we see that Q n n−1 in case i − j = 1 gives the required expression for Q n n−1 j, j−1 . On the other hand, by Proposition 4.10 and since J lc(P n )J = lc(P n ) by Corollary 4.9, it follows that J Q n (x)J = Q n (x). Therefore, we find the symmetry of the entries of the monic matrix-valued polynomials Q n (x) i, j = Q n (x) 2 −i,2 − j so that the case j − i ≥ 0 can be reduced to the previous case, and we get Q n n−1 j, j+1 = Q n n−1 2 − j,2 − j−1 .
Proof of Theorem 4.11 The explicit expression for X n follows from Lemma 7.4 and Lemma 7.5. By Theorem 4.8 and Corollary 4.9, we have the orthogonality relations since the matrices involved are diagonal and self-adjoint, hence pairwise commute. By the discussion in Sect. 2, we have Y n = G −1 n−1 lc(P n−1 ) 2 G n lc(P n ) −2 , and a straightforward calculation gives the required expression. (2)). This gives C 1 , 2 , n 1 ,n 2 ,n = C q ( 1 , 2 , ; n 1 , n 2 , n), where the righthand side is the notation of the Clebsch-Gordan coefficient as in [20, §3.4.2, (41)]. The Clebsch-Gordan coefficients are explicitly known in terms of terminating basic hypergeometric orthogonal polynomials, the so-called q-Hahn polynomials [26], see [20, §3.4].
Appendix 2: Proofs involving only basic hypergeometric series
In Appendix 2, we collect the proofs of various intermediate results only involving basic hypergeometric series. For these proofs, we use the results of Gasper and Rahman [15]. In particular, we follow the standard notation of [15], and recall 0 < q < 1.
Proofs of lemmas for Theorem 4.8
Here we present the details of the proof of Lemma 6.2, which is used in order to prove the explicit expression of the matrix entries of the weight in terms of Chebyshev polynomials in Theorem 4.8. We start with two intermediate results needed in the proof. Lemma 7.6 can be viewed as a q-analogue of Sheppard's result [2,Corollary 3.3.4].
The inner sum over i is a 3 ϕ 2 -series, and after some rewriting, it can be transformed using Lemma 7.6. The sum over i becomes We observe that the evaluation of the t-order q-difference operator yields only one non-zero term in the sum over i, namely the one corresponding to i = p − s − t. After simplifications, we have Reversing the order of summation using T = p − s − t proves the proposition. Suppose that p − k ≤ 0, k + p ≤ 2 and s ≥ 1 2 (k − p). Using Proposition 6.1, we find the expression By taking e s (k, p) = d s+ 1 2 (k− p) , Proposition 7.8 shows that d Comparing (9.6) with the explicit expression of α i (k, p) yields the statement of the lemma.
The proof of Lemma 6.4 follows the lines of the proof of [23, Lemma 2.7] closely.
Proof of Lemma 6.4 In order to evaluate the integral of Lemma 6.4, we observe that the weight function in the integral is the weight function (4.11) for the continuous q-ultraspherical (in base q 2 ) with β = q 2k+2 . Rewrite the product of the two continuous q-ultraspherical (in base q 2 ) with β = q 2k+2 as a sum over i of C m+n−2k−2i (x; q 2k+2 |q 2 ) using (9.8). Since the Chebyshev polynomials can be viewed as continuous q-ultraspherical polynomials, which in base q 2 is U m+n−2t (x) = C m+n−2t (x; q 2 |q 2 ), we can use (9.7) to write the Chebyshev polynomial as a sum of continuous q-ultraspherical polynomials with β = q 2k+2 . Plugging in the two sum-By a long but straightforward calculation, we can show that the coefficient of z n−m−2k , k ∈ {−1, 0, . . . , n − m + 1} equals zero. This proves the case i = 2.
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2022-12-23T14:42:13.364Z
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2016-06-21T00:00:00.000
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237938023
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pes2o/s2orc
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v3-fos-license
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The Many Faces of Enterococcus spp.—Commensal, Probiotic and Opportunistic Pathogen
Enterococcus spp. are Gram-positive, facultative, anaerobic cocci, which are found in the intestinal flora and, less frequently, in the vagina or mouth. Enterococcus faecalis and Enterococcus faecium are the most common species found in humans. As commensals, enterococci colonize the digestive system and participate in the modulation of the immune system in humans and animals. For many years reference enterococcal strains have been used as probiotic food additives or have been recommended as supplements for the treatment of intestinal dysbiosis and other conditions. The use of Enterococcus strains as probiotics has recently become controversial due to the ease of acquiring different virulence factors and resistance to various classes of antibiotics. Enterococci are also seen as opportunistic pathogens. This problem is especially relevant in hospital environments, where enterococcal outbreaks often occur. Their ability to translocate from the gastro-intestinal tract to various tissues and organs as well as their virulence and antibiotic resistance are risk factors that hinder eradication. Due to numerous reports on the plasticity of the enterococcal genome and the acquisition of pathogenic microbial features, we ask ourselves, how far is this commensal genus from acquiring pathogenicity? This paper discusses both the beneficial properties of these microorganisms and the risk factors related to their evolution towards pathogenicity.
Introduction
Enterococci are a diverse, species-rich group of lactic acid bacteria isolated from various environments, including from the digestive systems of humans, animals, and insects but also from natural biomes such as water [1,2], sewage [3], soil [4], and arable land [5]. Enterococci have also been isolated from plants such as olives [6] and are found on plants in the wild [7,8]. Some enterococci species are commensal, can stimulate the immune system, and have a significant influence on the maintenance of intestinal homeostasis [9,10]. Enterococci can be used as a factor to support the immune system in the form of a probiotic (diet supplement or therapeutic application). Likewise, enterococci play a role in food technology as the initiating culture involved in the fermentation of meats and cheeses [11] and the preservation of food [12][13][14][15]. On the other hand, enterococci can act as pathogens [16]. They are responsible for food contamination [12] and due to their sometimes present virulence and multi-drug resistance, they pose an epidemic threat in the hospital environment [17]. Research suggests they may have a role in the development of colon tumorigenesis as well [18]. Some countries disregard enterococci regardless of their positive features, while others accept them despite being a threat in certain situations. The approach to enterococci differs from country to country, so it is important to standardize the criteria that allow a strain to be considered beneficial for health. The advantages and disadvantages of enterococci applications are described in this review.
It is widely considered that without bacteria, there is no functioning immune system, as they are responsible for stimulating the immune system of the intestinal mucosa [10]. The gut microbiota is seen as a virtual endocrine organ [38], and microbes that are permanently associated with the gut microbiota are regarded as commensals [39]. E. faecalis plays an immunomodulatory role and is responsible for the activation of CD4, CD8 (CD-cluster of differentiation) cells, and B lymphocytes.
The GI tract has developed many defense mechanisms to control the gut microbiota. The intestinal lymphatic system (gut-associated lymphoid tissue) is the immune organ responsible for the production of secretory immunoglobulin A (sIgA). sIgA is an important element of the intestinal barrier, as it prevents the adhesion of microorganisms to the epithelium, neutralizes bacterial toxins, coats and agglutinates microorganisms, and has a bacteriostatic effect. The intestinal barrier is also shaped by enterocytes that form strong adherens junctions (AJs) (zonulae occludentes and tight junction). When these junctions are loosened, the problem of a so called "leaky gut" arises. Such relaxed junctions allow bacteria to pass through and initiate an immunological cascade. Enterococci, especially E. faecium and E. faecalis, subsequently cross the intestinal barrier, which can lead to bacteremia/sepsis in patients [40]. Therefore, maintaining the microbial balance in the gut is of utter importance. This maintenance especially applies to patients with blood cancer, as immunosuppressive drugs or antibiotic therapy change the intestinal environment and its permeability, facilitating the translocation of bacteria into the blood bed [41,42].
Enterococci as Probiotics
According to the Food and Agriculture Organization of the United Nations and World Health Organization, probiotics are live microorganisms identified at the strain level which, when given in an appropriate amount, have a beneficial effect on the health of the host [43]. They are often interpreted as "live biotherapeutics" for human use [44] and "direct-fed microbials" in animal feeds [45,46]. Probiotics can be single or multi-species, as some theorize that a mixture of probiotic bacteria not only interact or compete, but also influence each other's beneficial effects. This interaction means that while using each bacterium separately can yield results, taking them together may be less effective or not effective at all, highlighting the clinical significance of the relationship between bacterial species. Probiotics must meet certain requirements; for example, they should be isolated from the hosts that they are intended to be used for, should be able to survive in the GI tract, and should produce compounds with bacteriostatic activity. According to the Food and Drug Administration, probiotic bacteria should "Generally be Recognized as Safe" [47]. In Europe, a "Qualified Presumption of Safety" is responsible for recommending biological agents intentionally added to food or feed, and information is available in the European Food Safety Authority scientific panels [48].
The features describing a probiotic are shown in Figure 1.
Enterococcal Probiotic Strains
The use of enterococci in the treatment of various diseases, such as chronic and recurrent infections of the upper respiratory tract, skin lesions, or chronic diseases related to the sinuses (chronic sinusitis), was first described in the 1950s [49]. The first probiotic therapies, which entailed the application of Enterococcus faecalis following antibiotic therapy, was described by Heinz Kolb in 1955 [49]. Currently, probiotic preparations of E. faecalis, sometimes enriched with Escherichia coli and lactobacilli, are recommended for the treatment of diseases such as urinary tract inflammation, sinusitis or bronchitis, and the common cold. In Germany, Enterococcus faecalis (DSM 16431) is sold as a drug under the brand name Symbioflor 1 (SymbioPharm, Herborn, Germany) and is recommended for acute and recurrent sinusitis or bronchitis [13,50]. Likewise, this non-pathogenic probiotic bacterium has been fully sequenced, and the genome sequence has been deposited in the European Molecular Biology Laboratory database under the accession number HF558530. The circular genome (2,810,675 bp), with 37.72% GC content, consists of 2733 coding sequences and 63 tRNAs.
Enterococcal Probiotic Strains
The use of enterococci in the treatment of various diseases, such as chronic and recurrent infections of the upper respiratory tract, skin lesions, or chronic diseases related to the sinuses (chronic sinusitis), was first described in the 1950s [49]. The first probiotic therapies, which entailed the application of Enterococcus faecalis following antibiotic therapy, was described by Heinz Kolb in 1955 [49]. Currently, probiotic preparations of E. faecalis, sometimes enriched with Escherichia coli and lactobacilli, are recommended for the treatment of diseases such as urinary tract inflammation, sinusitis or bronchitis, and the common cold. In Germany, Enterococcus faecalis (DSM 16431) is sold as a drug under the brand name Symbioflor 1 (SymbioPharm, Herborn, Germany) and is recommended for acute and recurrent sinusitis or bronchitis [13,50]. Likewise, this non-pathogenic probiotic bacterium has been fully sequenced, and the genome sequence has been deposited in the European Molecular Biology Laboratory database under the accession number HF558530. The circular genome (2,810,675 bp), with 37.72% GC content, consists of 2733 coding sequences and 63 tRNAs.
Clone DSM 16431 carries 2 large mutations which eliminate the vanB operon and genes encoding virulence factors such as cytolysin L, gelatinase, hyaluronidase, bacteriocin, and efaA surface proteins. The genome contains a unique bacteriophage region (1,846,700-1,891,973) [51,52] as well. This strain contains features to facilitate its colonization in the digestive system. Specifically, the agg gene encodes an aggregating factor and facilitates gut colonization, while the esp and ace genes enhance adhesion and colonization. Additional features involved the ability to survive against acids (gastric acid) and proliferation within the intestinal epithelium [53]. Finally, a crucial property of this strain is its lack of any antibiotic resistance mechanisms.
The genome of E. faecalis Symbioflor 1 was compared to the first vancomycin resistant strain E. faecalis V583 isolated in the United States and has been completely sequenced (Accession No. NC_004668) [54]. E. faecalis Symbioflor 1 does not contain any pathogenic features or antibiotic resistance genes previously identified in E. faecalis V583, such as cytolysin, enterococcal surface protein, gelatinase, hyaluronidase, or the peptide antibiotic AS-48. These enterococcal virulence factors have been recognized as suitable markers for the risk assessment of strains used in food products or probiotics [54]. Clone DSM 16431 carries 2 large mutations which eliminate the vanB operon and genes encoding virulence factors such as cytolysin L, gelatinase, hyaluronidase, bacteriocin, and efaA surface proteins. The genome contains a unique bacteriophage region (1,846,700-1,891,973) [51,52] as well. This strain contains features to facilitate its colonization in the digestive system. Specifically, the agg gene encodes an aggregating factor and facilitates gut colonization, while the esp and ace genes enhance adhesion and colonization. Additional features involved the ability to survive against acids (gastric acid) and proliferation within the intestinal epithelium [53]. Finally, a crucial property of this strain is its lack of any antibiotic resistance mechanisms.
The genome of E. faecalis Symbioflor 1 was compared to the first vancomycin resistant strain E. faecalis V583 isolated in the United States and has been completely sequenced (Accession No. NC_004668) [54]. E. faecalis Symbioflor 1 does not contain any pathogenic features or antibiotic resistance genes previously identified in E. faecalis V583, such as cytolysin, enterococcal surface protein, gelatinase, hyaluronidase, or the peptide antibiotic AS-48. These enterococcal virulence factors have been recognized as suitable markers for the risk assessment of strains used in food products or probiotics [54].
Baccouri et al. recently [55] described two new strains of E. faecalis, OB14 and OB15, which were isolated from traditional Tunisian fermented dairy products, Testouri and Rigouta cheese, respectively. Genomic sequencing revealed that OB15 is genetically related to the E. faecalis Symbioflor 1 (DSM 16431) and displays potential as a probiotic, while the second OB14 strain is characterized by tetracycline resistance and high virulence due to the presence of the cytolysin gene. In another study [56], transcriptomic analysis of several clinical strains isolated from the urinary tract of patients was performed and compared to the probiotic strain E. faecalis Symbioflor 1. In particular, energy and nitrogen metabolism, cell stress, and metal acquisition were compared. Citrate and aspartate were important for the growth of both E. faecalis groups in urine, and related gene expression was similar in both groups. According to the authors, virulence factors are responsible for adaptation to an ecological niche and ultimately determine the pathogenic potential of bacteria.
E. faecalis is not alone in having probiotic properties; E. lactis [57], E. hirae [58], E. durans [59], and E. faecium [60] are also used as probiotics. The purpose of using these preparations is to improve the composition of the intestinal microbiota [61,62]. The probiotic strain E. faecium M-74 (Aberdeen, UK; the National Collections of Industrial Food and Marine Bacteria (NCIMB) registered no 11181) was isolated from the gastrointestinal tract of healthy Swedish children and exhibits immunomodulatory, antimutagenic [63][64][65], and hypocholesterolemic properties [66]. E. faecium strain 11181 is also currently used in animal feed as a supplement [67].
Other probiotic preparations exist and are recommended for the treatment of irritable bowel symptoms. The "Bioflorin" preparation includes one of the first cultured probiotic E. faecium SF68 ® strains [60]. E. faecium SF68 ® (the strain deposit of Enterococcus faecium NCIMB 10415 in Aberdeen, Scotland, registered trademark owned by Cerbios-Pharma SA) displays a wide clinical application for the treatment digestive tract disorders in humans [68]. Currently E. faecium SF68 ® is recommended for veterinary applications as a probiotic supplement (e.g., FortiFlora TM ). E. faecium SF68 ® has been described to prevent and treat diarrhea in pets and cats [69,70]. Probiotics such as Cylactins (Hoffmann-La Roche, Basel, Switzerland) and 85 Fargo 688s (Quest International, Naarden, The Netherlands) with E. faecium are also used for veterinary applications.
The Probiotic Importance of Enterococcus spp. and Applications
Pregnant women, newborns, and the elderly are typically at greater risk of infection due to undeveloped or weakened immune systems. Research has demonstrated that supplementation with probiotics in the elderly leads to the growth of potentially beneficial intestinal bacteria but also leads to the increased activation of a non-specific immune response [71]. In some Western European countries, a fermented milk drink called "Gaio" (yogurt) is available, containing bacteria called "Causido," which includes the E. faecium K-77D strain and two strains of Streptococcus termophilus. These bacteria come from the intestinal microbiota of elderly people living in Abkhazia in the Caucasus, an area known for longevity amongst its population [72].
Microbiota are important in maintaining the physiological balance of the intestine and have a significant role in immune homeostasis. Enterococci produce small peptides that belong to the bacteriocin group and have antimicrobial properties. These include enterocin A, B, P, ON-157 produced by E. faecium, and L50 made by E. faecalis [53]. Enterocins exhibit broad antimicrobial activity, inhibiting the multiplication of bacteria such as Staphylococcus spp., Bacillus cereus, Listeria monocytogenes, Clostridium spp., E. coli, Pseudomonas aeruginosa, and Vibrio cholera [73]. E. faecalis KT11, isolated from Kargı Tulum cheese, produces a bacteriocin with antimicrobial activity against Gram-positive (L. monocytogenes, S. aureus, B. subtilis) and Gram-negative (P. aeruginosa, K. pneumoniae, S. marcescens and E. aerogenes) bacteria and inhibits the growth of methicillin-and/or vancomycin-resistant bacteria [74].
Enterococci (e.g., E. faecium M-74, E. durans KLDS) are also characterized by their ability to lower cholesterol levels [63,75]. These bacteria produce a hydrolase which catalyzes the bile acid deconjugation process and assists in cholesterol integration into the bacterial cell wall or assists in precipitation if the environment is acidic [63,75]. Mego et al. demonstrated the use of the probiotic E. faecium M-74 in the treatment of gastrointestinal complications in patients with myeloid leukemia [66]. Another scientific report by Viaud et al. in a mouse model showed that Enterococcus hirae helps shape the anti-cancer immune response. [76]. The authors showed that cyclophosphamide (one of the drugs that stimulates anti-cancer immune response) changes the composition of the microbiota in the small intestine and induces the translocation of certain Gram-positive bacteria, including E. hirae, to the secondary lymphoid organs [76].
The importance of probiotic strains has been confirmed not only in humans, but also in animals. Benyacoub et al. [77] confirmed an immunomodulatory role of E. faecium SF68 on the intestinal mucosa and the development of the digestive system in young dogs. Another preparation, Cylactin ® , containing the strain E. faecium NCIMB 10415, has been used in pig and poultry farming as a feed additive in European Union countries instead of supplementary avoparcin to stimulate animal growth [46,48,78].
Enterococci can be found in food products such as untreated milk, cheese, meat [15,46], and plant products (fermented vegetables) [79]. Their presence in some products is considered desirable. Enterococci are mainly used for the production of regional foods in Mediterranean countries. In the food industry, selected enterococcal strains contribute to the improvement of the aroma, texture, and taste of fermented dairy products [13,80]. Probiotic strains can degrade proteins into peptides and amino acids, break down citrates, and produce aromatic substances with lipolytic and proteolytic properties. They are used as starter cultures in dairy products due to their ability to conduct the proteolysis, lipolysis, and metabolism of citrate and pyruvate [14,15]. Furthermore, enterococci produce substances such as acetaldehyde, acetoin, diacetyl, or 2,3-butanediol. In addition, these bacteria inhibit the proliferation of spoilage microbes by producing enterocins. Hence, bacteriocins can be used as food preservatives [12]. Due to their resistance to thermal treatment (cooking, pasteurization, or fermentation), they can be used as a hygienal indicator in food production as well. According to European guidelines, the producer is responsible for the safety of probiotics and starter strains. Producers therefore have an obligation to evaluate them to be safe for use.
Hospital-Acquired Infection
Enterococcal colonization is observed 10-20 times more often than the symptoms of infection [81], but some epidemiological studies conducted on carriers indicate a possible association between colonization and symptomatic infection [82]. Enterococci are opportunistic pathogens that, outside of their typical commensal habitats (GI tract), may be the cause of various infections (urinary tract infections, sepsis, bacteremia, and endocarditis) [83][84][85]. Infants [86] and people with diabetes may also be particularly at risk [85,87]. Generalized infections most often occur after surgery from burn wounds, leg ulcers, and pressure ulcer infections during diagnostic or therapeutic procedures in the urinary tract. Catheter-related infections, which can lead to meningitis, are reported especially in newborns and infants [88]. A dozen or Enterococcus species have been identified in human clinical samples. Among them, E. faecalis (80-90%) and E. faecium (5-15%) dominate, and these species are commonly associated with very serious complications and hospital infections [17].
A patient whose gastrointestinal tract is colonized by enterococci and undergoes diagnostic and therapeutic procedures during hospitalization, including antibiotic therapy, may be a source of drug-resistant Enterococcus isolates. The hospital is considered to be a reservoir of drug-resistant enterococci (e.g., high-level ampicillin resistance (Pbp5-R); highlevel aminoglycoside resistance; glycopeptides resistance (vancomycin and teicoplanin); oxazolidinones resistance) [37,84,86,89,90]. Medical personnel and specifically the hands of health care workers are considered a vector for these resistant bacteria and are likely the main sources by which enterococci spread throughout the hospital [37]. It is mainly patients hospitalized in the vicinity of already colonized people who are at risk of exogenous infections due to multidrug-resistant enterococci [84]. E. faecalis and E. faecium are two of the major etiological factors of urinary tract infections, especially in people with structural abnormalities or following catheterization [91]. By colonizing catheters (longterm catheterization: >28 days) with hospital strains, bacteriuria with catheter-associated urinary tract infection symptoms may occur. E. faecalis likely acts as a pioneering species which, by infiltrating catheters, creates a medium for the colonization of other bacterium such as P. mirabilis [92,93]. Finally, studies have shown that the secretory factors of E. faecalis enhance the pathogenicity potential of P. mirabilis and, as a co-occurring bacteria, contribute to the destruction of tissues and bacteremia [93].
Bacterial Translocation from the GI Tract to Organs
Translocation from the gastrointestinal tract to various organs has been demonstrated in the enterococcal microbiota [44,[94][95][96][97]. Both E. faecalis and E. faecium are invasive bacteria that pass through the intact mucosal epithelium and enter the host's tissues. The bacteria translocate through the lamina propria mucosae to the mesenteric lymph node and from there to the circulatory system. In vitro studies on HT-29 and T84 cell lines infected with Enterococcus strains have shown bacterial factors that favor colonization and aggregation into the extracellular matrix, such as aggregatory substances, enterococcal polysaccharides, Epa antigen (responsible for the survival of bacteria in phagosomes), and gelatinase, promote the migration of bacteria [98,99]. In patients undergoing intensive chemotherapy or long-term broad-spectrum antibiotic therapy, the intestinal barrier and oral mucosa are damaged. Competition among enterococci within the intestinal microbiota may lead to the enrichment of those bacteria that better adapt to the ecological niche. Even strains considered to be probiotic may pose a threat to the patient when considering dominance within a sterilized medium (such as in the intestine).
Cases of enterococcal translocation to the lymph nodes, blood, liver, and spleen have been described [96,97]. Vieira et al. demonstrated that the so-called E. gallinarum strain can cause autoimmunity in genetically predisposed hosts [100]. E. gallinarum-specific DNA was recovered from liver biopsies of autoimmune patients. Such strains are referred to as pathobiontic (pathogenic bacteria originating from the microbiota). The translocation of bacteria from the gut into the blood stream has been reported in oncological and immunosuppressed patients, mainly those involving E. coli [41,42]. Cases of enterococcal sepsis or endocarditis [94,[101][102][103] are less frequent and are the result of translocation from the gut [44,94,104]. In mice, Archambaud et al. [44] showed that the translocation of E. faecalis can occur across the intestinal mucosa and proved that the intestinal translocation of enterococci requires a threshold level of enterococcal hyperplasia in the intestinal lumen.
Mutagenic Effects and Theories of Tumorigenesis
Wang et al. [104] described that enterococci are also responsible for mutagenic effects. E. faecalis producing extracellular superoxide may induce chromosome breaking factors. Moreover, under experimental conditions on immortalized human and non-transformed murine colonic epithelial cells, E. faecalis can generate foci of aneuploidy, tetraploidy, and gamma-H2AX. In addition, the direct exposure of E. faecalis to these cells induced a G2 cell cycle arrest, hence the suggestion that commensals, including intestinal E. faecalis, may contribute to cellular transformation and tumorigenesis [104].
An association of E. faecalis translocation with colorectal carcinogenesis has been reported [105]. However, the role of enterococci in the development of colorectal cancer is still controversial [18]. Some authors suggest a protective role (e.g., Enterococcus faecium 137v (EF137v) [106], while others have indicated harmful effects. E. faecalis overgrowth usually occurs in the feces of colorectal cancer patients [107]. The involvement of E. faecalis in intestinal neoplasms can damage colonic epithelial cell DNA through the production of reactive oxygen and nitrogen species in the fermentation process [108]. Stimulation of macrophage activity [109] or changes in oxygen concentration can further activate oncogenes or inactivate tumor-suppressor genes [110,111]. The relationship between E. faecalis with various types of colorectal polyps thought to be a common cause of colorectal cancer have been documented as well [105]. Contrarily, in adenomatous polyposis coli mutant mice studies, the administration of a heat-killed strain of E. faecalis EC-12 reduced the development of polyps in the small intestine through the suppression of β-catenin signaling [105]. Grootaert et al. [112] demonstrated that E. faecalis grown on an aggressive colorectal cancer cell line (HCT-116) decreased the expression of FIAF protein similar to angiopoietin 4, which is typically detected in certain cancers.
Food-Borne Enterococci
The enterococci threat is not only observed in the hospital. The presence of enterococci in food is the result of contamination due to poor hygiene. E. faecalis and/or E. faecium are most often responsible for artisanal and traditional cheese contamination; however, other species have also been found (E. casseliflavus, E. durans, E. hirae and E. gallinarum) [113]. For example, in work by Gelsomino et al., E. casseliflavus and E. faecalis were isolated from food from a bulk-milk storage tank [12]. Poultry meat may also become contaminated with E. faecalis and E. faecium during processing and is frequently encountered [114]. The number of enterococci present in poultry meat range from 10 1 to 10 3 CFU/gr of raw chicken or turkey meat [15,115].
The widespread use of antimicrobials and intensive trade favors the emergence and spread of resistant microorganisms. The food chain is the key site where resistance is transmitted between the environment and humans. Moreover, a similarity in bacterial resistance profiles has been discovered between clinical material and food. Most frequently, these bacterial strains display resistance to streptomycin, erythromycin, tetracycline, and rifampicin [113]. The horizontal transfer of genes encoding resistance to aminoglycosides, tetracyclines, and macrolides in Enterococcus strains isolated from ready-to-eat dishes was documented by Chajęcka-Wierzchowska et al. [116]. The transfer of resistance to tetracyclines in enterococcal strains was also observed with a frequency ranging from 1.3 × 10 −6 to 8.7 × 10 −7 transconjugants/donor, for macrolides from 3.2 × 10 −6 to 2.4 × 10 −8 transconjugants/donor, and for genes encoding aminoglycosides from 1.7 × 10 −6 to 3.2 × 10 −8 transconjugants/donor. According to Haug et al., the high number of food-borne enterococci carrying resistance genes may significantly reduce the effectiveness of antibiotic therapy in intestinal infections [117,118]. Due to its highly adaptive capabilities, enterococci present in food are in a transient or permanent state to colonize the digestive tract, and this increases the danger of gene transfer to the intestinal microflora.
Enterococci can also cause food spoilage [119]. They produce thermostable amines such as tyramine, histamine, phenylethylalanine, cadaverine, and putrescine, which can cause allergic reactions or poisoning [15]. Problems are resistance to extremes used in food technology such as temperature as well as high pH and salinity.
Virulence Factors of Enterococcus spp. and Pathogenicity
E. faecalis and E. faecium strains are potentially pathogenic due to their special ability to adapt and survive in new environmental conditions [120]. These bacteria have developed mechanisms that facilitate and promote the colonization of biotic and abiotic surfaces and have the ability to evade the immune system [83], enabled by both the innate characteristics and the plasticity of their genome. Bacteria carry genes encoding virulence factors responsible for pathogenicity. Enterococci virulence factors can be grouped into several classes: (a) externally secreted, e.g., cytolysin, gelatinase, and serine protease; (b) surface proteins, e.g., Acm/Ace adhesins, Ebp pili, and extracellular surface protein Esp; (c) other virulence factors-hyaluronidase [121].
Many studies have confirmed that E. faecium, similar to E. faecalis, has the ability to bind collagen present on the surfaces of human cells. The microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) [122] are responsible for this. Some of the best characterized MSCRAMMs molecules are Ace (a collagen-binding protein) for E. faecalis [122] and Acm for E. faecium [123]. These proteins bind to type I collagen and, to a lesser extent, to type IV collagen, and support the early colonization of various tissues. The participation of the Ace protein of E. faecalis has been described in the colonization of the heart valves and, consequently, in endocarditis [124]. The acm gene is most often detected in clinical, multi-drug resistant strains of E. faecium [125]; however, collagen binding proteins have also been found in isolates from healthy vectors.
The aggregation substances (AS) are responsible for the adhesion of enterococci to eukaryotic cells. The aggregation substances of bacterial cells are of plasmid-born origin. The best known conjugation plasmids containing the genes encoding these proteins are pPD1-Asp1 protein, pCF10-Asc10 protein, and pAD1-Asa1 protein [126][127][128]. Interestingly, AS proteins have been shown to be involved in the adhesion to and the penetration into intestinal cells, indicating that they may play a role in the translocation of E. faecalis through the intestinal wall [129].
Another virulence factor is the extracellular surface protein Esp. Esp is an adhesin occurring in various forms, allowing it to avoid the host's immune defense mechanisms [130]. In addition, it participates in biofilm formation, which significantly increases the viability of bacteria in biopolymers (amyloid-like fibers) and may also be involved in antimicro-bial resistance [131]. Biofilm production allows enterococci to avoid phagocytic attacks and makes it difficult to eradicate them. The multispecies biofilm environment is also conducive to the exchange of genes related to virulence. The esp gene, detected in both E. faecalis and E. faecium, occurs on pathogenicity islands (PAIs) and can be transmitted by conjugation [132].
Pili are encoded by the ebp operon (endocarditis-and biofilm-associated pili) and are involved in biofilm formation as well. Similar to adhesins, pili allow for the binding of collagen, fibrinogen, and thrombocytes [84]. In addition to ebp, the virulence genes efaAfs and efaAfm, which encode adhesion-like endocarditis antigens for E. faecalis and E. faecium, respectively, also contribute to endocarditis [133,134].
Cytolysin (Cyl-β-haemolysin) is a bacteriocin encoded on pheromone-responsive plasmids or is located on pathogenicity islands within the chromosome [130]. Cyl lyses red blood cells and some human white blood cells and is active against some Gram-positive bacteria [135].
The participation of gelatinase (GelE) [136,137] and serine protease (SprE) [138] in pathogenesis has also been observed. The main role of these proteins is to provide nutrients to bacteria by breaking down the host tissue and by participating in biofilm formation [139]. In addition, gelatinase is important for the translocation of E. faecalis across human enterocytes and facilitates microbial invasion [99].
The increased virulence of clinical strains is due to the presence of hyaluronidase, an enzyme that acts on hyaluronic acid and breaks down connective tissue through the depolymerization of mucopolysaccharide moieties [31,140]. In conjunction with toxin secretion, this enables E. faecium to more easily spread throughout a host's tissues. The hyl gene encodes hyaluronidase, which is genetically programmed in megaplasmids and is present in many pathogenic enterococci.
Extracellular peroxides are an important factor for virulence and mainly occur in E. faecalis strains. They promote the survival of enterococci inside of the phagosome and damage the epithelium of the gastrointestinal tract, facilitating the exit of phagocytic cells from the intestine [108]. These strains are mainly isolated from patients with bacteremia [141]. A role has also been suggested for peroxidases in the formation of colorectal neoplasms [120].
The virulence factors are detected not only in clinical strains but also in bacterial strains from food. Genes encoding adhesion factors such as esp, asa1/agg, and efaA are highly prevalent among E. faecalis and E. faecium [142]. On the contrary, these genes are rarely reported in E. durans [143], E. hirae [144], and E. casseliflavus [145]. Finally, in foodborne strains, cyl, gel, and hyl are detected but with much lower frequency compared to clinic enterococci [143,146].
Another increasingly common feature of enterococci is the presence of pathogenicity islands, where the virulence genes involved in aggregation, cytolysin, or Esp as well as transcription factors regulating bile acid hydrolases are located. The formation and genetic instability of PAIs is the result of horizontal gene transfer (HGT), a process that is wellknown for its contribution to microbial evolution. Many of the discussed genes encoding virulence factors (e.g., as, cyl, hyl) are also located on conjugation plasmids. HGT as a mechanism for genetic variation through gene acquisition in PIAs and the role of mobile genetic elements in the evolution of E. faecalis have been proven many times [147]. HGT is involved in spreading unfavorable and risk-raising features of enterococci, increasing the chances of these commensal bacteria to become pathogenic [148]. Moreover, HGT is responsible for the transfer of mobile genetic elements (e.g., plasmids) to other unrelated species [149]. Vignaroli et al. [150] observed the transfer of the vanA and erm (3) genes from porcine E. faecium and E. durans isolates to human E. faecium.
It should be noted that some determinants of enterococcal virulence are desirable in probiotic strains. This includes aggregation factors, exopolysaccharide (EPS) production, and the proteolytic system. Aggregation substances improve the likelihood of probiotic strain adherence to the host's intestinal epithelium and are therefore an important feature for the efficient colonization of the GI tract along with indirectly affecting immunomod-ulation and providing protection against pathogens. The aggregation substances (ASs) on the cell surfaces of bacteria induce cell aggregation (and auto-aggregation) and are responsible for biofilm production. The enterococcal aggregation protein, AggE, is found in probiotic strains such as Enterococcus faecium BGGO9-28 and possesses high adhesive capabilities to collagen, fibronectin, and mucin [151]. The competitive formation of nonpathogenic biofilms promotes the elimination of harmful bacteria through pH alteration and competition for nutrients [151].
EPS is an exometabolite composed of β-1,6-linked poly-N-acetylglucosamine (polyGlcNAc)containing polymers. The production of EPS can be considered a virulence trait [152]; however, EPS also facilitates the adhesion of probiotic enterococci through cooperation and the aggregation of cells [153]. The synthesis of EPS allows for the movement of this non-motile bacterium to an environment with nutrients and allows it to escape stressful conditions (higher pH, temperature, osmolarity), toxic conditions (e.g., antibiotics, metal ions, bile salts, gastric and pancreatic enzymes), or even evade the human immune response [154]. EPS can exert antagonistic activity against Gram-positive and Gram-negative pathogens, but the potential mechanisms are difficult to explain. It is suggested that EPS accumulates metabolites that adversely affect other bacteria [155], and these metabolites may also disrupt the structure of peptidoglycan and block receptors and channels on the outer membrane of the Gram-negative bacteria [156,157].
From a practical point of view, EPS-production is desirable because it improves the viscosity and texture of dairy products and can be used by the food industry to control biofilm-production by bacteria [142].
Enterococcus strains also display proteolytic activity (producing of extracellular proteinases, intracellular peptidases, and transport enzymes) [158,159] and play an important role in bacterial growth [158]. Some of them, including extracellular-secreted (E) or cell envelope proteases are used in the fermentation of dairy products) [142,160].
Bacteriocins constitute a functionally diverse family of toxins that are ribosomally synthesized peptides or proteins. Enterocins are used in dairy products, meat, fish, and plant-derived products (enterocin RM6, CRL35, AS-48) as beneficial additives in food production. Currently, bacteriocins are also being considered as promising candidates to treat infections caused by multi-drug resistant pathogens, e.g., in GI-tract diseases (enterocin A, S760, E50-52) and skin infections (enterocin A-48) [158]. In conclusion, some features of enterococci, such as virulence factors, make them an intermediate between emerging pathogens and potential probiotics.
The Problem of Antibiotic Resistance
The uncontrolled prophylactic use of antibiotics in a hospital environment and on animal farms has resulted in a gradual build-up of resistance among enterococci [126,161]. It has been proven that the commensal genome of enterococci can evolve greater pathogenicity through adaptation to the hospital environment [84]. Likewise, the European Center for Disease Prevention and Control has estimated that 37,000 people die due to infection caused by multidrug-resistant bacteria as a result of Hospital Acquired Infections every year [162].
Research indicates the development of ampicillin resistance and associated resistance to ciprofloxacin is the main phenotypic marker of hospital E. faecium isolates, a marker that precedes resistance to glycopeptides by several years [49,163,164]. In some countries, tetracycline is one of the most frequently used antibiotics for human and animal infections due to its availability and low cost [165]. However, the extensive use of tetracyclines has often led to the emergence of resistant bacteria [166]. In one hospital in Italy, a 2-year retrospective analysis of antimicrobial drug resistance and the spread of nosocomial infection found that about 70% of E. faecalis isolated from clinical patients had resistance to tetracycline and erythromycin [164]. The most commonly encountered tetracycline-resistant determinant in enterococci is tet(M), which is mainly associated with a conjugative transposon, particularly Tn916 [151].
Resistance to vancomycin (mainly VanA and VanB phenotype) is also becoming more frequent [84]. VanA resistance is characterized by a high degree of vancomycin-and teicoplanin-induced resistance. It is most often found in E. faecium strains, but it is also found in E. faecalis and, to a lesser extent, in E. durans, E. raffinosus, E. hirae, E. avium, and E. gallinarum. The genes determining this type of resistance are found on the Tn1546 transposon, which may be on a plasmid or may integrate with the bacterial chromosome [167,168]. The most important factor in the outbreak of hospital vancomycin-resistant enterococci is the colonization of the excretory system, which almost always precedes bacteremia and is the main reservoir from which the spread of microorganisms in the hospital environment takes place.
Among multidrug-resistant treatments, linezolid was once the drug of last resort. Resistance to linezolid (linezolid-resistant Enterococcus) has now been observed for several years against clinical isolates of the genus Enterococcus. New medicinal products have been introduced, such as dalbavancin (a lipopeptide), oritavancin and telavancin (glycopeptides), and tedizolide (oxazolidinone, the successor of linezolide). However, the activity of these drugs against enterococci and their availability in different countries varies considerably [169]. Therefore, drug-resistant enterococci infections pose a significant epidemiological and therapeutic problem.
Antibiotics are used not only for therapeutic and prophylactic purposes; they are also to protect consumers against microorganisms that may contaminate farms and animal products [168]. Enterococci are also pathogens of farm animals, and the abuse of antibiotics in veterinary medicine by animal breeders and by food producers will contribute to the deepening of this multi-drug resistance phenomenon. Resistance to ciprofloxacin, norfloxacin, tetracyclines, and even linezolid have been found in strains isolated from sausage, cheese, fish, and fish products [170]. In foods of animal origin produced in Europe, isolates resistant to gentamicin and streptomycin are rare, while in the United States, they are quite common [171]. Antibiotic resistance in the enterococcal strains commonly used as starter cultures for biotechnological applications in the dairy industry has also been identified. It is known that these strains must be sensitive to relevant clinical antibiotics. In a study by Terzić-Vidojević et al., [172], enterococci (with predominant species: Enterococcus durans, Enterococcus faecalis, and Enterococcus faecium) isolated from dairy products from different regions of the Western Balkan countries of Serbia, Croatia, Bosnia, and Herzegovina showed resistance to various antibiotics. They found that 185 out of 636 isolates were susceptible to tested antibiotics, and five of them met the criteria for the starter cultures (without any gene encoding virulence factors and in the absence of biogenic amines). A significant portion of the strains isolated from dairy products turned out to be useless due to drug resistance.
Eating raw and processed food contaminated with multi-drug resistant microorganisms can pose a threat to human life and health. Jahan et al. [173] demonstrated that the gene determining resistance to tetracycline and streptomycin was transferred from food-derived E. faecium and E. faecalis strains to clinical strains. The overuse of feed antibiotics in breeding has also made Enterococcus bacteria cross-resistant to vancomycin and teicoplanin. Cylactin has been proposed as an alternative to antibiotics along with the probiotic strain E. faecium NCIMB 10415, which protects piglets against diarrhea by competing with pathogenic strains of E. coli and Salmonella spp. [174].
Zoonotic transmission of drug-resistant enterococci (E. faecium and E. faecalis, and much less frequently E. durans, E. casseliflavus and E. gallinarum) from animals to humans through contact with animal secretions and excretions (dogs are a more common reservoir of drug-resistant enterococci than cats) [175] represents another issue. Studies conducted in various countries show a close relationship between vancomycin-resistant enterococcal species isolated from dogs with isolates responsible for nosocomial infections in humans [176].
The use of probiotic Enterococcus strains is controversial despite their beneficial effects in humans and animals. The reason for this is because of the bacterial acquisition of genes encoding resistance to glycopeptide antibiotics (vancomycin) and resistance to high concentrations of aminoglycosides (high level aminoglycoside resistance). Horizontal gene transfer from pathogenic enterococci to strains of commensals and other species of bacteria constituting the physiological microflora of the gastrointestinal tract has been reported [177].
In this context, it should be noted that the digestive tract is an excellent environment for bacterial growth and for the exchange of genetic material between microbes.
Conclusions
Enterococci represent one of the most controversial groups of bacteria. They are mainly commensal organisms isolated from humans, animals, plants, and insects. These bacteria affect the intestinal balance and modulate the human immune system. Due to their beneficial effects, selected strains are used as probiotics in numerous therapies and are also of biotechnological importance in the food industry. However, it should not be forgotten that in high-risk patients, enterococci may show potential for pathogenicity, especially in a hospital environment. E. faecalis and E. faecium display complex mechanisms of virulence that enable their colonization in various host tissues. The epidemic importance of E. faecalis and E. faecium is not new to clinicians, nor is the threat of increasing antibiotic resistance. We should remember that enterococcal overgrowth in the intestine and biofilm formation facilitates communication between bacteria and gene exchange through HGT. The use of selected probiotic enterococcal strains to support the treatment of patients may be an effective therapy; however, it should be remembered that enterococci have many faces. Due to their plastic genomes, enterococcal treatments should be used with caution, especially in immunodeficient patients. The probable relationships between enterococci of various phenotypes (probiotic, commensal, and pathogenic) in the transition towards pathogenicity are shown in Figure 2. The advantages and disadvantages of enterococci are compiled in Table 1. Table 1. Biofilm is a key process in horizontal gene transfer (HGT). Transfer antibiotic resistance genes and virulence factors are facilitated. Commensal strains and probiotic strains can convert into pathogenic strains. For oncological patients and people with a weakened immune system, the translocation of pathogenic strains into the circulatory system is highly likely. HGT-horizontal gene transfer; Vfs-virulence factors, ARG-antibiotic resistance gene. Antibiotics, chemotherapeutics, and diet can alter the composition of the gut microbiota. The overgrowth of Enterococcus causes dysbiosis and often disorders homeostasis. Biofilm is a key process in horizontal gene transfer (HGT). Transfer antibiotic resistance genes and virulence factors are facilitated. Commensal strains and probiotic strains can convert into pathogenic strains. For oncological patients and people with a weakened immune system, the translocation of pathogenic strains into the circulatory system is highly likely.
Disadvantages of Enterococci Reference
Potential pathogens (e.g., urinary tract infections, endocarditis) [83][84][85]92,96,[101][102][103][104]133,134] translocation in the circulatory system (sepsis, bacteremia) [41,44,[94][95][96][97] Nosocomial infection/hospital outbreak [17,84,86,89,90] Virulence and resistance factors can be transmitted between species or genera by horizontal gene transfer-a problem in hospital settings [116,147,148,150,164,177] Responsible for allergic reactions [15] Food spoilage [15,115,119] Food poisoning (foodborne pathogens) [15] Polyp formation and colorectal cancer [18,108,110,111] Enterococci do not possess a Qualified Presumption of Safety status in the EU and are not generally regarded as safe in the USA. Hence, in order to ensure the safety of using Enterococcus as probiotics or starter cultures, further investigations on their genotypic and phenotypic characteristics need to be conducted before they are put into use. Currently, molecular biology techniques (e.g., PCR, whole genome sequencing) and classical susceptibility assays are used to detect virulence determinants and antibiotic resistance. In this way, producers can control enterococci for medical applications, as supplements, or in the food industry. In addition to detecting antibiotic resistance and virulence determinants, we should investigate useful features such as the hydrophobicity, auto-aggregation and co-aggregation ability, adhesion ability of strains to human intestinal cells, EPS production ability, antimicrobial activity, and the detection of genes encoding useful enterocins.
Conflicts of Interest:
The authors declare no conflict of interest.
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2021-09-28T05:30:04.615Z
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2021-09-01T00:00:00.000
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Voluntary Ambulation by Upper Limb-Triggered HAL® in Patients with Complete Quadri/Paraplegia Due to Chronic Spinal Cord Injury
Patients with complete paraplegia after spinal cord injury (SCI) are unable to stand or walk on their own. Standing exercise decreases the risk of decubitus ulcers, osteoporosis, and joint deformities in patients with SCI. Conventional gait training for complete paraplegia requires excessive upper limb usage for weight bearing and is difficult in cases of complete quadriplegia. The purpose of this study was to describe voluntary ambulation triggered by upper limb activity using the Hybrid Assistive Limb® (HAL) in patients with complete quadri/paraplegia after chronic SCI. Four patients (3 men, 1 woman) were enrolled in this study. The mean patient age ± standard deviation was 37.2 ± 17.8 (range, 20–67) years. Clinical evaluation before intervention revealed the following findings: case 1, neurological level C6, American Spinal Cord Injury Association impairment scale (AIS) grade B; case 2, T6, AIS A; case 3, T10 AIS A; and case 4, T11, AIS A. The HAL intervention consisted of 10 sessions. Each HAL session lasted 60–90 min. The HAL electrodes for hip and knee flexion-extension were placed on the anterior and posterior sides of the upper limbs contralaterally corresponding to each of the lower limbs. Surface electromyography (EMG) was used to evaluate muscle activity of the tensor fascia lata and quadriceps femoris (Quad) in synchronization with a Vicon motion capture system. The modified Ashworth scale (mAs) score was also evaluated before and after each session. All participants completed all 10 sessions. Cases 1, 2, and 3 demonstrated significant decreases in mAs score after the sessions compared to pre-session measurements. In all cases, EMG before the intervention showed no apparent activation in either Quad. However, gait phase dependent activity of the lower limb muscles was seen during voluntarily triggered ambulation driven by upper limb muscle activities. In cases 3 and 4, active contraction in both Quads was observed after intervention. These findings suggest that upper-limb-triggered HAL ambulation is a safe and feasible option for rehabilitation in patients with complete quadri/paraplegia caused by chronic SCI.
Patients with complete paraplegia after spinal cord injury (SCI) are unable to stand or walk on their own. Standing exercise decreases the risk of decubitus ulcers, osteoporosis, and joint deformities in patients with SCI. Conventional gait training for complete paraplegia requires excessive upper limb usage for weight bearing and is difficult in cases of complete quadriplegia. The purpose of this study was to describe voluntary ambulation triggered by upper limb activity using the Hybrid Assistive Limb ® (HAL) in patients with complete quadri/paraplegia after chronic SCI. Four patients (3 men, 1 woman) were enrolled in this study. The mean patient age ± standard deviation was 37.2 ± 17.8 (range, 20-67) years. Clinical evaluation before intervention revealed the following findings: case 1, neurological level C6, American Spinal Cord Injury Association impairment scale (AIS) grade B; case 2, T6, AIS A; case 3, T10 AIS A; and case 4, T11, AIS A. The HAL intervention consisted of 10 sessions. Each HAL session lasted 60-90 min. The HAL electrodes for hip and knee flexion-extension were placed on the anterior and posterior sides of the upper limbs contralaterally corresponding to each of the lower limbs. Surface electromyography (EMG) was used to evaluate muscle activity of the tensor fascia lata and quadriceps femoris (Quad) in synchronization with a Vicon motion capture system. The modified Ashworth scale (mAs) score was also evaluated before and after each session. All participants completed all 10 sessions. Cases 1, 2, and 3 demonstrated significant decreases in mAs score after the sessions compared to pre-session measurements. In all cases, EMG before the intervention showed no apparent activation in either Quad. However, gait phase dependent activity of the lower limb muscles was seen during voluntarily triggered ambulation driven by upper limb muscle activities. In cases 3 and 4, active contraction in both Quads was observed after intervention. These findings suggest that upper-limb-triggered HAL ambulation is a safe and feasible option for rehabilitation in patients with complete quadri/paraplegia caused by chronic SCI.
INTRODUCTION
Patients with complete paraplegia after spinal cord injury (SCI) are unable to stand or walk on their own. Standing exercise for patients with SCI decreases decubitus ulcers, osteoporosis, hip joint flexion, and adduction deformities and improves the performance of the cardiovascular and digestive systems (Karimi, 2011). Conventional gait training using orthoses for complete paraplegia requires locking of the knee joint in an extended position as well as excessive upper limb usage for weight bearing (Karimi, 2011), and it is difficult in cases of complete quadriplegia.
Robotic devices have recently been used in clinical settings for patients with chronic SCI. Exoskeleton robotic devices employed with a treadmill, such as the Lokomat (Hocoma, Switzerland) (Colombo et al., 2000) and LOPES (Veneman et al., 2007) and powered exoskeleton devices, such as the ReWalk (Robotics, Israel) (Miller et al., 2016), have angular sensors in the joints and pelvis as well as foot force pressure sensors. However, they have no sensors to detect the neuromuscular activation of the user.
In sensing a user's neuromuscular activation, our research has focused on another exoskeleton robot, the Hybrid Assistive Limb R (HAL, Cyberdyne Inc., Ibaraki, Japan). HAL is a wearable robot suit that assists the user in voluntary control of knee and hip joint motion by detecting signals from force/pressure sensors in the shoes or even very weak bioelectric signals on the skin surface. Power units on the bilateral hip and knee joints are comprised of angular sensors and actuators, and the control system consists of a cybernic voluntary control (CVC) mode, cybernic autonomous control (CAC) subsystem (Kawamoto and Sankai, 2005), and cybernic impedance control (CIC) mode. The HAL suit has a unique operating system, with a hybrid control system that includes the CVC, CAC, and CIC modes. The CAC mode can automatically move a user's leg using signals from the force-pressure sensors (Ikumi et al., 2017). The CVC mode can support the user's voluntary motion by providing assistive torque to each joint according to their voluntary muscle activity. The CIC mode can adjust to the user's motion while compensating for HAL's weight and joint viscosity.
We previously evaluated the application of HAL for single joints in a patient with complete C4 quadriplegia in order to restore elbow joint flexion using residual trapezius muscle activation. We found that the use of HAL that associated the residual muscle contraction to the elbow joint movement might activate the paralyzed muscle (Shimizu et al., 2017a).
We focused on remaining muscle activity in the upper limb in complete paraplegia. Previous studies have reported neural coupling of the upper and lower limbs in humans (Zehr and Duysens, 2004;Dietz, 2011;La Scaleia et al., 2014;Sylos-Labini et al., 2014). Arm-leg coordination was reported to be useful for gait assistive technology (Hassan et al., 2014;La Scaleia et al., 2014). Therefore, we concentrated on the structural analogy and symmetric motion of the shoulder and hip during gait. The lower extremities move synchronously and almost simultaneously with the contralateral upper extremities during natural locomotion. As we flex a shoulder, the contralateral hip flexes; and at the same time the other shoulder extends, with contralateral hip extension.
We hypothesized that triggering lower limb motion by upper limb muscle activity using HAL was feasible for the voluntary generation of gait composed of voluntarily driven hip and knee joint motions, in people with chronic SCI leading to complete paraplegia or quadriplegia with residual control over upper limb movement. In addition, we hypothesized that simulating the synergy of the upper and contralateral lower limbs in voluntary gait during HAL intervention may activate paralyzed lower limb muscles.
Here, we describe the feasibility and effects of upper limbtriggered HAL intervention for patients with complete paraplegia or quadriplegia caused by chronic SCI.
Participants
Four patients (3 men, 1 woman) were enrolled in this study. The mean patient age ± standard deviation was 37.2 ± 17.8 (range, 20-67) years. Clinical evaluation before the intervention revealed the following findings: case 1, neurological level C6, American Spinal Cord Injury Association (ASIA) impairment scale (AIS) (Kirshblum et al., 2011) grade B; case 2, T6, AIS A; case 3, T10, AIS A; and case 4, T11, AIS A. International Standards for Neurological and Functional Classification of Spinal Cord Injury (ISNCSCI) lower extremity motor score (LEMS) was 0 in cases 1, 2, and 3, and 2 in case 4. Participants' clinical data are shown in Table 1.
This study was conducted in accordance with the Declaration of Helsinki, with approval from the Ethics Committee of the Tsukuba University Faculty of Medicine (approval no.: H26-22). All participants provided written informed consent for participation and publication, including the use of accompanying images.
HAL Intervention
Participants underwent 10 HAL sessions. The frequency of sessions ranged from 2 per week to 1-2 per month. Intervention for cases 1, 3, and 4 was performed on an outpatient basis, and no additional therapies were implemented during HAL intervention. In case 2, HAL intervention was performed as part of an inpatient stay at our hospital in addition to standard physical therapy. Before the intervention, active hip flexion and active knee extension were evaluated using a Trigno TM Lab Wireless electromyography (EMG) system (Delsys Inc., Boston, MA, USA).
Upper Limb-Triggered HAL (UT-HAL)
We designed the HAL intervention based on patients' residual muscle activity. The primary goal of HAL intervention was (Zehr and Duysens, 2004;Dietz, 2011;La Scaleia et al., 2014;Sylos-Labini et al., 2014), voluntary ambulation with HAL using upper limb activation involved motion intention from the anterior deltoid and posterior deltoid for contralateral hip flexion and extension, respectively, and motion intention from the biceps and triceps brachii for contralateral knee flexion and extension, respectively. We referred to the upper limb-triggered HAL session as the "UT-HAL" method ( Figure 1). The electric motors for the hip and knee joints are controlled in real-time to generate joint torque computed as a weighted difference of the activation of the flexor and extensor muscles. In equation, Thip=Gda * Ada-Gdp * Adp and Tknee=Gb * Ab-Gt * At are, respectively, the hip and knee joint torques generated by the motors, where Gda, Gdp, Gb, and Gt are the gain, and Ada, Adp, Ab, and At are the filtered activation of the anterior deltoid, posterior deltoid, biceps, and triceps muscles, respectively. Gda, Gdp, Gb, and Gt are adjusted for the user's comfort.
Knee Extension HAL
The secondary goal of HAL intervention was to regain active knee extension. In patients who were able to flex their hips, a second component was added to each HAL session. In addition to the first component, which involved voluntary gait using the patient's upper limb activation, the second component involved active knee extension using hip flexor activation, focusing on the hip flexor as the site of remaining muscle activation in patients who could not contract the knee extensors.
HAL Session
A typical HAL session lasted 60-90 min, including the time required to attach and detach the device (20 min). The gait session was ∼40 min, including a 10-min period of rest. The knee extension session lasted about 30 min including evaluation (10 min) of muscle activity during five repetitions of each of the following motions: active hip flexion; active knee extension; and active combined hip flexion and knee extension, such as in a kicking motion, before and after each HAL session.
A physiatrist was present in case of an emergency, a therapist and two assistants attached and detached the HAL, and an engineer implemented the gait analysis. For safety reasons, a walking device (All-in-One Walking Trainer, Ropox A/S, Naestved, Denmark) with a harness was used to prevent falls.
For participants without a history of ambulation training (cases 1 and 2), we initially used the CAC and CIC mode along with heavy assistance of three therapists, and as they became accustomed to ambulation training, upper limbtriggered methods were introduced.
Assessments
Assessments were performed before the intervention and during each HAL session. A surface EMG system was used to evaluate the muscle activity of the tensor fasciae lata (TFL) for hip flexion and the femoral quadriceps (Quad) for knee extension on both sides. We chose the TFL for the evaluation of hip flexion because the main hip flexor, the iliopsoas muscle, is located in the deep layer and thus is difficult to detect by surface EMG. We chose the vastus lateralis in the Quad because it is a simple knee extensor. The activity of each muscle was evaluated using EMG collected at 2,000 Hz and filtered with a 30-to 400-Hz band-pass filter using scripts on MATLAB 8.2 (Mathworks, Natick, MA, USA). Motion capture (Vicon MX with 16 T20S cameras, Vicon, Oxford, UK) was used to evaluate foot motion in synchronization with EMG. Following the Vicon plug-in gait marker set, autoreflective markers were placed bilaterally on the feet, head of the second metatarsal bone of the toe, lateral malleolus of the ankle, and posterior peak of the calcaneus of the heel. The swing phase and stance phase within a gait cycle were extracted according to the movement trajectory of the markers. Heel strikes were detected as the lower peaks of the height of the heel markers, and toe lifts were detected as the lower peaks of the toe markers. The swing phase started with a toe lift and ended with the subsequent heel strike on the same side. The stance phase started with a heel strike and ended with the subsequent toe lift (Ivanenko et al., 2007).
We also evaluated the walking distance and bilateral modified Ashworth scale (mAs) score (range: 0-24; hip abduction, knee extension, and ankle dorsiflexion; Bohannon and Smith, 1987) before and after each HAL session. The mAs scores before and after each HAL session were compared with the Wilcoxon signed-rank test. All analyses were performed using JMP R 10.0.2 (SAS Institute Inc., Cary, NC, USA); P < 0.05 were considered significant. Any adverse events associated with HAL intervention were also recorded.
RESULTS
All participants completed all 10 sessions. The only observed adverse event was redness caused by contact between the skin and harness in cases 1, 2, and 3 which was avoidable by using a cushion. Figure 2 shows the walking distance in all cases; improvement was observed in cases 3 and 4 as the HAL intervention progressed. The decrease of the mAs score was statistically significant from before to after session; the each post-session mAs score decreased significantly compared to the pre-session mAs score in case 1 (from 9.9 ± 1.5 to 5.1 ± 2.2; P = 0.004), case 2 (from 15.3 ± 1.6 to 9.1 ± 2.5; P = 0.029), and case 3 (from 7.0 ± 1.2 to 6.2 ± 0.8; P = 0.063; Figure 3). In cases 1, 2, and 3, activation was observed in both Quads in the stance phase (Figures 4-6), which became more apparent as the sessions progressed. However, in all cases, surface EMG before the intervention showed no apparent activation in either Quad (refer to Figures 6, 7 for cases 3 and 4). In these graphs, we chose sessions that were representative of the progression of each subject throughout the UT-HAL sessions.
In cases 3 and 4, knee extension sessions were performed using the hip flexor as the trigger of knee extension. In both cases, active contraction in both Quads was observed after intervention. Table 2 shows the neurological findings in all cases after intervention.
CASE DESCRIPTIONS
Case 1 ( Figure 1B) A 20-year-old male with complete quadriplegia and chronic cervical SCI due to cervical vertebral dislocation (C5/6) underwent HAL intervention with a total of 10 sessions, 1-2 per month, beginning 3 years and 2 months post-injury. He had the ability to perform daily activities independently with the help of a wheelchair, including transfer from/to the wheelchair and intermittent self-catheterization. Before intervention, manual muscle test (MMT) of the upper limb muscles was 5/5 for both deltoids, 5/5 for both biceps brachii, 2/2 for both triceps brachii, and 1/1 for both wrist flexor muscles. The muscle strength of his elbow extensors and wrist flexors had shown recovery; however, his lower limbs had remained in complete paraplegia. AIS was C6 B, ISNCSCI upper extremity motor score (UEMS) was 26/50, and LEMS was 0/50. On evaluation before HAL intervention, there was no muscle activation in either TFL or Quad. As the patient had no history of ambulation training, in order to become accustomed to ambulation training, he started HAL sessions using CAC or CIC mode accompanied by heavy assistance from three therapists with an overhead harness. Activation in both Quads was observed in the stance phase from the first session. From the latter half of the fifth session, his upper limb was used as a trigger for lower limb movement, and activation became more apparent in the UT-HAL session than in previous sessions. Figure 4A shows surface EMG during the fifth session using CAC mode. There was some activation in both Quads, but no periodic activation in the right Quad. More periodic activation, from the end of swing phase to early stance phase, was observed during UT-HAL gait in the same fifth session (Figure 4B). More apparent activation was present in the sixth session, predominantly on the right side ( Figure 4C).
Walking distance increased from 73.4 m (first session) to 200 m (tenth session; Figure 2). The mAs score decreased after each HAL session compared to pre-session measurements; the mean scores before and after the sessions were 9.9 ± 1.5 and 5.1 ± 2.2, respectively (P = 0.004; Figure 3).
Case 2
A 67-year-old male with complete paraplegia and chronic SCI underwent HAL intervention 2 years and 3 months after the onset of complete paralysis caused by pyogenic spondylitis in the fifth and sixth thoracic vertebrae. He was admitted to our hospital to undergo HAL intervention. His main complaint was spasticity. The mAs scores on admission were as follows: total, 16; hip abduction, 3/2; knee extension, 3/2; and ankle dorsiflexion, 3/3; for the right and left sides, respectively. He underwent HAL sessions twice per week. He had the ability to perform daily activities with the help of a wheelchair and his wife, including transfer from/to the wheelchair. Before intervention, AIS was T6 A, ISNCSCI UEMS was 50/50, and LEMS was 0/50. There was no activation in either TFL or Quad.
As the patient had no history of ambulation training, he started HAL sessions using CAC or CIC mode and the heavy assistance of three therapists. Activation in both Quads was observed in the stance phase from the first session. UT-HAL sessions were started from the latter half of the third session, and more apparent activation was observed in the UT-HAL session than during CIC mode (Figures 5A,B).
Walking distance increased from 40 m (first session) to 77 m (tenth session; Figure 2). The mAs score after each HAL session decreased compared to pre-session measurements; the mean scores before and after each session were 15.3 ± 1.6 and 9.1 ± 2.5, respectively (P = 0.029; Figure 3).
Case 3
A 32-year-old female with complete paraplegia and chronic SCI due to spinal cord infarction underwent HAL intervention 6 years and 3 months after spinal cord infarction. She had the ability to perform daily activities with the help of a wheelchair. She had a history of ambulation training using long leg braces. She underwent 1-2 HAL sessions per month.
Before intervention, AIS was T10 A, and ISNCSCI LEMS was 0/50. She was able to contract the right hip adductor slightly. She could not contract either TFL or Quad.
On evaluation before HAL intervention, there was no muscle activation in either TFL or Quad. From the first session, we used the upper limb as the trigger for limb motion. Some aperiodic activation in both TFLs and the right Quad was observed in the first session ( Figure 6C).
On evaluation before the second session, she could contract the right TFL; therefore, we added a knee extension session using the right hip flexor as the trigger for right knee extension from the second session. Figure 6A shows surface EMG in both TFLs and Quads before, during, and after the third knee HAL session. Before the HAL session, there was periodic activation in the right TFL and Quad. During the HAL session, activation was observed in both TFLs and Quads. After the session, there was rhythmic activation in the right TFL and Quad. Before the ninth session, periodic activation was observed in both TFLs and the right Quad; during the HAL session, activation was more apparent in both TFLs and Quads; and after the session, more obvious activation was observed in the right Quad. Some activation was also seen in the left Quad during hip flexion and knee extension ( Figure 6B). Figures 6C,D shows surface EMG during the first and the tenth UT-HAL sessions, respectively. In the tenth session, more rhythmic activation was observed in both Quads during the stance phase, predominantly on the right side.
FIGURE 6 | EMG of both TFLs and Quads in case 3. (A) Before, during, and after the third knee HAL session. Before this session, there was periodic activation in the TFL and Quad only on the right side. During the HAL session, activation was observed in both TFLs and Quads. After the session, there was rhythmic activation in the right TFL and Quad. (B) Before, during, and after the ninth knee HAL session. Before the session, there was periodic activation in both TFLs and the right Quad; during the HAL session, activation was more apparent in both TFLs and Quads; and after the session, more obvious activation was observed in the right Quad, and some activation was seen in the left during hip flexion and extension. (C) During the first session in UT-HAL gait, there was some aperiodic activation in both TFLs and the right Quad. (D) During the tenth session in UT-HAL gait, there was more rhythmic activation in both Quads during the stance phase, predominantly on the right side. (E) During voluntary ambulation with a walker and overhead harness without HAL after the entire intervention, there was rhythmic activation in the right quad from the swing mid-phase to the early stance phase. Figure 6E shows surface EMG during voluntary ambulation with a walker and overhead harness without HAL after the entire intervention. Rhythmic activation in the right quad was observed from the swing mid-phase to the early stance phase.
Walking distance increased from 80 m (first session) to 234 m (tenth session; Figure 2). After the intervention, the patient's hip flexor and knee extensor MMT score improved from 0/5 to 1/5 on both sides, and LEMS improved from 0 to 4 (Table 2). (C) Before, during, and after the tenth knee HAL session. Before and during the session, there was periodic activation in the left Quad; during the session, the right Quad was also periodically activated. After the session, the left Quad was more periodically activated than before it. The mAs score was reduced after each HAL session compared to pre-session measurements; the mean scores before and after each session were 7.0 ± 1.2 and 6.2 ± 0.8, respectively (P = 0.063; Figure 3).
Case 4
A 30-year-old male with complete paraplegia and chronic SCI due to twelfth vertebral burst fracture underwent the HAL intervention 1 year and 7 months post-injury. He had the ability to perform daily activities with the help of a wheelchair. He had a history of ambulation training with long leg braces or the assistance of two therapists. He underwent HAL sessions twice per week.
Before intervention, AIS was T11 A, and ISNCSCI LEMS was 3/50. MMT of the hip flexor was 1/5 on the right and 2/5 on the left. There was no voluntary contraction in either Quad ( Figure 7A).
From the first session, we used both hip flexors as the trigger for knee extension in the knee extension HAL session, and the upper limb as the trigger of limb motion during the HAL ambulation session. Activation was observed in both TFLs during the gait session, predominantly in the swing phase, but not in either Quad (Figure 7D). On evaluation before the fifth session, voluntary contraction was observed in the left Quad ( Figure 7B). Therefore, we placed electrodes at the recommended sites on both limbs in the latter five sessions and performed knee extension sessions triggered by both knee extensors and HAL ambulation sessions triggered by activation of both lower limbs. Figure 7C shows surface EMG in the tenth knee extension session. Before and during the session, there was periodic activation in the left Quad; during the session, the right Quad was also periodically activated. After the session, the left Quad was more periodically activated than before it. Figures 7D-F show EMG in the first, fifth, and tenth HAL ambulation sessions, respectively. There was activation in both TFLs from the first to tenth sessions, and no rhythmic activation in either Quad was observed in the first or fifth gait sessions. During the tenth gait session, there was slight periodic activation during the swing phase. Figure 7G shows voluntary ambulation with a walker and overhead harness without HAL after the entire intervention. Rhythmic activation was observed in both TFLs, with no periodic activation in either Quad.
Walking distance increased from 100 to 338 m (Figure 2). The mAs score remained 0 before and after each session. After the intervention, the patient's hip flexor MMT score improved from 1/5 to 2/5 on the right, and from 2/5 to 3/5 on the left. Knee extensor MMT also improved from 0/5 to 1/5, and LEMS improved from 3 to 7 (Table 2).
DISCUSSION
In this study, we focused on residual upper limb muscle activity in complete quadri/paraplegia and coordination between the upper limbs and contralateral lower limbs in natural gait. In addition, we used voluntary contraction of the hip flexors as a trigger for knee extension. We observed neurological changes in two cases after the intervention.
For all cases, including patients with cervical SCI and high thoracic SCI who were considered ineligible to perform conventional walking training with orthoses, it was possible to perform movement of the paralyzed joints with voluntary control of the relevant muscle using HAL. Considering that the method was feasible in case 1, whose injury level was the highest of the among the patients presented in the study, this method is feasible for patients who has at least voluntary control the triceps muscle with MMT score greater than or equal to 1.
The operating system of HAL directly maps in real-time the detected neuromuscular activity to the assistive torque on the joints of the lower limbs, and therefore directly reflects the user's motion intention into the generated motion. By contrast, other exoskeleton robots such as the Lokomat (Colombo et al., 2000), LOPES (Veneman et al., 2007), and ReWalk (Miller et al., 2016) move paretic limbs automatically; therefore, the limbs are passively moved. We focused on exploiting the residual voluntary muscle activation of paraplegia patients. Using the HAL motion assist function in the context of upper-lower limb coordination during gait, voluntary ambulation was possible, and the improvement of muscle activities was observed.
The HAL has been reported to be a feasible tool for some types of neuromuscular disorders Kubota et al., 2013Kubota et al., , 2017Sakakima et al., 2013;Aach et al., 2014;Nilsson et al., 2014;Sczesny-Kaiser et al., 2015Wall et al., 2015;Fujii et al., 2017;Ikumi et al., 2017;Shimizu et al., 2017b) and to improve ambulation in patients with chronic SCI (Aach et al., 2014;Sczesny-Kaiser et al., 2015;Wall et al., 2015;Ikumi et al., 2017;Kubota et al., 2017;Shimizu et al., 2017b). HAL is able to detect very weak neuromuscular activities, if any, using its surface electrodes and to provide motion assistance. In this sense, HAL is an effective tool for gait training in more severe cases of SCI. However, the conventional method using the CVC mode of HAL is not applicable in patients with complete paraplegia who have difficulty in detecting bioelectrical signals. The CAC mode uses a foot pressure sensor as a motion trigger, and pressure sensors are not effective for patients who cannot place their weight on both feet. Therefore, we chose upper limb motion as the voluntary trigger for lower limb movement during ambulation.
In utilizing upper extremity muscle activation, we focused on the structural analogy and symmetric motion between upper and contralateral lower limbs in natural gait. The upper and contralateral lower limbs move in synchrony and almost simultaneously during natural locomotion. Based on this contralateral relationship of upper and lower limb movement during locomotion, we placed electrodes on the biceps and triceps brachii to drive contralateral knee flexion and extension, respectively, and on the anterior deltoid and posterior deltoid to drive contralateral hip flexion and extension, respectively.
A previous study reported that the paralyzed lower limb muscles of patients with SCI can be activated by passive leg movements, suggesting the residual function of the rhythmic pattern generator circuit after SCI (Kawashima et al., 2005). The rhythmic pattern generator is highly relevant to quadrupedal coordination during locomotion in humans (Dietz et al., 1995;Zehr and Duysens, 2004;Dietz, 2011). In fact, recent research has indicated that voluntary upper limb alternate movement generates locomotor-like movements and enhances muscle activation in the lower limbs (de Kam et al., 2013), and this property can be used in the control of legged exoskeletons (La Scaleia et al., 2014). From this perspective, we hypothesized that synergistic coordination of the upper and lower limbs during voluntary gait using HAL may activate lower limb muscles.
UT-HAL-assisted gait in SCI patients incorporates coordinated voluntary movement of the multiple joints of the upper and lower limbs. Since UT-HAL connects each of the upper limb muscles to the corresponding lower limb joint movements independently, the coordination of lower limb movement is realized solely by the coordinated voluntary activation of the upper limb muscles. Our hypothesis is that gait phase-dependent, voluntary, and coordinated activation of the upper limb muscles, in coherent combination with the voluntarily generated gait pattern of the lower limbs, may re-activate gait phase-dependent muscle activity in the lower limbs. This is difficult to implement by other assistive devices such as passive orthoses, other robots, or current functional electric stimulation systems (Karimi et al., 2014).
Our protocol included voluntary knee extension. Patients with paralysis of knee extensors use long leg braces in the knee-locked position for walking exercises; therefore, it is difficult for them to train with knee extensor movement during gait exercise. Gait with the HAL enabled users to extend their knee periodically in the stance phase according to the gait cycle without knee locking (Shimizu et al., 2017b).
In case 3, the patient was unable to contract the hip flexor before intervention. However upper limb-triggered HAL ambulation appeared to activate her paralyzed and disused hip flexors. Subsequently, we used improved muscle activation of the hip flexors as the trigger for knee extension.
We speculate that muscle activation acquired during voluntary ambulation using motion intention conferred by residual activity of the upper limb muscle and during voluntary knee extension using residual activity of the hip flexor may contribute to the improvement of paralyzed Quad muscle activation. Both sessions-knee extension with the hip flexor and locomotion with upper limb muscle activation-were effective in improving muscle activity in cases 3 and 4.
In addition, in case 3 (lower panels of Figure 6), the patient contracted the right TFL in the knee extension HAL session, while in the UT-HAL locomotion session the TFL activation was absent. In our opinion, it was easier to contract the TFL in the knee extension session than in the UT-HAL locomotion because during sitting, TFL contraction is accompanied by a simple single-joint motion of hip flexion, and it was easy for her to focus her attention on the motion. On the other hand, while walking, complex coordinated motion involving hip and knee flexion and extension is required, incorporating coordinated activation of multiple muscles. She could generate voluntary gait using UT-HAL and activate the right Quad in phase with the gait; however, she could not achieve the level of coordinated multiple muscle control during gait.
A similar discussion may apply to case 4 (lower panels of Figure 7). Quad activation was present during the knee extension HAL session, while in the UT-HAL locomotion session it was absent. The patient could contract both TFLs and left Quad independently. During walking, he could generate voluntary gait using UT-HAL and activate both TFLs in phase with the gait. However, he could not reach the level of including the newly activated left Quad in the coordinated activation of the muscles. We consider HAL intervention was effective for achieving voluntary contraction of single-joint muscles; however, a longer-term intervention might have been necessary in order for this patient to be able to perform coordinated muscle control including the newly activated muscles during gait.
Cases 1 and 2 had not experienced conventional walking training before the HAL intervention. They had complete paralysis in both lower extremities; however, during HAL ambulation, there was some activation in the TFLs and Quads. In addition, there was more apparent activation in UT-HAL sessions than in CAC sessions. We consider that ambulation with voluntary control have positively influenced periodic activation.
After intervention, the neurological levels in cases 1 and 2 were unchanged; however, in these cases, spastic paralysis evaluated by the mAs score after each session decreased from pre-session values. As spasticity was the main concern in case 2, this change represented meaningful clinical data. Regarding the reduction of spasticity, we previously reported a decrease of mAs after HAL gait training for a patient with C4 SCI (Ikumi et al., 2017). Spasticity negatively influences quality of life for many SCI patients by causing pain, joint contracture, and restricted activity of daily living (Adams and Hicks, 2005;van Cooten et al., 2015). Therefore, this method may be useful to reduce spasticity and related concerns.
One limitation of this protocol is that it is not suitable for patients who are unable to control upper limb muscles. In addition, it is challenging for patients with severe joint deformities or orthostatic hypotension. It is important for medical staff to evaluate skin problems or bruising in paralyzed areas because of their sensory deficiency. Moreover, although neurological improvement was observed in two cases in this study, we did not confirm the underlying mechanism behind this change. Future perspectives include neurological evaluation of this protocol using, for example, functional MRI of the spinal cord (Stroman et al., 2004) or spinal cord mapping of motor activity during gait (Ivanenko et al., 2008).
The ambulation protocol using HAL and voluntary upper limb muscle activation was feasible for complete paraplegic patients. Moreover, patients with injury to the cervical cord or upper thoracic cord, who have difficulty performing conventional gait training with orthoses, may experience decreased spasticity and activation of paralyzed muscles. It is applicable for patients with both spastic and flaccid-type paralysis and represents a novel option for such patients to perform voluntary controlled ambulation similar to natural gait by using their residual neuromuscular activities. Therefore, this HAL protocol may contribute to broadening gait rehabilitation for complete SCI patients.
CONCLUSIONS
This study reports the safety and feasibility of upper-limbtriggered HAL ambulation for patients with complete quadri/paraplegia caused by chronic SCI. Improvement of activation in both TFLs and Quads was observed for two cases, and decreased spasticity after each session was also observed for three cases with spastic-type paralysis. Therefore, voluntary upper limb muscle activation using HAL is a potential option for rehabilitation in complete quadri/paraplegic patients.
AUTHOR CONTRIBUTIONS
All authors participated in the design, execution, and analysis of these studies and have seen and approved the final version of the manuscript. YuSh and HK participated in the study design and drafted the manuscript. YuSh, HK, SK, and YuSo executed HAL interventions and performed the data analysis. YoS conceived the device and helped to draft the manuscript. KS, TU, TA, YH, and MY participated in the study design and helped to draft the manuscript. MY is the principal investigator of this study and participated in the design and coordination of the study. Furthermore, this manuscript has been revised by a professional editor whose first language is English.
FUNDING
This study was supported by Industrial Disease Clinical Research Grants of the Ministry of Health, Labor, and Welfare in Japan (14060101-01).
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2017-11-21T18:07:56.377Z
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2017-11-21T00:00:00.000
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Calculation of the Characteristics of the Multi-gap Seal of the Centrifugal Pump, in Depend-ence on the Chambers' Sizes
Oleksandr Pozovnyi1 , Andriy Zahorulko1, Jan Krmela2,3, Artem Artyukhov1, Vladimíra Krmelová3 1Faculty of Technical Systems and Energy Efficient Technologies, Sumy State University. RymskogoKorsakova st.,2, 40007, Sumy. Ukraine. E-mail: aleksandrpozovnyi@gmail.com, a.zagorulko@omdm.sumdu.edu.ua, a.artyukhov@pohnp.sumdu.edu.ua 2Faculty of Mechanical Engineering, J. E. Purkyně University in Ustí nad Labem. Pasteurova 3334/7, 400 01 Ustí nad Labem. Czech Republic. E-mail: jan.krmela@ujep.cz 3Faculty of Industrial Technologies in Púchov, Alexander Dubček University of Trenčín. I. Krasku 491/30, 02001 Púchov. Slovak Republic. E-mail: jan.krmela@tnuni.sk, vladimira.krmelova@tnuni.sk
Introduction
Providing the necessary sealing of centrifugal pumps is still a significant issue. Gap seals are widely used as multi-stage, and in some cases, as end seals. Besides that, throttles can be a part of the designs of modern sealing systems performed to balance devices. In recent years, many studies have been aimed at increasing the overall efficiency of centrifugal pumps by reducing a leakage through the end and intermediate seals at high values of pressure variations and rotor speeds. Therefore, to reduce the mass flow rate, the seal with two or three throttle gaps as the front seals of the impeller are used. The construction of such a seal is maintained by gradually located throttles connected with each other by chambers (Fig. 1), the size of which affects the hydrostatic stiffness of the seal and can cause the rotor vibration.
A number of scientific articles are devoted to the study of single-gap seals of centrifugal pumps [1 -6], nevertheless, and for the reduction of the volume flow rate, multi-gap seals are used. The analysis showed that such types of constructions on hydraulic resistance are equivalent to two or three gradually placed throttles, therefore, at the same axial overall dimensions the mass flow rate is reduced. In work [1], the possibility of multi-gap seals that cause increased vibration of the rotor is described. Though, the study of the effect of cameras' size on the hydrostatic force of seals is limited and should be considered.
An analytical calculation of the hydrodynamic cha-racteristics of seals is associated with certain difficulties [7]. Solving equations describing the three-dimensional flow of liquid through the seal construction, at present time, cannot be obtained in a closed analytical form. Each researcher in solving the given task is forced to introduce assumptions and neglect the influence of various factors.
Recently, more and more computer modelling and computational researches have been increasingly used by software complexes based on numerical finite-volume methods, mass transfer, and experimental planning [8,9]. Thus, it may be noted that the experimental and numerical analysis of multi-gap seals is currently relevant.
Numerical analysis of the hydrostatic forces of multi-gap seals requires solving the hydrostatic task in three-dimensional formulation with the help of methods of computational hydrodynamics with the corresponding verification of the obtained results of numerical studies by comparing them with the results of the physical experiment.
Numerical calculation
Modelling of fluid flow in a multi-gap seal using the finite volumes' method: during the calculation research with the help of the ANSYS CFX software complex the problem of hydrostatic fluid flow through the multi-gap seal was solved. The complete model of fluid flow, which includes three consecutively placed throttles, connected by two chambers, was considered ( Fig. 1). The calculation grid was created in indexed on: http://www.scopus.com the ANSYS Meshing program using the Sweep method, which allows to construct a deployed grid on a model that rotates around the axis where the output and target boundaries have a common topology [7,12]. 30 elements were set according to the thickness of the gap. As a result, the calculation grid consisted of about 7.5 million elements. The thickening of the calculation grid in the parietal layers was carried out. The quality of the grid was controlled from the analysis of the distribution of the dimensionless parameter Y + along the walls of the flowing part, which varied from 20 to 50, and the visual control, in order to prevent the presence of excessively elongated or wry elements. For calculation, high Reynold's k-ε model of turbulence with an intensity of 5 % input was used. This model is semi-empirical and uses two differential equations for the closure of the Reynolds equations, describing the transfer of the kinetic energy of turbulence k and the velocity of dissipation of turbulent energy ε. The boundary conditions are set: the water temperature is 20 °С, the pressure of the liquid supply at the inlet is 1 MPa, the pressure at the outlet with the seal is 0 MPa. The walls remained at stationary state.
During the calculation, the dimensions of the chamber (Fig. 2, l 2 = l 4 ) changed from 0.4 to 4 mm and at the radial displacement of the shaft -0.08 mm. The main thermodynamic properties of water: temperature, coefficient of dynamic viscosity and density are assumed to be constant [11 -14].
Results of numerical calculation
At first, the model of a three-gap seal with the equal radial dimensions of the gaps I-variant (H 1 = H 2 = H 3 in Fig. 2) was considered. But due to the fact that the pressure on the first chamber from the inlet is aligned unsatisfactorily (Fig. 3) and there may be rotor vibrations due to changes in the hydrostatic force value that should balance the rotor, further the model of the seals with a two times bigger second gap II-variant (H 2 = 2H 1 = 2H 3 in Fig. 2) was considered in relation to the first and third (Fig. 4) in order to equalize pressure on it at a given displacement of the shaft e = 0.08 mm.
Fig. 3 Pressure distribution in a three-gap seal with gaps I-variant, with eccentricity (e = 0.08 mm, l 2 = l 4 = 3 mm): 1 -a shaft wall, 2 -a stator wall
Also, the influence of the chambers' size on the hydrostatic forces and the leakage from the seal was considered (chambers' indication -K was introduced, where l 2 = l 4 , Fig. 2). Values of calculations from the ANSYS software system are at Tab. 1. By the value of radial forces, a graph was constructed, depending on the axial size of the chambers, and the approximation by the least squares' method was carried out.
From the graph, it is evident that with the size K ≤ 0.5 mm the hydrostatic force has a positive value, that is, it decenters the rotor, so it is necessary that the axial size of the chambers is more than 0.5 mm. At values 0.5 < K <1 mm the magnitude of the hydrostatic force increases sharply, and at K > 1 mm its value increases not so intensive, so the difference between forces at K = 1 mm and K = 0.9 mm 20 %, at K = 1 mm and K = 2 mm is 16.7 %. For values K > 3 mm, the force is almost unchanged.
Experimental research
Tests of multi-gap seal were carried out on the basis of the research and development laboratory of vibration-reliability and leak proofness of centrifugal machines of the Department of General Mechanics and Machine Dynamics of Sumy State University on an experimental stand for the study of hydrodynamics of throttles in accordance with the program and test methods. The necessary conditions for the installation operation are provided by a special system of preparation and supply of sealing medium -liquids (water). To ensure the effectiveness of experimental researches, taking into account the need for registration of fleeting processes and based on the capabilities of modern electronic computers, the stand is equipped with a specially created a universal automated measuring system. It allows to record automatically and accumulate the results of measurements of all parameters controlled during the research process. Before assembling the experimental unit, the geometry of the seal was measured and two pressure sensors were installed in the first chamber. Fig. 6 shows the scheme of the experimental unit of the installation with a three-gap seal. A pair of seals is created by the stator bushing 2, fixed in the cylinder of the test head, and the rotor bushing 6, fixed by a nut on the shaft. The rotary bushing has threaded holes at the ends for fastening the weights that allow to create an artificial imbalance, to ensure that the center of the mass of the rotor shifts within 0 -0.2 mm. Pressure measurements were carried out at the inlet and in the first chamber in two places, depending on the eccentricity. Tab. 2 shows the results of the physical experiment with a two times bigger second gap II-variant (H2 = 2H1 = 2H3 in Fig. 2) in relation to the first and third in order to equalize the pressure on it with a given displacement of the shaft e = 0.08 mm.
Conclusion
The paper considers the effect of the change of the axial size of the chambers of the multi-gap seal on the operation of the centrifugal pump by means of experimental research and numerical experiment. The experimental research allowed to obtain the performance characteristics of the seal at different operating modes, depending on the change in the size of the chambers, as a result of which the pressure distribution and the leakage from the seal also changed. As a result of the numerical experiment, radial hydrostatic forces were obtained and the boundary of the radical change of radial force was determined, this occurs when the values of the axial size of the chambers are K < 1 mm.
Comparison of the results of the experiment and the numerical calculation showed their satisfactory matching, the maximum divergence of the leakage is 5.1 %, and the values of pressure is 7.1 %.
Analysis of the research results showed that there is a possibility of increasing the hydrostatic power of the multi-gap seal without the increasing the amount of volumetric losses due to the change in the axial size of the chambers.
|
2020-09-10T10:02:11.799Z
|
2020-09-07T00:00:00.000
|
{
"year": 2020,
"sha1": "5d101d42d0993987a0bdf4219b2a4e697e174644",
"oa_license": "CCBYNC",
"oa_url": "http://journalmt.com/doi/10.21062/mft.2020.048.pdf",
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222050460
|
pes2o/s2orc
|
v3-fos-license
|
Relationship of Antral Follicle Count, Plasma Estradiol and Progesterone Levels with Super Ovulatory Response and Embryo Production in Sahiwal Cows
The production and reproduction potential of dairy cows is going to be severely affected due to global warming. Therefore, dairy cows having potential to produce milk at higher environmental temperature are required to mitigate effect of global warming. Sahiwal is considered as the best milch breed of indigenous cattle. These cows are better adapted to tolerate high temperatures prevailing in tropical climate due to higher number of sweat glands (Khan et al., 2008). However, population of Sahiwal cows has decreased to a drastic level due to adoption of cross breeding programs. Therefore, efforts are required to improve and propagate this breed at farmer level. International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 6 (2020) Journal homepage: http://www.ijcmas.com
Introduction
The production and reproduction potential of dairy cows is going to be severely affected due to global warming. Therefore, dairy cows having potential to produce milk at higher environmental temperature are required to mitigate effect of global warming. Sahiwal is considered as the best milch breed of indigenous cattle. These cows are better adapted to tolerate high temperatures prevailing in tropical climate due to higher number of sweat glands (Khan et al., 2008). However, population of Sahiwal cows has decreased to a drastic level due to adoption of cross breeding programs. Therefore, efforts are required to improve and propagate this breed at farmer level.
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 9 Number 6 (2020) Journal homepage: http://www.ijcmas.com The study was designed to find correlation between antral follicle count (AFC), plasma levels of estradiol and progesterone with superovulatory response and embryo production in Sahiwal cows. Pluriparous Sahiwal cows (n=12) were administered estradiol-17β (2 mg, i.m.) along with intravaginal placement of progesterone implant (TRIU-B) at random day of the estrous cycle (day 0). On day 4, cows were administered Folltropin-V @ 100 or 200 mg of NIH FSH PI, divided in 8 tapering doses administered at 12 hourly intervals. PGF2α was given on day 6 and day 6.5; Implant TRIU-B was withdrawn with last FSH injection. Cows were fixed timed inseminated at 24 and 36 hours of last FSH dose. Results showed that the AFC at start of FSH administration (Day 4) had high positive and significant correlation with superovulatory response (r = 0.863, p = 0.0003). Mean plasma estradiol was non significantly higher on day of estrus (Day 8) compared to the day of start of FSH treatment (Day 4) and day of embryo collection (21.8±3.27, 26.4±3.32 and 21.6±2.99 pg/ml, respectively). The number of anovulatory follicles (>10 mm) present on the day of embryo collection had negative and significant correlation with number of transferable embryo (r = -0.609; P = 0.0355). Plasma progesterone had a weak negative correlation with >10 mm follicles (r = -0.311, P>0.05) on day of estrus and a weak positive correlation with superovulatory response on the day of embryo collection (r = 0.332, P>0.05). In conclusion, the study indicated that antral follicle count on the day of ovarian stimulation could be used to predict superovulatory response and persistence of anovulatory follicles till the day of embryo collection had adverse effect on embryo quality in Sahiwal cows.
The reproductive biotechniques like multiple ovulation & embryo transfer (MOET) could play an important role in faster dissemination of available superior Sahiwal germplasm. However, success of any embryo transfer program depends on successful induction of superovulatory response in selected donor cows which is quite variable and unpredictable.
Numerous intrinsic and extrinsic factors viz. physiological stage of donor animal, breed, nutritional status, type of gonadotrophin used, dose, season, farm management etc. effect superovulatory response in cattle. In addition, antral follicles count (AFC) at the beginning of FSH treatment, circulatory plasma levels of ovarian hormones viz. Estradiol (E2) and Progesterone (P4) play an important role in determining superovulatory response as well as production of transferable embryos. Antral follicle count (AFC) at a given point of time reflects the ovarian follicular reserve and has been positively correlated with superovulatory response and embryo production in the donor cows (Ireland et al., 2008 andSantos et al., 2016). Similarly, a positive significant correlation between concentrations of circulating ovarian steroids at the start of stimulatory treatment and at estrus with number of ovulations has been reported in crossbred cows (Arosh et al., 2001). However, similar information on Sahiwal cows is scanty. Therefore, the present study was undertaken to evaluate relationship between antral follicle count, plasma estradiol and progesterone concentrations with superovulatory response and embryo quality in Sahiwal cows.
Experimental animals
The study was conducted on pluriparous Sahiwal cows (n=12), non-pregnant, normally cyclic, weighing between 370 to 450 kg having body condition score (BCS) >3 on scale of 5, maintained at Dairy farm, DLF, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana. The selected cows were maintained under loose housing and fed with chaffed green fodder, concentrates, wheat straw, mineral mixture, common salt and ad libitum drinking water.
Superstimulatory treatment
The cows were administered 2 mg of Estradiol-17β + 50 mg of progesterone (Sigma Aldriche, India) along intravaginal Progesterone implant; TRIU-B having 958 mg of progesterone (Virbac Animal Health India Pvt. Ltd., India) on the random day of estrous cycle (Day 0). On Day 4, Follicle stimulating hormone; Folltropin-V @100 or 200 mg NIH-FSH-P1, divided in 8 tapering was administered at 12 hourly intervals for 4 days. Cloprostenol, Inj. Estrumate -Intervet India Pvt. Ltd.; a PGF2α analogue @ 500 µg per cow was injected on day 6 th and 6.5. Intravaginal progesterone implant; TRIU-B was removed at the time of last FSH injection. Luitenizing hormone; (Lutropin 12.5 mg -Bioniche Animal Health, Inc., Belleville, On, Canada) was given at 12 hours after the last FSH Injection. Cows were inseminated twice i.e. at 24 and 36 hour after last FSH injection using frozen thawed semen of high fertility Sahiwal bulls.
Ultrasonography
Trans-rectal ultrasonography was performed using ultrasound scanner (Exago, ECM, France) equipped with B-mode linear array transrectal probe using 7.5 MHz frequency to record the size, location, number of the follicles and corpus luteum (CL). Disappearance of a large follicle (>10 mm) of previous day scan was considered as ovulation. The superovulatory response was determined by counting number of corpora lutea (CLs) on the day of embryo collection i.e. Day 15 of the protocol.
Embryo collection and evaluation
Embryos collection was done non-surgically on day 15 of the protocol (Day 7 post AI) by two way Worrlein catheter (Minitub GmbH, Germany) with Dulbecoo's phosphate buffered saline supplemented with bovine serum albumin (BSA, 0.1 percent) and Gentamicin @50 µg/ml. Ova/embryos were searched under stereomicroscope and recovered embryos were graded according to the criteria proposed by Robertson and Nelson (1998).
Blood sampling and hormone assay
Blood samples were collected from jugular vein in heparinised vials on day 0, 4, 6, 8 and Day 15 of the protocol. Following centrifugation of blood at 2000 RPM for 15 minutes, plasma was collected and stored frozen at -80°C until assay. Plasma estradiol was estimated using a commercially available direct immune enzymatic assay kit (Bovine Estradiol (E2) ELISA Kit), manufactured by Shanghai Yehua Biological Technology Co., Ltd., China. Cat. No: YHB0065Bo. Plasma progesterone was measured using enzyme immunoassay Bovine Progesterone (PROG) ELISA kit, manufactured by Genxbio Health Sciences Pvt. Ltd, China. Cat.No:GXBB01297.
Statistical analysis
The data was analyzed for means and standard errors (SE) for all variables. After confirming the normality of data and homogeneity of variance, Student`s t test (two tailed) was applied to compare mean values of variables.
The relationship of AFC, Plasma estradiol and progesterone with different embryo production parameters was evaluated by Pearson's correlation coefficient. Probability values <0.05 were considered significant.
Antral follicle count and embryo production
Results of the study showed that the number of antral follicles measuring 3 to 5 mm on the ovaries of donor cows at the time of start of super stimulatory treatment ranged from 13 to 40; and the mean was 23.08±2.26 follicles per cow. The AFC at the time of start of FSH administration (i.e. Day 4) had high positive and significant correlation with the superovulatory response (r = 0.863, p = 0.0003) indicating that cows having higher AFC at the start of FSH developed more number of CLs. A positive but very low correlation of AFC was observed with number of embryos recovered and transferable embryos (r1 = 0.192, r2 = 0.122; p1 & p2 >0.05). Various factors like ovulation rate, size of superstimulated ovaries, nutritional status, individual animal variations etc. could be attributed for the non-translation of high correlation observed between AFC and superovulatory response into total and number of transferable embryos recovered. Variable and unpredictable superovulatory response is one of the major impediments in application of embryo transfer technology at farmer level. Some donor cows consistently produce large number of embryos while the others despite similar age, weight, breed, management etc. produce very low or no embryos. The difference in ovarian activity at the time of FSH treatment has been attributed as one of the major reason for such type of variation in superovulatory response (Rico et al., 2009). Singh et al., (2004 reported that AFC was highly variable between individuals but highly repeatable between different follicular waves of the same animal in cattle. Various studies had shown that AFC was associated with superovulatory response, indicating reliability of AFC in assessing number of functional follicles that can respond to exogenous FSH treatments, in cattle and humans (Singh et al., 2004 andHendriks et al., 2005). The objective of exogenous gonadotrophin hormones (FSH or PMSG) is to support small antral follicles to grow and mature and undergo ovulation, resulting in multiple ovulations. Therefore, superovulatory response of a donor should depend on pool of small antral follicles present at the time of initiation of stimulation. In previous studies using ultrasound to determine antral follicle count, Ireland et al., (2007) reported that cows with high follicle numbers had more embryos recovered and had more transferable embryos. Whereas, donor cows with few growing antral follicles had a low ovulatory response and low number of recovered embryos (Kanitz et al., 2002). Our study corroborated the findings that evaluation of AMC (3 to 5 mm follicles) on the ovaries at time of initiation of superovulatory treatment could be used as an alternate to predictor of superovulatory response in Sahiwal cows. AFC based prediction of superovulatory response for selecting donor could be a more practical approach as many practitioners use ultrasound machines for embryo transfer work.
Plasma estradiol, anovulatory follicles and embryo production
The results of present study showed that the mean estradiol plasma concentration on Day 4, 8 and 15 was 21.8±3.27, 26.4±3.32 and 21.6±2.99 pg/ml, respectively. Although, the mean estradiol plasma concentration was higher on day of estrus (Day 8) compared to day of start of FSH treatment (Day 4) and day of embryo collection (Day 15), the differences were non-significant (P>0.05). The number of anovulatory follicles (>10 mm) present on day of embryo collection had negative but significant correlation with number of transferable embryo recovered (r = -0.609; P = 0.0355). Based on the presence of number of anovulatory follicles measuring >10 mm on the day of embryo collection; cows were grouped into two groups i.e. Cows having no anovulatory follicle (>10 mm follicles ≤2) and cows having anovulatory follicles (>10 mm follicles >2) as shown in Table-3.
Out of the twelve superovulated Sahiwal cows, five cows showed >2 anovulatory follicles on the day of embryo collection and seven cows had ≤2 follicles measuring on the day of embryo collection. Based on number of anovulatory follicles present on Day 15, the difference was significant between the two groups (P<0.001) No significant difference in AFC at the start of FSH treatment (Day 4) and at the time of estrus (Day 8) were observed between the cows having ≤2 vs. >2 anovulatory follicles. The mean number of CLs and embryos recovered were comparatively lower in Sahiwal cows having >2 anovulatory follicles compared to cows having ≤2 anovulatory group (15.4±2.87 & 7.6±3.04 vs. 16.6±2.40 & 11.4±3.44, respectively. Although the difference was statistically non-significant but the cows having ≤2 yielded more number of transferable embryos compared to cows having >2 anovulatory follicles (4.9±1.56 vs. 1.4 ± 0.75, respectively). Plasma estradiol levels at the start of FSH treatment (Day 4) and on the day of estrus (Day 8) were higher in Sahiwal cows having ≤2 anovulatory follicles compared to cows having >2 anovulatory follicles; the difference tended to be significant on Day 4 (26.9 ± 4.65 vs. 15.4 ± 2.80 pg/ml, respectively, P=0.09) and numerically higher on Day 8 (29.7±5.19 vs. 21.9±2.7 pg/ml, respectively, P=0.267).
On the day of estrus (Day 8), plasma progesterone concentrations were significantly lower as compared to the other days under the study. Highest plasma progesterone concentrations were observed on day of embryo collection (Day 15) which was significantly higher than the day of estrus (2.73±0.11 vs. 0.68±0.05, respectively; P <0.05). A weak negative correlation (r = -0.311, P>0.05) was observed between plasma concentration on day of estrus (Day 8) and ovulatory sized follicles (>10 mm follicles) whereas, a positive but weak correlation (r = 0.332, P>0.05) was recorded between plasma progesterone concentration on day of embryo collection and superovulatory response. The mean progesterone concentrations on the day of estrus and embryo collection observed in the present study were lower than in the Ongole cows (2.3 ± 0.3 and 9.6 ± 3.15 ng/ml) reported by Duddukuri (2015). Mutha Rao et al., (2005) and Siddiqui et al., (2011) also reported similar pattern of increase in concentrations of progesterone from day of estrus to day of embryo collection. Goto et al., (1988) reported that higher levels of P4 on the first treatment day resulted in significant higher superovulatory response, total embryos and viable embryos. However, the present study did not find any relationship between P4 concentration on the day of treatment and ovarian response. This was in agreement with earlier reports of Walton and Stubbings (1986) and Lopes et al., (2001). Wiley et al., (2019) reported that the start of superstimulatory treatment during low level of endogenous progesterone due to regressing CL could decrease the overall embryo production. Number of transferable embryos recovered in the present study was comparatively low compared to superovulatory response and total embryo recovery which warrants further investigation. Brar and Nanda (2008) reported that better management practices, housing, nutrition supplemented with minerals and vitamins had shown to improve reproductive performance of Bos indicus cows. The results of the present study showed that antral follicle count on the day of ovarian stimulation could be used to predict superovulatory response and persistence of anovulatory follicles till the day of embryo collection had adverse effect on embryo quality in Sahiwal cows.
|
2020-07-30T02:07:30.615Z
|
2020-06-10T00:00:00.000
|
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"oa_url": "https://www.ijcmas.com/9-6-2020/Nidha%20Imtiyaz,%20et%20al.pdf",
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|
247614809
|
pes2o/s2orc
|
v3-fos-license
|
Profilin is involved in G1 to S phase progression and mitotic spindle orientation during Leishmania donovani cell division cycle
Profilin is a multi-ligand binding protein, which is a key regulator of actin dynamics and involved in regulating several cellular functions. It is present in all eukaryotes, including trypanosomatids such as Leishmania. However, not much is known about its functions in these organisms. Our earlier studies have shown that Leishmania parasites express a single homologue of profilin (LdPfn) that binds actin, phosphoinositides and poly- L- proline motives, and depletion of its intracellular pool to 50%of normal levels affects the cell growth and intracellular trafficking. Here, we show, employing affinity pull-down and mass spectroscopy, that LdPfn interacted with a large number of proteins, including those involved in mRNA processing and protein translation initiation, such as eIF4A1. Further, we reveal, using mRNA Seq analysis, that depletion of LdPfn in Leishmania cells (LdPfn+/-) resulted in significantly reduced expression of genes which encode proteins involved in cell cycle regulation, mRNA translation initiation, nucleosides and amino acids transport. In addition, we show that in LdPfn+/- cells, cellular levels of eIF4A1 protein were significantly decreased, and during their cell division cycle, G1-to-S phase progression was delayed and orientation of mitotic spindle altered. These changes were, however, reversed to normal by episomal expression of GFP-LdPfn in LdPfn+/- cells. Taken together, our results indicate that profilin is involved in regulation of G1-to-S phase progression and mitotic spindle orientation in Leishmania cell cycle, perhaps through its interaction with elF4A1 protein.
Introduction
Profilin is a key regulator of actin dynamics that plays a central role in almost all vital cellular processes, including endocytosis, motility, signal transduction, metabolism, cell division, etc. [1][2][3]. It is a 16kDa actin-binding protein that has been recognized as one of the crucial proteins for cell survival [4]. Besides binding to actin, profilin also binds to a large array of actinbinding membrane proteins that contain poly-L-proline (PLP) motives in their structure, and
Pull-down assay and mass spectrometry
Full-Length profilin was cloned and expressed with GST-tag (GST-LdPfn), as described earlier [11]. For the pull-down assay, purified recombinant GST-LdPfn or GST alone was incubated with 100μL of Glutathione-Sepharose affinity beads for 2 hours. The unbound proteins were removed by centrifugation at 1000 x g for 10 minutes at 4˚C and the recombinant proteins associated with the beads were incubated with 2mg protein of clear lysate of L. donovani promastigotes (clear supernatant obtained after sonication and centrifugation) overnight at 4˚C. The next day, unbound proteins were removed, and the beads were washed five times with 1x PBS. Finally, the bound proteins were eluted by boiling the beads at 96˚C for 5 min in 50ul of SDS-poly acrylamide gel electrophoresis (SDS-PAGE) loading buffer (250mM Tris, 10% SDS, 0.5% bromophenol blue, 50% glycerol, and 500mM 2-mercaptoethanol). Eluates were run on 12% polyacrylamide gel. Three biological replicates of each of GST and GST-LdPfn pull-down samples were stained with silver nitrate, the gel was rinsed thrice with double distilled water; the respective lanes were excised, kept in labelled Eppendorf tubes submerged with double distilled water, and submitted at the mass spectrometry facility (Institute for Stem Cell Regeneration and Medicine, NCBS-TIFR Campus, Bellary Road, Bangalore Karnataka, India) for analysis, using LC-MS (Orbitrap Fusion™ Tribrid™ Mass Spectrometer). Only the protein hits with a score cut off 25, significance threshold p<0.05 were considered. The data thus-obtained were used for searching the www.tritrypdb.org database (version 51), using the Leishmania donovani (BPK282A1) strain as reference. Data has been deposited at PRIDE-ProteomeXchange Consortium [12] and can be accessed with the identifier: PXD026036. Among the three biological replicates, the proteins present in all three replicates or at least two replicates of GST-LdPfn, but not present in any of the replicates of GST control, were taken into consideration.
Total RNA isolation and library construction
Total RNA from three independent biological replicates was isolated from LdPfn +/+ (control) and LdPfn +/promastigotes using TRIzol reagent (Ambion, Life Technologies), according to the manufacturer's instructions. RNA samples were treated with DNase I (2μg) and the RNA concentration was determined, using a spectrophotometer at A260/280 (Nanodrop ND1000, Thermo Scientific, USA). In addition, the RNA integrity was evaluated using TapeStation (Agilent) and Qubit (Invitrogen). Using NEBNextUltra TM II RNA Library Prep Kit for Illumina, Poly (A) mRNA magnetic isolation was performed. Library preparations were carried out, using the NEBNextUltra TM II RNA Library Prep kit (Illumina), according to the manufacturer's instructions.
RNA-Seq and data analysis
Paired-end reads (2 x 150 bp) were obtained using the Illumina HiSeq 2500 platform at the Bio-IT centre at the Institute of Bioinformatics and Applied Biotechnology. Raw data were generated for each of the libraries from 6 samples. The quality of the produced data was analysed using FastQC by Phred quality score. Reads with Phred quality scores lower than 20 were discarded. Reads were aligned to the L, donovani (BPK282A1) genomic data obtained from TriTrypDB version 51 (www.tritrypdb.org), using Bowtie2 (-x option) [13,14]. All analyses were carried out using the Tophat pipeline with the following versions: Tophatv2.1.1, Bowtie2 v2.3.51 [15]. Tophat is a fast splice junction mapper for RNA-Seq reads. It aligns RNA-Seq reads to genomes, using the ultra-high throughput short read aligner Bowtie and then analyses the mapping results to give the transcript counts. The gene expression level values were calculated from the transcript counts. Gffread tool [16] was used to convert gff files to gtf files for read-count calculation using HTSeq. The HTSeq version 0.12.4 (htseq-count -f option) was used to count the number of reads aligned to protein-coding genes. HTSeq [17,18] is a Python package that gives the infrastructure to processed data from high-throughput sequencing assays. HTSeq calculates the number of mapped reads to each gene. DeSeq tool was used for differential gene expression analysis between samples in protein-coding genes. DeSeq [18,19] is an R package to measure variance-mean dependence in count data from high-throughput RNA-Seq assays and test for differential expression depending on a model applying the negative binomial distribution. Differentially expressed (DE) genes were classified as genes with a Benjamini-Hochberg multiple testing p-value of <0.05. Principal component analysis plot (PCA plot), clustered heat map, and volcano plots were created using base R plot PCA function (or base R prcomp function), gplotsheatmap.2 functions and ggplot function of gglplot2 package respectively. Functional annotation was performed using GO (gene ontology) and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) and Metabolic Pathways from all Domains of Life (MetaCyc) using the L.donovani (BPK282A1) GO annotations provided in TriTrypDB.
Real time quantitative polymerase chain reaction (RT-qPCR) validation assays
DNase-treated RNA (2μg) was reverse transcribed with MMLV reverse transcriptase (NEB). Equal amounts of cDNA were assessed in triplicate in a total volume of 10ul containing SYBR Green (Kapa Biosystems) and the primers used were given in S1 Table. The mixture was incubated at 95˚C for 20 sec, 55˚C for 30 sec, and 72˚C for 1 sec for 40 cycles. Negative controls were included in the RT-qPCR assays to detect DNA contamination in RNA samples. The fold-change in expression of up and down regulated genes was determined, using RT-qPCR. Reactions were carried out on a fast RT-qPCR system (Applied Biosystems). The results were quantified by the delta-delta CT method with actin as a reference gene and β-tubulin as an endogenous control to normalize each sample. The specificity of the reaction was verified by melt curve analysis. All the experiments were conducted at least three times and the results were expressed as mean ± SEM of three independent experiments.
permeabilized, using 0.5% (v/v) Triton X-100 for 15 minutes, and washed with PBS-glycine. The washed cells were blocked with 3% bovine serum albumin in PBS for 2 hours at 25˚C and labelled with anti-LdPfn antibodies [11] (1:100) and or anti-tubulin antibodies (Santacruz biotechnologies cat no. sc-5286 and Sigma cat.no. T7816) (1: 500) overnight at 4˚C. The labelled cells were washed with 0.5% bovine serum albumin in PBS to remove non-specifically bound antibodies and again labelled with Alexa Fluor 568 -conjugated goat anti-mouse IgG (Thermo Fisher Scientific, cat no. A21043) or Alexa Fluor 488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, cat no. A32731) or Fluor 488-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, cat no.A32723) or Alexa Fluor 568-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, cat no.A21069) secondary antibodies depending on the experiment. The coverslips were mounted using prolong diamond anti-fade mounting media containing 4,6-diamidino-2-phenylindole (DAPI, Invitrogen). To analyse the nuclear division patterns, the coverslips were treated with RNase (5mg/ml) followed by labelling with propidium iodide (PI). Images were captured on Nikon laser scanning confocal microscope c2 using a 100X1.4 NA (oil) plan apochromatic lens.
Cell cycle analysis
For cell cycle analysis, Leishmania cultures (5 x 10 7 cells) were synchronized by incubating them with 200 mg ml -1 of N-hydroxyurea (HU) (Sigma) overnight (12-14 hours). The cells were washed and then re-suspended in DMEM media containing 10% FCS without HU. Aliquots of cell suspension were drawn at 0 hour to 10 hours at regular time intervals of 2 hours. The cells were washed with cold PBS and resuspended in 50μl of PBS. The cell suspension was mixed with 150μl of fixative solution (1% Triton X-100, 40mM citric acid, 20mM sodium phosphate, 200mM sucrose) and incubated at 25˚C for 5 minutes. After incubation, 350μl of diluent buffer (125mM MgCl 2 in PBS) was added to it and stored at 4˚C until further use. Before the cell cycle analysis, the fixed cell suspension was treated with 50μg RNase (5mg ml -1 in 0.2M sodium phosphate buffer, pH7.0) for 2 hours at 37˚C and then incubated with 50μg PI (5mg ml -1 in 1.12% sodium citrate) for 30 minutes at 25˚C. The samples were analysed in Gallios flow cytometer (Beckman coulter) and proportions of the G1, S, and G2M populations were determined using ModFit LT software (Verity Software House, Topsham, ME, USA).
Statistical analysis
All the experiments were conducted at least three times and the results were expressed as the standard error of the mean (mean ± SEM) of three experiments. The data were statistically analysed by ANOVA test with replication. A p-value of <0.05 was considered significant.
Leishmania profilin interacts with a large number of cellular ligands, including elF4A1
To map the ligand binding profile of LdPfn, we performed a pull-down assay in lysates of midlog phase Leishmania promastigotes, using glutathione S-transferase (GST)-tagged LdPfn [11] and GST protein alone (negative control), as a bait, as described in 'Materials and Methods'. LdPfn bound to its ligands was isolated by using glutathione-Sepharose affinity beads. The beads were washed thoroughly to remove unbound proteins and the bound proteins were eluted by boiling the beads in SDS-polyacrylamide gel electrophoresis (PAGE) sample loading buffer. The eluates from three independent pull-down experiments of both GST-LdPfn and GST-alone were resolved on SDS-PAGE. The gels were silver-stained, excised, and subjected to trypsin digestion followed by liquid chromatography-mass spectrometry (LC-MS) analysis ( Fig 1A). The proteins present in all three replicates (4 proteins) and those present in at least two replicates (34 proteins) of GST-LdPfn pull-down, but not present in any one of the GSTalone pull-down controls are listed in Table 1. A summary of each protein hit is given in S2 Table. The LdPfn ligands thus detected were classified according to their deduced function or cell location, and the percent of proteins representing each group are shown in Fig 1B. Some of the LdPfn ligands, such as actin (LdBPK_041250.1), valosin-containing protein (LDBPK_361420.1), mitochondrial outer membrane protein porin (LDBPK_020430.1), and eIF4A1 (LdBPK_010790.1), have earlier been identified as the potential ligands also of the T. cruzi profilin [8]. Interestingly, unlike T. cruzi [8], we have also identified gamma-tubulin complex component 3-like protein (LdBPK_362370.1), a key component of the microtubule-organizing centre [20], as one of the potential ligands of LdPfn. While valosin-containing protein VCP/p97 has been shown to be essential for the intracellular development of Leishmania [21], T. brucei porin (Tb927.2.2510) has been reported to be the main metabolite channel in the mitochondrial outer membrane and is required to support efficient oxidative phosphorylation [22]. Further, the eukaryotic initiation factor 4A1 (eIF4A1) is abundantly present in Leishmania cytoplasm [23], and this protein has been suggested to be the main translation initiation factor involved in protein synthesis in T. brucei [24]. To further confirm the presence of this protein in the LdPfn interactome, we analysed the pull-down eluates from GST-LdPfn and GST-alone by western blotting, using anti-eIF4A1 antibodies. Results given in Fig 1C and S1 Fig show that eIF4A1 (45.3kDa) was present in the input lysates used in both the GST-LdPfn and GST-alone pull-down assays and also in the GST-LdPfn pull-down eluates and pass-through fraction from the GST-alone pull-down assay, but it was completely absent in the pass-through fraction from GST-LdPfn pull-down and GST-alone eluate. These results strongly indicate that LdPfn interacts with multiple cellular ligands, a majority of which constitute the proteins that may play an important role in regulating mRNA processing, translation initiation, cell metabolism and mitochondrial functions.
As LdPfn appeared to interact with proteins involved in mRNA processing (LDBPK_353150.1 and LdBPK_322350.1) and protein translation initiation (elF4A1), we performed transcriptomic analysis of mRNA isolated from mid-log phase LdPfn +/+ and LdPfn +/cells, employing high throughput RNA sequencing technique (RNA-Seq), to identify differentially expressed genes in LdPfn +/cells.
Transcriptomic data analysis revealed that the genes involved in DNA transcription, mRNA translation, cell cycle regulation, membrane transport, and mitochondrial activity were differentially expressed in LdPfn depleted cells L. donovani (BPK282A1) contains 36 chromosomes and has a haploid genome size of 32.44 Mb, which encodes a total of 8135 genes (8023 protein-coding genes and 112 non-proteincoding genes) [25]. These parasites display a unique way of controlling their gene expression in that the mRNAs are made from polycistronic precursors by SL-trans splicing and polyadenylation [26][27][28]. Many protein-coding genes of unrelated functions are arrayed in long clusters on the same DNA strand. Intergenic regions of polycistronic pre-mRNAs are cotranscriptionally processed by two reactions: polyadenylation of the upstream gene and trans- splicing of the capped mini exon to the downstream gene [29], thus generating monocistronic units ready for degradation or translation. It is presumed that all polycistronic precursor RNAs are transcribed approximately at the same rate. As a consequence, the regulation of gene expression occurs most entirely post-transcriptionally [30]. To identify differentially expressed genes in profilin depleted Leishmania promastigotes, we analysed the transcriptomes of both the mid-log phase LdPfn +/+ and LdPfn +/-Leishmania promastigotes, using high-throughput RNA sequencing technique (RNA-Seq). Total RNA from three independent biological replicates of each LdPfn +/+ and LdPfn +/promastigotes was extracted and after library preparation, RNA sequencing was carried out on Illumina HiSeq 2500. The RNA-Seq datasets generated in the analysis with each sample have at least 30 million read pairs (S2 Fig and S3 Table). Principal Component Analysis (PCA) was used to analyse the relationship between the samples (S3 Fig), showing a clear separation between LdPfn +/+ and LdPfn +/samples. RNA-Seq data were aligned to the L. donovani genome (L. donovani BPK282A1, NCBI taxon ID: 981087). 8135 transcripts were identified in the data set, which correlated with the data reported earlier [25]. RNA-Seq data revealed that 254 genes were differentially expressed (DE) having an adjusted p-value <0.05. Based on the DE genes, volcano plots were generated (Fig 2A), showing distribution of the transcripts by comparing the fold change in the expression (log 2 ) of each group with the corresponding adjusted p-value (-log 10 ). Among these DE genes, 112 genes (~44%) were hypothetical with unknown functions. The complete list of differentially expressed (DE) genes with known functions either upregulated or downregulated and those with unknown functions along with their respective transcript IDs and log2 fold change are listed in S4 Table. Further, a clustered heat map was generated with the top 30 up and top 30 down-regulated genes to evaluate the reproducibility of the biological replicates ( Fig 2B). The data obtained from RNA-Seq was validated by RT-qPCR of 11 genes, 5 up-regulated and 6 down-regulated including profilin transcript (Fig 2C). Results of the RT-qPCR analysis showed a strong correlation with the RNA-Seq data, thus validating the RNA-Seq results ( Fig 2C).
To evaluate the probable effects of differentially expressed genes in LdPfn +/cells, we performed gene ontology (GO) analysis of 254 DEGs to identify the cellular component, molecular functions, and biological processes enriched due to profilin depletion. Fig 3A shows that the transcripts encoding proteins were mainly localized to the nuclear component, mitochondrial complex, and TORC2 complex. The molecular functions (Fig 3B) enriched by these transcripts were mainly predicted to include the transmembrane transporter activity, phosphatidylinositol binding, kinase activities, and ubiquitin-protein transferase activity. Biological processes that were enriched due to depletion in the LdPfn levels are shown in Fig 3C. From RNA-Seq data, we identified some of the genes enriched in GO biological processes and then validated their mRNA expressions by RT-qPCR. Fig 3D shows findings from the RNA-Seq data (with log 2-fold change, cut off above +0.8 for upregulated and below -0.8 for down regulated genes) were grouped based on their cellular activities ( Table 2) that could have been affected due to LdPfn depletion. Results given in Table 2 clearly indicate that expression of genes that are involved in regulation of mitochondrial activity The Hsp60/10 complex is believed to be responsible for accelerating the folding of polypeptides imported into mitochondria, as well as reactivation of denatured proteins, and diminishing aggregation of non-native polypeptides and partially unfolded kinetically trapped intermediates [82] (Continued ) (such as ATOM14, ATOM11), cell division (such as CYC2-like cyclin, cell division cycle protein 20 (CDC20) and kinesin 13.1), DNA transcription (such as histone-lysine N-methyltransferase), mRNA translation (such as eIF4A1), and amino acids and nucleosides transport was significantly reduced in LdPfn depleted cells.
Depletion of intracellular pool of LdPfn results in significantly reduced levels of eIF4A1 protein in LdPfn +/cells
The translation initiation factor 4A1 (eIF4A1), a DEAD-box RNA helicase, is a component of the translation initiation complex eIF4F, which binds to the cap structure of eukaryotic mRNA and helps in recruiting the small ribosomal subunit. As elF4A1 is a component of the LdPfn interactome ( Table 1) and expression of its gene is significantly down-regulated in the LdPfn +/cells (Table 2), we examined whether the intracellular levels of elF4A1 protein have also been affected in these cells. For this, we probed the SDS-electrophoretograms of lysates of mid-log phase LdPfn +/+ , LdPfn gene complemented LdPfn +/cells (LdPfn +/-comp ) and LdPfn +/cells by western blotting, employing anti-LdPfn and anti-eIF4A1 antibodies (Fig 4A and S1 Fig). Results presented in Fig 4B clearly show that depletion of the intracellular pool of LdPfn results in about 50% reduction in cellular levels of eIF4A1 (Fig 4B) in Leishmania promastigotes. That the decreased expression of elF4A1 is caused due to depletion of LdPfn in Leishmania cells, was confirmed by episomal expression of GFP-LdPfn gene in the LdPfn +/cells (Fig 4A), which restored the elF4A1 levels to normal.
PLOS ONE
As cellular levels of key cell cycle proteins are strictly controlled by tightly regulated synthesis of such proteins during the G1-phase of the cell cycle [31][32][33], it may be inferred that the reduced expression of translation initiation factor 4A1 in LdPfn +/cells may adversely affect protein synthesis and consequently the cell division cycle in LdPfn +/cells. To confirm this conclusion, we analysed the cell division cycle in LdPfn +/+ , LdPfn +/-comp and LdPfn +/cells, using flowcytometry and immunofluorescence microscopy.
LdPfn is involved in regulation of the G1-to-S phase progression
As the gene expression in trypanosomatids is largely controlled at the post-transcriptional level, the main control points in Leishmania gene expression should therefore be mRNA degradation and translation [34]. This means that, the poor mRNA processing and translation would reflect poor gene expression in LdPfn +/cells. To test this possibility, we analysed the cell division cycle in LdPfn +/-, LdPfn +/+ and LdPfn +/-comp cells. The cells were synchronized by treating them for about 12 hours with HU. Approximately 70% of the cells were synchronized at the G1/S border by HU treatment. After releasing the HU block, the cells were stained with PI to probe the total DNA content and then the samples were processed for the cell cycle analysis by flow cytometry. The LdPfn +/+ and LdPfn +/-comp cells immediately entered the S-phase attaining the peak at 2 hours, and the G2/M peak was achieved at 4 hours after releasing the HU block. However, the LdPfn +/cells lagged in entering from the G1-to S-phase by at least 1 hour and remained mostly in the S-phase up to 6 hours (Fig 5A, S4A Fig). These results clearly revealed that profilin plays an important role in G1-to-S phase progression in Leishmania cell cycle. To further analyse this finding, we performed quantitative flow cytometric analysis with BrdU incorporation in LdPfn +/+ , LdPfn +/and LdPfn +/-comp cells to determine the number of cells present in different phases of cell cycle at different time points, after removal of the HU block. There were significantly higher number of LdPfn +/cells (71.7%), as compared to LdPfn +/+ (53.1%) or LdPfn +/-comp cells (59.5%), in the G1-phase at 2 hours after releasing the HU block, whereas at the same time point, considerably lesser number of LdPfn +/cells (6.0%), compared to LdPfn +/+ (30.3%) or LdPfn +/-comp (24.1%) cells, were present in the Sphase. Similarly, the number of LdPfn +/cells in the G1-phase was much higher (61.4%) than that of the LdPfn +/+ (31.9%) or LdPfn +/-comp cells (25.6%) at 4 hours after release of the HU block, while only 24.6% LdPfn +/cells, compared to 53.9% LdPfn +/+ and 56.2% LdPfn +/-comp cells, were in the S-phase at the same time period (Fig 5B and 5C).To rule out the possibility of toxic effects of HU on Leishmania promastigotes during synchronization conditions, we performed cytometric assay with BrdU incorporation in asynchronously growing mid-log phase Leishmania cells, without HU treatment, at 2 and 4 hours. In these conditions also, a significantly lesser number of LdPfn +/cells, compared to LdPfn +/+ and LdPfn +/-comp cells, transited from the G1-to-S phase at both the time points (S4B and S4C Fig). These results demonstrate that LdPfn is involved in regulation of G1-to-S phase progression during Leishmania cell division cycle.
LdPfn is involved in regulation of the mitotic spindle orientation
Kinesin 13-1 has been shown to be exclusively an intranuclear protein that regulates spindle assembly during mitosis, and its depletion leads to abnormalities in spindle structure and nuclear division [35,36]. As shown in Table 2, depletion of LdPfn in Leishmania cells resulted in a significant down regulation of expression of the kinesin 13-1 transcript, we considered it of interest to investigate the segregation pattern of the nucleus during mitosis (Fig 6A). For this, the cells collected from the mitotic phase were labelled with anti-tubulin antibodies, anti-LdPfn antibodies, and DAPI or PI and then examined under the fluorescence microscope.
Fig 5. Retardation of cell cycle progression in LdPfn +/cells. (A)
Representative flow cytometry data of LdPfn +/+ , LdPfn +/and LdPfn +/-comp cells. The samples were collected, after releasing hydroxyurea (HU) block, at 2 hours interval for up to 10 hours. 20,000 events were analysed at every time-point. Three independent experiments were performed, and one representative dataset is shown here. G1 (first red peak), S (grey peak) and G2/M (second red peak) phases are indicated in the histogram itself along with percent of cells in each phase. LdPfn +/+ cells entered into S-phase at 2 hours after release of HU block. However, transition of LdPfn +/cells from G1-to S-phase was considerably delayed, compared to LdPfn +/+ and LdPfn +/-comp cells. (B) Representative flow cytometry data with BrdU incorporation in LdPfn +/+ , LdPfn +/and LdPfn +/-comp cells. The cells were collected, after releasing the HU block, at 2 hours interval for up to 10 hours, and then labelled with anti-BrdU antibodies, as described in 'Materials and Methods'. 10,000 events were analysed at every time-point. Three independent experiments were performed, and one representative dataset of 2 hours and 4 hours is shown. G1, S and G2/M phases are indicated in the histogram along with the percent of cells in each phase. In LdPfn +/cells, a significantly lesser number of cells (6.01% and 24.61%, respectively, at 2 hours and 4 hours after releasing HU block) exhibited BrdU incorporation in S-phase, as compared to LdPfn +/+ cells (30.26% and 53.93%, respectively, at 2 hours and 4 hours after releasing HU block), and LdPfn +/-comp cells (24.08% and 56.23%, respectively at 2 hours and 4 hours after releasing HU block). (C) Bar diagram showing considerably lesser number of BrdU labelled LdPfn +/cells (green bar) in S-phase at 2 hours and 4 hours after releasing the HU block, compared to LdPfn +/+ cells (red bar) and LdPfn +/-comp cells (blue bar) at the same time points. p-value ��� <0.001 at both 2 hours and 4hours after releasing the HU block.
https://doi.org/10.1371/journal.pone.0265692.g005 The cells were stained with anti-tubulin (red) and anti-LdPfn (green) antibodies and DAPI (blue). Analysis of dividing cells revealed that the plane of the nuclear division during karyokinesis in the LdPfn +/+ and LdPfn +/-comp cells was positioned parallel to the flagellar base, leading to the formation of laterally arranged spindle between the two dividing nuclei. In contrast, in the LdPfn +/cells the dividing nuclei were arranged nearly perpendicular to the flagellar base, leading to a longitudinally formed mitotic spindle. Scale: 2μm; F, Flagellar base; K, Kinetoplast; N, Nucleus. (C) (a) The cells were alternately labelled with tubulin (green) and the nucleus and the kinetoplast with propidium iodide (red). In this case also, the nuclei division plane orientation in dividing LdPfn +/cells was altered, as compared to dividing LdPfn +/+ cells. Scale: 2μm. Results revealed that the plane of the nuclear division during karyokinesis in the LdPfn +/+ cells was positioned parallel to the flagellar base, leading to the formation of a laterally arranged spindle between the two dividing nuclei. In contrast, in about 70% LdPfn +/cells, the dividing nuclei were arranged nearly perpendicular to the flagellar base, leading to a longitudinally formed mitotic spindle (Fig 6B). However, the division pattern in LdPfn +/-comp cells was similar to that of the control LdPfn +/+ cells, indicating that the defect in the arrangement of dividing nuclei and orientation of spindle in LdPfn +/cells was a specific defect due to depletion in intracellular levels of profilin. To further confirm, we labelled the cells with ant-tubulin antibodies (green) and their nucleus and kinetoplast with PI (red) and then analysed them under a fluorescent microscope (Fig 6C). Quantification of the spindle positioning (lateral or longitudinal) in the dividing cells revealed that about 66% of LdPfn +/cells (n = 140) possessed nearly longitudinally positioned spindle, while only 11% of LdPfn +/+ cells (n = 152) and 10% of LdPfn +/-comp cells (n = 146) showed such aberration (Fig 6C). These results demonstrate that profilin is involved in regulation of the mitotic spindle orientation and nucleus positioning during the mitotic phase in dividing Leishmania cells. However, quantification of the percent of dividing cells with division furrow (Fig 6D) showed no significant difference between the two cell types, suggesting cytokinesis has not been significantly affected.
Discussion
Profilin is a ubiquitous actin-binding protein present in all eukaryotic cells, including unicellular eukaryotic organisms, such as Plasmodium, Toxoplasma, Acanthamoeba Dictyostelium, Tetrahymena, etc. It plays an important role in a number of cellular activities such as cell growth, intracellular trafficking, gene transcription, cell division, cell signalling, etc. [1][2][3][4][5][6]. Profilin has been suggested to be vital for the invasive blood stage Plasmodium falciparum and its complete deletion has been shown to have severe effects on viability of the parasites [37,38]. In related parasite Toxoplasma gondii, this protein is known to be involved in cell motility, host cell invasion, immune evasion, and virulence [39]. Further, Acanthamoeba castellanii contains three profilin isoforms, each one of which exhibits different subcellular localization, diverse ligand binding properties and biological functions [40]. Furthermore, Dictyostelium amoebae lacking profilin isoforms I and II increase in cell size by up to 10 times, their motility is adversely affected, and a wide ring of filamentous actin accumulates beneath the plasma membrane, which blocks their development [41]. Besides this, in Tetrahymena thermophila, loss of profilin affected nuclear positioning, stomatogenesis, and cytokinesis [2]. Results of the present study clearly reveal that profilin is involved in regulation of G1-to-S phase progression and mitotic spindle orientation during Leishmania cell division cycle.
Cell division cycle may be visualised as a developmental process wherein a cell duplicates itself in to two progeny cells. During this process the cell first grows in size, replicates its chromosomes, segregates a full set of chromosomes to each of two new nuclei, and then divides into daughter cells [32]. In eukaryotes, the cell cycle comprises four discrete phases: G1, S, G2, and M. During the G 1 phase, the cell congregates the building blocks of chromosomal DNA and the associated proteins, and also sufficient energy reserves for completing the replication process of each chromosome in the nucleus. Whereas in the S phase, DNA replication occurs to form identical pairs of DNA molecules. The building blocks of proteins and nucleic acids, namely amino acids and nucleobases/ nucleosides, are largely imported by the Leishmania parasite from the host environment through the plasma membrane by specific transporter furrow in LdPfn +/cells (n = 118), as compared to LdPfn +/+ (n = 125) and LdPfn +/-comp (146) cells, indicating that the cytokinesis was not much affected by LdPfn depletion in Leishmania cells. https://doi.org/10.1371/journal.pone.0265692.g006 proteins [42][43][44]. As depletion of LdPfn intracellular pool in Leishmania cells resulted in downregulation of genes that encode amino acids and nucleosides transporters, it may severely limit availability of the DNA and protein building blocks in G1 phase, which, in turn, may result in slowing down the progression of the G1-to-S phase transition. This is strongly supported by our quantitative flow cytometry data, which showed that the number of cells transited from G1-to-S phase at different time periods considerably decreased in LdPfn depleted Leishmania promastigotes, Furthermore, it is consistent with our observation that expression of genes that encode CDC20 and CYC2-like cyclin, which are known to play a critical role in regulation of trypanosomatids cell cycle [45][46][47], is markedly reduced in LdPfn +/cells.
Purines and pyrimidines are basic building blocks of nucleic acids. Distinct from their mammalian and insect hosts, Leishmania parasite lacks the metabolic machinery to produce purine nucleotides de novo and mainly relies on the acquisition of preformed purine nucleobases and nucleosides through its plasma membrane-bound specific permeases from the host [43]. The first such permease, designated LmaNT3, was identified in L. major, which showed about 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1), and is, thus, a member of the equilibrative nucleoside transporter (ENT) family [44], which seems to be key elements controlling nucleoside and nucleotide pool for DNA synthesis [48]. As expression levels of the genes that encode nucleoside transporter 1 (LdBPK_151260.1.1; LdBPK_151250.1.1) was considerably reduced in LdPfn +/cells, it may be inferred that the decreased availability of purine nucleobases and nucleotides would significantly affect the DNA and RNA synthesis, and consequently the DNA replication, during the 'S' phase of the cell cycle.
DNA replication in Leishmania is primarily determined by the active transcription [49]. In this group of organisms RNA polymerase II transcription is polycistronic and individual mRNA are excised by trans-splicing and polyadenylation [34]. The lack of individual gene transcription control is mainly compensated by post-transcriptional mechanisms, including tight translational control and regulation of mRNA stability/translatability by RNA-binding proteins [50]. As LdPfn interacts with the proteins that are involved in mRNA processing and translation initiation, these interactions would be adversely affected in LdPfn depleted cells, which in turn could affect the mRNA processing and translatability.
Translation of mRNA into proteins is initiated with the binding of the multimeric translation initiation complex elF4F to the cap structure of mRNA present at its 5' end. The elF4F complex is comprised of the cap binding protein, elF4E, the highly conserved mRNA helicase, elF4A, and the large scaffolding protein having binding sites for both elF4E and elF4A, elF4G is responsible for recruiting ribosomal subunits to the initiation codon of mRNA. Although several potential elF4F homologues have been identified in the L. major database, only three elF4E, two elF4A and one elF4G (elF4G3) have so far been characterized [23]. While elF4A1 and elF4E3 have been reported to be abundantly present, the other proteins are either moderately abundant or not detected in L. major promastigotes [23]. Similar to L. major, elF4A-1 is very abundant and predominantly cytoplasmic protein also in T. brucei, and its depletion to 10% of regular levels dramatically decreases protein synthesis one cell cycle following doublestranded RNA induction and blocks cell proliferation [24]. Based on these findings, it has been suggested that only the elF4A1 protein is involved in protein synthesis [24]. As the expression of elF4A1 was found to depend on the intracellular levels of LdPfn, the decreased availability of elF4A1 in LdPfn +/cells should affect the protein translation process.
The direction in which a cell divides is determined by the orientation of its mitotic spindle at metaphase. The spindle orientation in eukaryotes is controlled by a conserved biological machine that mediates a pulling force on astral microtubules. Constraining the localization of this machine to only certain regions of the cortex can thus determine the orientation of the mitotic spindle [51]. Kinesins and dyneins are microtubules-dependent motor proteins that transport cargo in opposite directions along microtubules. However, the kinesin 13 family of proteins in trypanosomatids do not possess cargo transporting property, but they do depolymerize microtubules at their ends, and thereby control microtubule length [36]. It has been shown that in kinesin 13 family of proteins, kinesin 13-1 is exclusively intranuclear in both T. brucei and L. major, where it mainly localizes to the mitotic spindle and spindle poles and plays a central role in regulating the spindle assembly during mitosis [35,36]. As expression of the gene that encodes kinesin 13-1 was significantly down regulated in the LdPfn depleted Leishmania cells, we speculate that LdPfn might have been involved in regulating the spindle orientation by controlling the expression of kinesin 13-1 protein in these cells.
Based on our proteomic and transcriptomic data analyses, it appears that besides having a role in cell cycle regulation, LdPfn may also be involved in regulating the Leishmania mitochondrial activity. Leishmania has a single large mitochondrion, which is distributed in branches under the subpellicular microtubules and in a specialized region, called kinetoplast, it houses its unusual genome, known as kDNA [52]. Mitochondria in eukaryotic cells are responsible for oxidative phosphorylation, and harness energy from numerous substrates through electron transport chains. They are semi-autonomous cell organelles which have their own DNA and protein synthesizing machinery [52,53]. However, Leishmania mitochondrion is completely devoid of tRNA-encoding genes and hence, it imports nucleus-encoded tRNAs for protein synthesis [54] through a receptor-mediated pathway [55,56]. Several mitochondrial proteins that are involved in the electron transfer mechanisms and also the outer membrane protein, porin, which is the main metabolite channel and is required to support efficient oxidative phosphorylation in T. brucei [22], interact with LdPfn in Leishmania promastigotes, it may be envisaged that activity of these proteins would be adversely affected by depleting intracellular pool of LdPfn. This is consistent with our finding that expression of transcripts that encode for proteins involved in mitochondrial electron transport activities, mitochondrial protein folding, and protein import across the mitochondrial outer membrane was significantly decreased in LdPfn +/cells. Further, protein import in trypanosomatid mitochondrion is mediated by the archaic translocase of the outer membrane (ATOM) complex, consisting of six subunits [57,58]. Among them, expression of transcripts encoding ATOM14 and ATOM11 was significantly reduced in the LdPfn +/cells. As knockdown of ATOM14 in T. brucei has been shown to result in growth arrest and drastically reduced protein and tRNA import into the mitochondrion [59] and ATOM11, which is exclusively present in trypanosomatids and is known to have a direct role in mitochondrial tRNA import [60], These results strongly suggest that LdPfn could play an important role in regulating the protein synthesis and oxidative phosphorylation in Leishmania mitochondrion.
Finally, our earlier studies have shown that LdPfn binds actin, PLP motif-containing proteins and membrane phosphoinositides, especially PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3 [11]. As membrane phosphoinositides play a central role in regulation of cell signalling, membrane trafficking, actin remodelling, nuclear events and intracellular transport [61][62][63], depletion of LdPfn intracellular levels should affect LdPfn interactions with these lipids, which in turn should affect phosphoinositide metabolism and consequently their cellular functions, including regulation of actin dynamics, cellular metabolism and membrane signalling. This is well supported by our present and earlier findings that depletion of LdPfn in Leishmania cells not only affected the intracellular transport [11], but it appeared to affect also the expression of proteins that regulate cell signalling, membrane transport, and actin remodelling. It has earlier been suggested that cortical actin remodelling plays a key role during cytokinesis and early mitosis in eukaryotic cells [64]. However, in Leishmania cells, the role of actin dynamics has been shown only in the basal body and kinetoplast separation, cleavage furrow progression and flagellar pocket division [65]. Although no such aberrations were observed during cytokinesis of the LdPfn depleted cells, based on the presently available data, we cannot completely rule out the role of actin remodelling in cell division cycle of these cells.
Supporting information S1 Table. Primer list used for performing quantitative real-time PCR. (XLSX) S2 Table. Potential binding partners of L.donovani profilin. Four proteins were identified as present in all three GST-profilin pulldown assays, while 34 proteins were identified in at least two GST-profilin pulldown assays. Only proteins that were not detected in any of the GSTalone pulldown controls were considered. The protein IDs were given according to L. donovani BPK282A1 strain as downloaded from the TriTrypDB (version 51) database (www. tritrypdb.org). (XLSX) S3 Table. Statistics for RNA-Seq data sets. Reads alignment was done by Bowtie2, using the L. donovani genome (Leishmania donovani BPK282A1, NCBI taxon ID:981087) [17].
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2022-03-24T05:13:52.933Z
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2022-03-22T00:00:00.000
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}
|
202814600
|
pes2o/s2orc
|
v3-fos-license
|
Awareness of Chronic Kidney Disease among Patients Attending Tertiary Care Hospital in Bangladesh
Background: Patients with chronic kidney disease (CKD) are at increased risk of morbidity & mortality. Educational interventions aimed at empower-ing patients are successful in chronic disease management including CKD. Objective: To explore the awareness regarding CKD among patients attending in a tertiary care hospital in Bangladesh. Methodology: This was a descriptive observational study, which includes 100 adult patients attending the department of Medicine in Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh from January 2013 to June 2013. Data were collected on a pre-tested questionnaire by face-to-face interview to investigate awareness toward: 1) basic knowledge of personal health; 2) perceptions of factors increasing the risk of CKD; 3) knowledge of therapies to slow CKD progression; 4) perceptions of CKD increasing the risk of other medical conditions and 5) Among the participants, 32.8% had knowledge of increasing risk factor of CKD, 30.8% had knowledge of the method of slow progression of CKD, 30.3% had knowledge of conditions for increase risk of CKD and 41.7% respondent had knowledge of treatment of CKD. Conclusion: Most of the study participants had inadequate knowledge of CKD. Lack of CKD screening and educational programs have contributed to the inadequate patient knowledge about the condition.
Introduction
Chronic kidney disease (CKD) is increasingly being recognized as a global public health problem. The declaration of World Kidney Day and its annual observance remained us that CKD is common and harmful for almost all cross section of people [1]. There is some convincing evidence that CKD is treatable [2]. Prevalence of CKD has reached epidemic proportions with a range of 10% -13% population of USA [3], Canada [4], Japan [5], China [6], Taiwan [7], Iran [8], and India [9]. In some countries of Europe, the prevalence of CKD is 8% -10% [10] [11]. Recent data on the prevalence of early stage of CKD in India showed that 15% apparently healthy-looking Indian Central government employees are suffering from kidney disease [9].
As CKD is a silent disease which is treated as one of the leading causes of death worldwide, many developed countries have studied CKD awareness and developed guidelines and educational programs accordingly. Education to improve knowledge on CKD has been documented to play an important role in reducing this particular problem regardless of whether it is primary, secondary or tertiary prevention [12]. Varied risk factors have been reported in the awareness study on chronic kidney disease in different countries.
Bangladesh being a densely populated developing country, its health care budget is only 1.4% of gross national product (GNP) with the priority areas as population control, provision of clean drinking water and eradication of communicable disease. The treatment of non-communicable disease like chronic kidney disease (CKD) has low priority in Bangladesh because of government health policy and high cost of treatment [13]. Development of awareness through screening and educational programs is still in the stage of infancy. The important causes of CKD leading to kidney failure in South Asian region are chronic glomerulonephritis, diabetes and hypertension [13]. In Bangladesh, leading causes of end-stage renal disease (ESRD) are chronic glomerulonephritis (40%), diabetes (34%) and hypertension (15%) [14]. Patients are not aware of the importance of good control of these risk factors. Survey in a few rural, urban, disadvantageous population suggested that 18 Bangladesh [14]. About 30,000 patients are reaching end-stage renal failure every year in this country they need either dialysis or transplantation of kidney [14]. Out of 18% kidney patient, 11% have milder to severe form of kidney failure [14]. Increased CKD awareness over time in different countries and a recent increase in nephrology referrals suggested that these efforts may have some positive impact [15] [16] [17].
It has been observed that physicians other than nephrologists are less likely to recognize CKD and sometimes differ in their clinical evaluation of CKD [18]. A significant number of CKD patients are referred to nephrologists much later than it would have been appropriate [19]. Late evaluation of CKD patients by nephrologists, especially those presenting in end-stage renal disease (ESRD), is associated with suboptimal pre-dialysis care and treatment which ultimately increase mortality [19] [20] [21] [22].
Cancer screening studies have shown that patients with more knowledge and awareness of their diseases are more likely to follow methods that slow progression of the disease [23]. Disease educated patients are more likely to follow proper treatment and cope more successfully with their diagnosis and participate in health care decisions that affect their health [24].
Study on awareness and education of CKD is scarce in Bangladesh. Thus the present study was conducted to explore the awareness regarding CKD among patients attending in a tertiary care hospital in Bangladesh. The study was focused on the relationship between the awareness of CKD with age, occupation and educational status. The information generated out of this study will be useful in implying awareness program for CKD patients. The developed awareness program of the present study is expected to reduce CKD through early adoption of treatment and thereby will contribute to a considerable extent to prevent it from progression towards ESRD. Each of the patient's correct answer was given a score of 1 and therefore the maximum total score was 11. Knowledge scouring was leveled as; no knowledge = 0 score, low knowledge = 1 -5, moderate knowledge = 6 -9 and high knowledge = 9 -11 score. Those who answered 5 or fewer questions correctly were considered to have low knowledge on chronic kidney disease while those who answered 6-8 questions correctly were considered to have moderate knowledge while those who answered 9 -11 questions correctly were considered to have high knowledge on CKD. After editing and coding, the coded data were directly entered into the computer by using SPSS software release for Windows, version 16.0 (SPSS, Inc. Chicago. III). Data cleaning validation and analysis were performed using the SPSS software. Categorical data were presented as frequency,
Methodology
percentage and continuous variable were expressed as mean ± SD (standard deviation
Results
A total of one hundred (100) participants were included in this study. Of them, forty (40) were male and sixty (60) were female, among females most of them were housewives and male to female ratio was 1:1.5. Almost one third (32.0%) respondents were in 3 rd decade. Majority (43.0%) patients came from lowermiddle-income family. In all age groups, the majority (60%) respondents had low knowledge of CKD.
About the association between knowledge score with different age group, it was observed that majority patients (60%) had low knowledge score which was 9.0% in ≤20 years age group, 21 High knowledge score was found 5 (12.5%) in male patients and 6 (10.0%) in female patients respectively. The mean knowledge score was found 6.5 ± 6.7 in male patients and 6.0 ± 5.9 in female patients. The mean score difference was not statistically significant (p > 0.05) between two groups ( Table 2).
This study reveals that 82.0% of the participants believe that renal transplantation is the treatment of choice in CKD, 22.0% knows about dialysis is a treatment option of CKD, and as 21.0% believe that CKD can be treated by drugs (Table 8). Table 9 shows that 32.8% respondents believe that there are some factors that increase the risk of CKD, 30.8% know the issues that slow down the progression of CKD, 30.3% are aware of the conditions for increase of CKD, and 41.7% have knowledge about treatment of CKD. Table 6. Participants believe that factor increases risk of CKD (n = 100).
Discussion
This descriptive observational study was carried out with an aim to determine About the association between knowledge score with different age group it was observed that majority of the study patients (60%) had low knowledge score which was 9.0% in ≤20 years age group, 21.0% in 21 -30 years age group, 16.0% in 31 -40 years age group, 10.0% in 41 -50 years age group and 4.0% in >50 years age group patients. The mean knowledge score differences were not statistically significant (p = 0.240) among knowledge scores with different age groups in this current study. Leng C.W., et al. [25] showed majority of the respondents answered 3 to 5 questions correctly giving a mean score of 3.44 ± 1.53 and a median score of 3; Eighty (5.6%) respondents had no knowledge of kidney disease; Of these 80 respondents, most of them were > 40 years old (72.1%). Tan A.U., et al. [26] reported that only younger age was independent predictors of overall knowledge score. The current study is consistent with these above studies.
In this present study, it was observed that majority (31.0%) respondents were passed primary education level followed by 29.0% passed college & University level, 23.0% passed high school and 16.0% respondents had no education level.
Tan A.U., et al. [26] reported that nearly all participants (96%) in their study had at least completed high school education. In another study, Leng C.W., et al. [25] observed that 40.7% and 37.6% had secondary level and above secondary level of education respectively. Erick W. [27] obtained that 70% of the participants had acquired formal education of less than high school level. This low level of education could account for the inadequate patient's knowledge. Studies documented that a high correlation between educational attainment and health outcomes, as educated patients are more likely to allow proper treatment and cope more successfully with their diagnosis and participate in health care decisions that affect their outcome.
In this present study, it was observed that 31.0% respondents mention that they believe diabetes increases the risk of CKD, 32.0% hypertension, 12.0% fam- [28]. Most of the participants believed that a family history of chronic kidney disease increases the risk for CKD but a previous study in the United States by Tan A.U., et al. [26] showed that although most chronic kidney disease patients had a family history of chronic renal failure, they did not believe that it predisposed to chronic kidney disease. Tan A.U., et al. [26] 22.5% and 20.0% in male and female respectively and high knowledge score was found 5 (12.5%) in male patients and 6 (10.0%) in female patients. The mean knowledge score was found 6.5 ± 6.7 in male patients and 6.0 ± 5.9 in female patients. The mean score difference was almost similar between male and female participants. This result differs from the findings of Turner S. et al. [29], who had observed that younger and female participants had greater knowledge and awareness of CKD.
Regarding the associations between knowledge score and educational status, it was observed in this current study that low knowledge score was found in 10.0% respondents who had no institutional education, in 17.0% had primary educa- To summarize, the current study found that majority (60%) of the participant tion. This could be due to inadequate mass media involvement.
Conclusion
This study was undertaken to explore the awareness regarding CKD among the patients attending in a tertiary care hospital. Most of the respondents were in 3 rd decade and female predominant and they were mostly housewife. Majority of the study participants had inadequate knowledge of CKD. Lack of CKD screening and educational programs have contributed to the inadequate patient knowledge about the condition.
Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.
Limitations of Study
It was a single centre study with relatively small sample size.
|
2019-09-17T02:47:58.123Z
|
2019-08-08T00:00:00.000
|
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122014251
|
pes2o/s2orc
|
v3-fos-license
|
CANARD CYCLES AND HOMOCLINIC BIFURCATION IN A 3 PARAMETER FAMILY OF VECTOR FIELDS ON THE PLANE
Let the 3-parameter family of vector fields given by (A) y ∂ ∂x + [x + μ + y(ν0 + ν1x + x )] ∂ ∂y with (x, y, μ, ν0, ν1) ∈ R2 × R3 ([DRS1]). We prove that if μ → −∞ then (A) is C0-equivalent to (B) [y − (bx + cx − 4x + x)] ∂ ∂x + ε(x − 2x) ∂ ∂y for ε ↓ 0, b, c ∈ R. We prove that there exists a Hopf bifurcation of codimension 1 when b = 0 and also that, if b = 0, c = 12 and ε > 0 then there exists a Hopf bifurcation of codimension 2. We study the “Canard Phenomenon” and the homoclinic bifurcation in the family (B). We show that when ε ↓ 0, b = 0 and c = 12 the attracting limit cycle, which appears in a Hopf bifurcation of codimension 2, stays with “small size” and changes to a “big size” very quickly, in a sense made precise here.
Introduction
Let χ the set of C ∞ vector fields on the plane and χ 0 ⊂ χ, the set of vector fields with a singularity at (0, 0).
Consider the equivalences introduced by the following definitions.
a) X, Y ∈ χ are called C 0 -equivalent if there exists a homeomorphism h : R 2 → R 2 sending X-orbits to Y -orbits in a sense preserving way.b) X, Y ∈ χ 0 are called C ∞ -equivalent if there exists a diffeomorphism g : R 2 → R 2 , fixing (0, 0 Definition 1.2. a) A k-parameter family of vector fields on R 2 , X λ , with λ ∈ R k denoting the parameter, is defined to be a vector field a(m, λ) ∂ ∂x + b(m, λ) ∂ ∂y , m = (x, y) ∈ R 2 , where the coefficient functions a and b are C ∞ with respect to (m, λ) ∈ R 2 × R k .We say that X λ is a k-parameter unfolding of X 0 .b) X λ and Y µ , µ, λ ∈ R k , are (fibre) C 0 -equivalent if there exist homeomorphisms µ = φ(λ) and h λ : R 2 → R 2 such that h λ is a topological equivalence between X λ and Y φ(λ) .
. W 1 is the set of vector fields with hyperbolic singularity at (0, 0).One obtains for generic k-parameter families (with any k) the so called generalized saddle-node bifurcation of codimension k which is an unfolding of X 0 ∈ W 2 but whose restriction to center manifold starts with non-zero terms of order k.For generic k-parameter families (k ≥ 2) one finds the generalized Hopf bifurcation where such Hopf bifurcation of codimension k is a k-parameter unfolding of X 0 ∈ W 3 and whose radial component of the normal form in polar coordinates starts with non-zero terms of order 2k + 1.
The study of the unfoldings of X 0 ∈ W 4 starts with the Bogdanov-Takens bifurcation ( [RW]).If X 0 ∈ W 4 then, according [DRS1], If a = 0 we say that X 0 ∈ W 4 has singularity of the kind cusp.For the case a = 0, see [DRS2].
Figure 1 shows the intersection of the bifurcation set with the sphere H is composed of two parts separated by the curve H 2 (Hopf bifurcation of codimension 2).In one of them, when the parameter value crosses H, the focus changes the stability and we have the appearance of one repelling limit cycle.In the other part, the change of the stability gives the appearance of one attracting limit cycle.
When the parameter value crosses H 2 we have the appearance of two limit cycles, the inner one is attracting and the other is repelling.If (µ, ν 0 , ν 1 ) ∈ L (homoclinic bifurcation) then X µ,ν0,ν1 has a homoclinic orbit.The parameter values on L for which ∂P ∂x + ∂Q ∂y ( √ −µ, 0) = 0 give the curve L 2 .L is composed of two parts separated by the curve L 2 (homoclinic bifurcation of codimension 2).In one of them, the attracting limit cycles which appear along H disappear along L. In the other part, the repelling limit cycles which appear along H disappear along L.
The cycles which appear along H 2 disappear along L 2 .There exists a surface C where the attracting limit cycle and the repelling limit cycle coalesce.If (µ, ν 0 , ν 1 ) ∈ C then X µ,ν0,ν1 has a semistable cycle.For the parameter values on the region limited by H, L and C, X µ,ν0,ν1 has two limit cycles.On b 1 (δ) and b 2 (δ) we have the Bogdanov-Takens bifurcations corresponding to the cases (5 + ) and (5 − ).For the parameter values on H ∩ L we have simultaneous Hopf bifurcation and homoclinic bifurcation.
In this work we study the surfaces H and L for µ → −∞.
In section 2 we make some coordinate changes, such as the Liénard transformation, and prove that if µ → −∞ then (8 We denote F b,c the function given by ( 10) In section 2 we study the bifurcation set of the functions defined by (10).It is illustrated in Figure 2. In section 3 we study the singularities of the family (9).If ε = 0 then the singularities, with (b, c) fixed, are on the curve Except for the critical points, all the points on L b,c are normally hyperbolic singular points.The phase portrait is illustrated in Figure 3. and for (ε, b, c) = (0, 0, 4).
We have a special care to classify the singularity (0, 0) when b = 0 because in this case the associated eigenvalues have real part equal zero.We prove in section 3 the existence of Hopf bifurcation on H = {b = 0, c = 12} and Hopf bifurcation of codimension 2 on H 2 = {b = 0, c = 12}.The cycles are so that inner one is repelling and the other is attracting.We study the behaviour of the cycles for ε ↓ 0.
) and Γ n ⊂ R 2 , limit cycle (or homoclinic orbit) of X εn,bn,cn , such that Γ n → Γ b,c , for the Hausdorff distance between the compact sets in R 2 .For (ε, b, c) → (0, 0, 12) the possible limit periodic sets are those for which y is constant or y = F b,c (x).The limit periodic sets are known by "Canard Cycles".We use the term "canard" because the shape of the limit periodic sets of the Van der Pol's equation is like a duck ( [E]).Using the method introduced in [DR1], which consists in global desingularization and center manifolds, we prove Theorem 1.1.Let the family given by ( 9) and Γ 32 0,12 the compact set illustrated in Figure 5.There exist ε 0 > 0, δ > 0 and a surface give a homoclinic orbit which approaches Γ 32 0,12 for the Hausdorff distance.
The divergence at (2, F b,c (2)) determines the stability of the homoclinic orbit.We have We say that the homoclinic orbit is non-generic if the divergence at (2, F b,c (2)) is zero.
Theorem 1.2.Let the family given by ( 9) and Γ 0,4 the compact set illustrated in Figure 5.There exist give a homoclinic orbit L ε,b,c which approaches Γ 0,4 .Besides the limit of the parameter values for which X ε,b,c has nongeneric homoclinic orbits for ε ↓ 0 is (0, 0, 4).
The surface L is such that for b < −4c + 16, L ε,b,c is a repelling homoclinic orbit and for b > −4c + 16, L ε,b,c is an attracting homoclinic orbit.
Multiply ( 16) by −1 T and denote ε = 1 Multiply by 4 and put To simplify the desingularization, make the change and denote x and y again to get (9).Proof: The equation ( 21) is Figure 2 shows two sets of bifurcation: one defined by the equality of critical points and other by the equality of zeroes.
The Singularities of (9)
If ε = 0, the singularities of the vector field X ε,b,c , given by ( 9), are the points (0, 0) and ( 2 The equation of the eigenvalues is For ε > 0 the singularity (2, 2b + 4c − 16) is a saddle.The saddle is a critical point of F b,c for the parameter values in the plane given by (12).Theorem 3.1.Let X ε,b,c given by ( 9).We have: Proof: Make the change and denote y = y 1 to get Make the change The phase portraits of ( 30) are the graphics of We have Thus, according [ALGM], we get (36) Define the returning map π : R + → R by ( 37) The Lyapunov coefficients are given by ( 38) The computation, using maple, gives When b = 0 we have V 1 = V 2 = 0 and V 3 = 0 if and only if c = 12.Besides V 3 > 0 for c > 12 and V 3 < 0 for c < 12. Thus X ε,b,c has a weakly attracting focus for c > 12 and a weakly repelling focus for c < 12.For c = 12 we have (40) Thus, (0, 0) is a weakly attracting focus.
The Phase Portrait of X ε,b,c
First we are going to study the phase portrait of X ε,b,c given by ( 9) when ε = 0.In this case we have For b and c fixed the singularities of ( 41) are the points (x, y) such that y = F b,c (x).Except for the the critical points, all the points are normally hyperbolic singular points.Figure 3 shows the phase portrait for some values of (b, c).
For ε = 0, when x = 0 and x = 2 cross transversally the graphic of y = F b,c (x) in non-critical points we can use the R. Lutz and M. Goze lemma ( [LG]) to sketch the phase portrait of X ε,b,c .The trajectories are so that ( 42) Thus the vector field is almost horizontal for ε ↓ 0.
The main difficulty to obtain the whole phase portrait is the number of limit cycles.
Lemma 4.1 (Copell). Let the vector field given by
Then there is no limit cycle in ξ 0 < x < β.
The proof of the Copell's Lemma uses a symmetry in the line where the divergence is zero ( [DR2]).
Consider b = 0 and c ∈ R and suppose that X ε,b,c has a limit cycle Γ ε , then either the limit cycle disappear or the limit periodic set will be (0, 0) for ε ↓ 0. In fact, if b = 0 the singularity (0, 0) is not a critical point.
The Desingularization of
given by ( 47) There exists X on [0, T ] × K such that ϕ * ( X) = X ε,b,c .Dividing X by u the vector field resulting X will be "the desingularized vector field" in [0, T ] × K.
The desingularization at
Consider the map δ : [0, T ] × S 4 + → R 5 given by ( 69) For b = c = u = 0 we have It has no singular point.Using again the same steps we have for θ 1 and θ 2 solutions of s + 2c 2 = 0.
Let the change Thus ( 56) becomes Let F (X, Y ) given by ( 79) We have that F (X, Y ) is a first integral of (78).In fact Let G(x, y) and H(x, y) given by ( 81) We have ( 82) is the integrating factor and the hamiltonian H is given by ( 81).Now we consider P (1,0,0) defined in section 5. We have that P (1,0,0) is a 3-dimensional space and we can look a point in P (1,0,0) with coordinates (u, v, θ), indicates in the Figure 6.For u = 0, we denote D the set associated to v = 0. Let R a rectangle on P (1,0,0) with one side r on D and such that R is transversal to ∂P .We take R such that the connection c joing (θ 1 , 0) and (θ 2 , 0) is transversal to r at its middle point.Let S a subrectangle in R with one side s on D and such that c ∩ r is the middle of s.
. We take C N , the sature of N , that is, the closure of the union of segments of orbits of X (u,v,θ) through the points on N and taken between the first intersection of this trajectory with S in negative time and with R in positive time.
Let w u,b,c the dual 1-form associated to (48 The homoclinic orbits γ associated to the parameter values on S are attracting because the parameter is near of (0, 0, 12) (see ( 12)).In order to study the stability of the limit cycles γ associated to the parameter values on S 32 we must to compute γ div X ε,b,c .
We aproach the integral (92) The computation, using maple, gives The values given by (93) attest that the limit cycles are attracting for the parameter values (ε, b, c) ∈ S 32 .
Remark 6.1.The "Canard phenomenon" consists in a rapid variation of the shape of the periodic orbits in function of the variation of the parameter.According Theorems 1.1 and 3.1 one can find X ε1,b1,c1 and X ε2,b2,c2 two perturbations of X 0,0,12 such that: a) The phase portrait of X ε1,b1,c1 has two limit cycles contained in a small neighbourhood of (0, 0), the inner one is repelling and the other is attracting.b) The phase portrait of X ε2,b2,c2 has an attracting limit cycle contained in a small neighbourhood of Γ 32 0,12 (see Figure 5).
Proof of Theorem 1.2.The surface L is obtained implicitly of the same way that canard surface.To make it we consider γ = {(2, F b,c (2)) | b, c ∈ R, 0 ≤ ε ≤ ε 0 } composed by saddle points of X ε,b,c .Saturing γ by the flow of X ε,b,c we have that the intersections of the manifolds define the homoclinic bifurcation surface.
We need to precise the codimension of the non-generic homoclinic loops.We start with the desingularization of X ε,b,c in (x, y, ε, b, c) = (0, 0, 0, 0, 4).Let the change The equation R(h) = 0 defines the surface L and besides R (h) is finite if and only if the divergence at the saddle point is zero [R].Thus R (h) is finite if and only if b = −4c.To prove that the homoclinic loop is of codimension 2 we need to prove that R (h) = 0. We use the map R(h) = b + u I3(h) I1(h) and we have (100) R (h) = u I 3 (h) I 1 (h) .
With a similar argument used in [CW], one can prove that (101) I 3 (h) I 1 (h) < 0 for h < e.It follows that R (h) < 0.
c = 0 and b = 0 or a = c = 0 and b = 1
Figure 2 .
Figure 2. The Bifurcation Set of F b,c .
|
2018-12-05T16:58:56.188Z
|
1999-01-01T00:00:00.000
|
{
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"oa_url": "https://ddd.uab.cat/pub/pubmat/02141493v43n1/02141493v43n1p163.pdf",
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233699772
|
pes2o/s2orc
|
v3-fos-license
|
An Adult Case of Heart Failure due to Left Main Coronary Artery Atresia
Left main coronary artery (LMCA) atresia is a rare congenital heart disease and can be fatal in pediatric patients. We report an adult case of LMCA atresia, in which heart failure developed without episodes suggesting angina. A 40-year-old man presented with difficulty breathing. Echocardiography revealed diffuse hypokinesis of the left ventricle with an ejection fraction of 22% in the absence of significant valvular disease. A diagnosis of heart failure was made, and diuretics, enalapril, bisoprolol and warfarin were administered. Coronary angiography demonstrated no trace of the ostium of the LMCA in the sinuses of Valsalva; the middle to distal part of the LMCA was visualized by rich collateral flow from the right coronary artery to the left anterior descending coronary artery and left circumflex coronary artery. No trace of the ostium of the LMCA from the aorta or main pulmonary artery was detected on computed tomography angiography or echocardiography. The patient underwent coronary artery bypass grafting and a final diagnosis of congenital atresia of LMCA was made. The clinical course was uneventful and computed tomography angiography, performed 5 days after surgery, showed a patent bypass graft. This case demonstrates the importance of considering LMCA atresia even in the absence of chest symptoms suggesting angina in patients with heart failure.
Introduction
Congenital anomalies of the coronary arteries are not uncommon and are non-fatal in most cases such as ectopic origin of the coronary arteries from the aortic sinus of Valsalva or separate ostia of the left coronary arteries [1,2]. However, attention should be paid to anomalies related to the left main coronary artery (LMCA) because it plays an essential role in providing blood supply to the majority of the left ventricle. We report an adult case of LMCA atresia in which heart failure developed without episodes suggesting angina.
Case Report
A 40-year-old man was referred to the department of cardiology of our hospital for difficulty breathing. The patient had been in his normal state of health until approximately 2 weeks before presentation, when exertional dyspnea developed and gradually progressed. His previous medical history included hypertension, dyslipidemia and gout. He did not take any medications. The patient did not drink, smoke, or use illicit drugs, and had no known allergies. There was no family history of cardiovascular diseases.
On examination, he was alert and well. His vital signs were normal except for a blood pressure of 130/111 mm Hg. The jugular venous pressure was not high, but gallops were audible at the apex. No pulmonary rales were heard on auscultation and there was no edema in the legs. Electrocardiography demonstrated left ventricular hypertrophy and left atrial dilation. Chest radiography revealed cardiomegaly with a cardiothoracic ratio of 55% without pleural effusion. The complete blood cell counts were normal. Levels of total bilirubin, aspartate aminotransferase, alanine aminotransferase, creatinine, uric acid and C-reactive protein were 1.7 mg/dL, 110 U/L, 132 U/L, 1.67 mg/dL, 33 mg/dL and 0.43 mg/dL, respectively. Although the creatinine kinase level was 540 U/L, the MB enzyme level was 20 U/L and the high-sensitivity cardiac troponin T level was 0.020 ng/mL (reference value < 0.100). The level of brain natriuretic peptide was high at 734.9 pg/mL (reference value ≤ 18.4).
On echocardiography, the left ventricle was diffusely hypokinetic with an ejection fraction of 22% and enlarged with an end-diastolic diameter of 56 mm, accompanied by mild mitral regurgitation, findings consistent with dilated cardiomyopathy. The transmitral E/A ratio was 2.73 and the E/early diastolic mitral annular velocity ratio of the intraventricular septum was 22.77. The peak velocity of the tricuspid regurgitant was 3.7 m/s and the systolic pulmonary artery pressure was estimated to be 63 mm Hg. The left atrial volume index was high at 48 mL/m 2 (reference: 17 -32).
A diagnosis of heart failure was made, and diuretics, enalapril, bisoprolol and warfarin were administered. The contractility of the left ventricle improved to an ejection fraction of 52% and pulmonary hypertension disappeared. Warfarin was The patient underwent coronary artery bypass grafting via an in situ left internal thoracic artery to the mid-left anterior descending artery at another hospital. This surgery was performed through an off-pump approach via left mini-thoracotomy with no signs of myocardial ischemia during the surgery and a final diagnosis of congenital atresia of LMCA was made. The clinical course was uneventful and CT angiography, which was performed at 5 days post-surgery, demonstrated a patent bypass graft and no LMCA with collateral arteries from the right coronary artery (Fig. 4).
Discussion
The current patient was diagnosed with heart failure at the age of 40 years. Dilated cardiomyopathy was initially suspected as the etiology, but congenital LMCA atresia was later con- firmed. This case is notable because the patient was diagnosed in adulthood, there was a lack of chest pain suggestive of myocardial ischemia and scintigraphy findings were abnormal. Occlusion of the LMCA can be congenital or acquired. Considering the incidence in the general population and his lipid profile, atherosclerosis was initially considered the most likely cause for the LMCA occlusion despite the absence of chest pain. However, no stump of the LMCA ostium in the sinuses of Valsalva was detected on multiple modalities, including echocardiography, CT angiography and invasive angiography. Furthermore, no atherosclerotic lesion was observed in the coronary arteries except for the LMCA. A diagnosis of congenital LMCA atresia was made based on findings obtained during coronary artery bypass grafting.
The incidence of coronary artery anomalies is estimated to be less than 1% in the general population, although it varies depending on the definition such as including separate ostia of the left anterior descending and circumflex arteries without the LMCA, myocardial bridge, a high take-off of the coronary ostium and a hypoplastic right or circumflex coronary artery [3][4][5]. Although the exact incidence of LMCA atresia remains unclear, this condition is considered a markedly rare coronary anomaly that differs from a single coronary artery [6]. LMCA atresia has retrograde blood flow via collateral circulation from the right coronary artery, whereas the blood flow is antegrade in patients with a single coronary artery. In a series of more than one hundred thousand coronary angiograms, coronary anomalies were observed in 1.33%, including 0.04% in a single coronary artery, but no case of LMCA atresia was found [7].
Bland-White-Garland syndrome or ALCAPA syndrome (i.e., anomalous origin of the left coronary artery from the pulmonary artery) should be included in the differential diagnosis for LMCA atresia [8,9]. In the current case, no evidence suggesting the presence of the left coronary artery from the pulmonary artery was obtained on echocardiography focused on the pulmonary artery or cardiac CT. However, the right coronary artery may provide extensive collateral flow to the left anterior descending and circumflex arteries, leading to the disappearance of the LMCA originating from the pulmonary artery if patients survive to adulthood [10,11]. This possibility was excluded in this case based on the findings during coro- Figure 3. On dual isotope scintigraphy, the Tl-201 bull's-eye map shows reduced tracer uptake in the anterior region to the apex on the initial image with fill-in on the delayed image or 3 h after injection, findings consistent with ischemic but viable myocardium in the region. Similarly, on the I-123 BMIPP bull's-eye map, decreased fatty acid metabolism (i.e., myocardial ischemia) is noted in the same area. Note that reduced tracer uptake on the initial images is milder with I-123 BMIPP than with Tl-201.
Adult Case of HF due to LMCA Atresia J Med Cases. 2021;12(6):233-237 nary artery bypass grafting. LMCA atresia is considered to be associated with a poor prognosis because the blood flow to the left coronary arteries is insufficient for the left ventricle [6]. The majority of patients with LMCA atresia are symptomatic, and sudden cardiac death occurred in 10% of pediatric patients and 7% of adult patients [11]. The current patient developed heart failure with reduced ejection fraction, which was managed by medication. Based on the myocardial scintigraphy findings, recovered left ventricular ejection fraction and lack of chest pain, myocardial ischemia was considered minimal with optimal medical treatment. This may be explained by rich collateral flow from the right coronary artery to the left coronary arteries on angiography. Furthermore, this may explain why the current patient survived without symptoms until the age of 40 years. Similarly, there are reports of adults diagnosed with LMCA in their 40s or later [12,13].
In this case, the uptake of the I-123 BMIPP tracer was greater in the initial images than that of Tl-201. This phenomenon is called paradoxical mismatch and considered to result from an artifact, especially when observed in the inferior wall due to attenuation and infra diaphragmatic scatter [14]. This is also observed in the non-hypertrophied regions of patients with hypertrophic cardiomyopathy [15]. Both conditions are less likely to explain the paradoxical mismatch between I-123 BMIPP and Tl-201 observed in the current case. Of note, the myocardial damage assessed by dual scintigraphy using Tl-201 and I-123 BMIPP was less severe in the current patient than in patients with Bland-White-Garland syndrome [16,17], although the exact mechanisms remain to be elucidated. Further studies are required to evaluate the utility of dual scintigraphy for differentiation.
In conclusion, this case demonstrated the importance of considering LMCA atresia even in the absence of chest symptoms suggesting angina in patients with heart failure.
|
2021-05-05T00:08:21.488Z
|
2021-03-24T00:00:00.000
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232294580
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pes2o/s2orc
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v3-fos-license
|
Preparation of Cellulose/Laponite Composite Particles and Their Enhanced Electrorheological Responses
Cellulose, as a natural polymer with an abundant source, has been widely used in many fields including the electric field responsive medium that we are interested in. In this work, cellulose micron particles were applied as an electrorheological (ER) material. Because of the low ER effect of the raw cellulose, a composite particle of cellulose and Laponite was prepared via a dissolution–regeneration process. Scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) were used to observe the morphologies and structures of the composite particles, which were different from pristine cellulose and Laponite, respectively. The ER performances of raw cellulose and the prepared composite were measured by an Anton Paar rotational rheometer. It was found that the ER properties of the composite were more superior to those of raw cellulose due to the flake-like shapes of the composite particles with rough surface. Moreover, the sedimentation stability of composite improves drastically, which means better suspension stability.
Introduction
Electrorheological (ER) fluids are a kind of smart complex fluid which can respond to an electric field and show electric-field-controllable viscosity [1,2]. They usually consist of dielectric particles and insulating oil. The significant increase in viscosity of ER fluids is aroused by polarization and alignment of the dielectric particles in the direction of the electric field, which also results in a state transition from fluid-like to solid-like upon the stimuli of the electric field. These electric-field-controllable properties of ER fluids give them application potential in the fields of shock absorbing [3], damping [4], braking [5], finishing [6] etc.
In ER fluids, silicone oil is normally applied as the carrier liquid due to its good stability and diverse accessible viscosities. While the range of dielectric particles applied in ER fluids are wide, covering many kinds of materials like inorganics [7,8], semi-conducting polymers [9], natural polymers [10] and polyelectrolytes [11], for real applications, the field-induced change in viscosity or shear stress, which is called ER effect, is the main evaluation criterium of ER fluids. Thus, high and stable ER effect is what researchers want to pursue in the study of ER Fluids. For this purpose, one of the strategies is to design hybrid or composite materials to improve the dielectric polarizability of the dispersed particles. In addition, easy accessibility or facile synthesis of dispersed particles is also necessary for the wide use of ER fluids. From this point of view, natural polymers are attractive candidates as ER materials because of their abundant source, low price, available chemical modification and biodegradability. To date, various natural polymers including cellulose [12][13][14], chitosan [15,16], starch [17,18], and algae [19,20] particles have been applied as ER materials. However, most of them exhibited relative low ER effects compared with some outstanding inorganic ER materials. To improve the ER effect of natural polymerbased ER materials, chemical modification is usually used. Choi et al. reported phosphate cellulose as an anhydrous ER material which showed high ER effect [21]. Ko et al. also applied acid and acid/urea complex groups to modify chitosan particles to improve their ER effect [22]. It is also reported that the ER effect of chitosan depends on the degree of deacetylation [23].
Comparing with chemical modification, introducing inorganic nanomaterials into polymer matrix (or particle blending) is a facile and effective way to improve the ER properties of polymers, which also brings other superiorities, for example enhanced thermal stability [24]. When semi-conducting polymer is used as the matrix, in situ polymerization [25] and Pickering emulsion polymerization [26,27] have been widely used in preparing inorganics blended composite ER materials. In addition to silica and titania nanoparticles, 2-dimentional nanosheets such as clay and graphene (or graphene oxide) are the mostly applied inorganics. For natural polymer-based ER materials, blending with inorganics is rarely used because of the solid nature and high molecular weight of the natural polymers. Hu et al. reported a new ER material of chitosan-decorated graphene nanosheets by microwave-assisted treatment [28]. It implies the possibility of preparing inorganics-blended natural polymer particles as ER material.
Cellulose is a polysaccharide with linear chain of D-glucose units and grafted -OH groups. Because of its abundance of origin and sustainability, accessibility of chemical modification, low cost and non-toxicity, cellulose has attracted much attention both in research and engineering applications. Polar groups of -OH in cellulose provide it with electro-responsive properties as well as accessibility of reaction. That is why cellulose has been applied as ER materials. In the recent development in the processing of cellulose, dissolution-regeneration has become a useful way to prepare cellulose/inorganics composite materials by introducing inorganic nanomaterials into the cellulose solution [29,30], which also gives us inspiration for the preparation of new cellulose-based ER materials. In this study, we introduce Laponite, a synthetic clay type with 2-dimentional disk shape (thickness: 1 nm; diameter: 25 nm), into cellulose particles by the dissolution-regeneration process of cellulose. In a previous study, Laponite as well as many other kinds of clays were used as ER materials separately or blended with polymers [31][32][33]. The special layered structures of clays played significant roles in preparing composite particles with intercalated, exfoliated, and even core-shell structures [34][35][36]. Herein, because of the existence of Laponite nanosheets, the arrangement of cellulose polymer chains in regeneration process is hindered, which resulted in different crystal structures in the composite. In addition, the dielectric properties of Laponite can also contribute to the ER effect of the composite particles.
Results and Discussions
The morphologies of cellulose, Laponite and the cellulose/Laponite composite particles are shown in Figure 1 Because of the fast precipitation and vigorous stirring, the regenerated cellulose particles easily form porous structures with rough surfaces which has been observed in previous study [37]. Herein, the composite particles of cellulose/Laponite shown in Figure 1c are flake-like with larger particle size than raw cellulose. In addition, compared with the relatively smooth surface of raw cellulose, those composites are extremely rough with many pores and nanoparticles on the surface. [38][39][40]. According to the 2θ of (001), the basal spacing of Laponite is calculated to be 2 nm, which is much larger than the theoretical value of 0.96 nm. This may be due to the moisture that is absorbed in the interlayer of Laponite. After compounding, the main peaks of the composite are at 2θ = 12.1 • , 19.9 • and 21.8 • , indicating that, after alkali treatment, cellulose I transforms into the form of cellulose II [37]. The sharp peak observed in the XRD pattern of Laponite at 2θ = 4.4 • disappears, which means the cellulose chains are intercalated into the interlayers of clays. Other characteristic peaks of Laponite are observed in the XRD pattern of the composite, which confirms the successful blending of Laponite with cellulose. Another obvious characteristic of the composite is that there is an apparently sharp less peak at 35 • compared with the peak that is shown by raw cellulose. Based on the Scherrer Formula, a sharper peak means a higher degree of crystallinity and better orientation. The reduced diffraction intensity is due to the introduction of Laponite in the matrix of cellulose, which hinders the re-generation of highly oriented crystalline areas of the cellulose matrix. The thermogravimetric curves of Laponite, raw cellulose and their composite are shown in the Figure 2b. For the three samples, less than 5% weight loss is observed before 120 • C caused by the loss of absorbed water. As the temperature reaches 400 • C, large weight loss up to 50% and 70% appears in the curve of the cellulose/Laponite composite and raw cellulose, respectively, due to the degradation of the carbohydrate backbone. For Laponite, there is no obvious weight loss in this temperature range because of the high heat resistance of phyllosilicates. As the temperature increases to 800 • C, the residual mass for each sample is about 88% (Laponite), 43% (cellulose/Laponite) and 22% (cellulose). The higher residual mass of the composite than that of raw cellulose confirms that Laponite is introduced to the cellulose matrix. Figure 3 presents the shear viscosity of the ER fluids measured in the shear rate range of 0.01-1000 s −1 . Before application of an electric field (E = 0 kV/mm), the solid cellulose or cellulose/Laponite particles randomly dispersed in silicone oil. Thus, both ER fluids are more like Newtonian fluid with a constant viscosity when shear rate exceeds 1 s −1 . At higher shear rate, the viscosity of the ER fluid of cellulose/Laponite composite is lower than that of the cellulose ER fluid, which may be related to the flake-like morphology of the composite or the affinity between the composite and silicone oil. As the electric field is applied, the suspended particles are polarized and align in the direction of electric field driven by the electrostatic interaction between particles. The inner fibril structures result in sudden increase in shear viscosity of the ER fluids. When the rotor starts to shear, the particle chains or columns incline to the direction of shear flow gradually and are sheared into chain segments as the shear rate becomes higher. That is the reason both ER fluids show obvious shear thinning phenomena in shear viscosity curves. For each ER fluid, the value of shear viscosity is enhanced significantly by increasing electric field strength. When comparing the two ER fluids, it can be seen that, at an electric field strength, the viscosity curve of the composite ER fluid is much smoother and higher, indicating that the composite particles can form more robust chains under an electric field. Figure 4 shows the shear stress of two ER fluids as a function of shear rate (0.01-1000 s −1 ). When the electric field is zero, the same as has been observed in viscosity curves, two ER fluids show the characteristics of Newtonian liquids in that the shear stress has a linear relationship with the shear rate. When an electric field is applied, the shear stress of cellulose ER fluid (Figure 4a) shows a plateau value in the low shear rate region (0.01-1 s −1 ) and then increase with the shear rate in the high shear rate region (1-1000 s −1 ). This is because low shear rate cannot destroy the particle chains totally due to the disrupt-rebuild competition process of particle chains. The increase in shear stress at high shear rate is attributed to the strong hydrodynamic force of shear flow which depends on shear rate and breaks the particle chains totally. For the ER fluid of the cellulose/Laponite composite (Figure 4b), the shear stress curves are independent of the shear rate. A steady plateau region over the whole shear rate range is observed for each shear stress curve. It means the electrostatic force between the composite particles dominates in the steady shear process with controlled shear rate. It is different from that which has been observed in the cellulose ER fluid. In addition, the shear stress values for the ER fluid of cellulose/Laponite are much higher than that of the cellulose ER fluid. To analyze the shear stress curves further, we use a constitutive equation to fit the shear stress curves of the two ER fluids. It is a six-parameter-equation named the Cho-Choi-Jhon (CCJ) model and has played a significant role in analyzing flow curves of ER fluids in previous studies, especially for the flow curves with typical shear rate dependent characteristics [41,42]. The equation of the CCJ model is written as follows: where τ y and η ∞ present the dynamic yield shear stress and viscosity at infinite shear rate, t 2 and t 3 are both time constants, α and β are the parameters to adjust the shape of the fitting curves. The fitting values of these important parameters are shown in Table 1. It can be seen that the CCJ model can fit the flow curves of the two ER fluids very well regardless of whether the curves depend on shear rate or not. The dynamic yield stress (τ y ) obtained from the fitting results is the crucial criterion to evaluate ER fluids. It greatly depends on the components of ER fluids and also exhibits close relation with electric field strength (E). As shown in Figure 5, the dynamic yield stress increases stepwise with E. It has been found to be a power law dependence, described as τ y ∝ E m with a power exponent m [43,44]. The parameter m in this equation represents the ability of response to the electric field, the value of which ranges from 1.0 to 2.0 is mainly associated with the characteristics of the suspended particles. The dynamic yield stresses of the two ER fluids are replotted as a function of E in Figure 5. After fitting by the power law equation, the power exponent m is 1.45 for the raw cellulose ER fluid and 1.85 for the cellulose/Laponite composite ER fluid. It indicates that the composite has a higher field-dependent increase in dynamic yield stress. Therefore, it is obvious that both yield stress and electric-field-dependence of the composite ER fluid are enhanced compared with the ER fluid of raw cellulose. It may be related to the morphological and chemical structures of the particles. It can be observed from Figure 1 that the composite particles have a rough surface which will enhance the interfacial polarizability of the particles. In addition, the intercalated Laponite nanosheets in cellulose also have a positive effect on electro-responsive properties of the composite particles because of their own ER properties [45,46], which has been confirmed in a previous study [36]. Figure 6 illustrates the rheological properties of the ER fluids of cellulose and cellulose/Laponite composite particles, measured in a dynamic amplitude sweep mode with a constant frequency of 10 rad/s. The storage modulus (G') and loss modulus (G") can be obtained in the amplitude oscillatory tests. According to the dependence of G' and G" on amplitude, the linear viscoelastic (LVE) region of the ER fluids under the electric field are defined. It is the strain range where G' is independent of strain because of the elastic deformation of the system. As shown in Figure 6a,b, when the electric fields are not applied, the values of G' and G" are very low and almost equivalent before the strain of 0.1%. It means the ER fluid without the stimuli of the electric field is more like a viscoelastic liquid. As mentioned above, after applying an electric field, the particles in ER fluids form chains or columns [47,48]. That is why G' increases by several orders of magnitude and remains constant until strain amplitude reaches a certain value, which is the LVE region. A similar LVE region up to 0.03% is detected for the two ER fluids. In addition, compared with G", G' becomes predominant before a critical strain, indicating that the system performs like viscoelastic solid because of the fibril structures formed by the particles. As the electric field is higher than 0.5 kV/mm, both G' and G" of the cellulose/Laponite ER fluid are higher than that of the cellulose ER fluid, which also implies that more robust chain structures are constructed by the composite particles under the same electric field strength. The results of the frequency sweep are illustrated in Figure 7. A constant strain of 0.003% in the LVE range is used to make sure that the structure of the viscoelastic solid is not destroyed in the oscillatory shear. The tests are conducted in the angular frequency range of 1-100 rad/s. On one hand, without electric fields, the G" increase with frequency and is higher than G' in the whole frequency range (Figure 7a) or after a critical value (Figure 7b), showing the liquid characteristic of ER fluids. On the other hand, G' and G" of the ER fluids are nearly constant at an applied electric field and G' is much higher than G" over the entire frequency range, showing the viscoelastic solid properties. It is also observed that the G' and G" of the cellulose/Laponite ER fluid are clearly higher than those of the raw cellulose ER fluid, indicating that stiffer chains or columns are formed in the cellulose/Laponite ER fluid. The instant response of ER fluids can be gained through square-wave pulse voltage measurements. Figure 8 shows the shear stress of cellulose and cellulose/Laponite ER fluids at a shear rate of 1 s −1 and a square-wave pulse electric field (0.5-3 kV/mm) to observe the switching effect or sensitivity of the ER fluids to electric field. Because of the reversible liquid-solid transition in the ER fluid, the square-wave pulse in shear stress or viscosity can be observed according to the electric field in the same pattern. It can be found that the shear stress of the cellulose/Laponite ER fluid jumps up and down immediately at the switching point of the electric field, and shows a stable plateau value during each switch-on and switch-off period. For the raw cellulose ER fluid, climbing points are observed in the shear stress curve once the power supply is turned on, which is followed by lower and unstable shear stress. It confirms that the composite particles are more sensitive to the electric field than the raw cellulose particles. Sedimental stability is a significant performance of ER fluids in practical application. Because of the density mismatch between the suspended particles and carrier liquid, the particles of ER fluids are apt to settle down towards the bottom of container. Herein, the density of the raw cellulose and cellulose/Laponite particles are 1.62 and 1.55 g/cm 3 , both of which are higher than that of silicone oil (density: 0.96 g/mL). However, the density of the composite particles is lower. As shown in Figure 9 (inset), both raw cellulose and the composite ER fluids are white liquids at the beginning. Then it is found that the cellulose particles settle down immediately in a few minutes and an upper supernatant part (a) is observed. The sedimentation ratio is defined as the percentage of the lower opaque part (b) to the total height of the liquid (a + b). For the composite ER fluid, an obvious supernatant layer is not observed until 40 min passes. While in this period, the sedimentation ratio of the raw cellulose ER fluid decreases rapidly and reaches 70%. Then in the next stage (40-160 min), the sedimentation ratio approaches nearly 50% for the raw cellulose ER fluid and is more than 90% for the cellulose/Laponite composite ER fluid. It is obviously that the sedimentation rate of the composite particles is much slower than that of raw cellulose particles, implying that the ER fluid of cellulose/Laponite has better suspension stability. One of the reasons for the improved anti-sedimentation property of the cellulose/Laponite particle is its lower density. The other is its flake-like shape and rough surface, both of which enhance the resistance suffered by the particle in the settling process.
Preparation of Cellulose/Laponite Composite Particles
An amount of 1.0 g of Laponite was added into 100 mL water in batches at room temperature with the condition of intense stirring to form the stable sol system. An amount of 5.0 g of pristine cellulose was dispersed in an aqueous solution of NaOH (10 wt%) and precooled at the −10°C. After vigorous stirring, cellulose swelled in the alkali solution and formed a semitransparent and homogeneous solution. The Laponite hydrates were added into the cellulose system drop by drop at the stirring speed of 500 r/min. The composites of cellulose/Laponite were precipitated by adding alcohol and washed with deionized water several times. After freeze-drying, a white powder was obtained. A schematic process for preparing the cellulose/Laponite composite particles is shown in Scheme 1.
Characterization and Rheological Measurement
Morphologies of the raw cellulose, Laponite and the composite particles were observed by a scanning electron microscopy (SEM) (S4800, Hitachi, Tokyo, Japan). The crystal structures of the samples were characterized using a powder X-ray diffraction pattern (XRD) (MAX-2500PC, Rigaku, Tokyo, Japan) with a Cu-Kα radiation source. The thermal stability of the samples was determined using a thermogravimetric analyzer (TGA) (STA4993, Netzsch, Germany) with a heating rate of 10 • C/min in the temperature range of 25 to 800 • C in an air atmosphere.
Before rheological measurement, ER fluids of raw cellulose and cellulose/Laponite particles were prepared by dispersing the particles in silicone oil, respectively. Two uniform suspensions with the same mass fraction of 20% were obtained after ultrasonication. The rheological properties of the ER fluids were measured by a commercial rheometer MCR 502 (Anton Paar, Graz, Austria) with a concentric cylinder (CC) geometry and a DC power supply. The ER fluid was loaded between the gaps of the CC geometry. As the electric field was applied between the gaps, the field-induced change in rheological properties of the ER fluids could be sensed by the rotor of the rheometer. Steady shear flow and dynamic oscillation tests were used to observe the viscosity, shear stress and dynamic modulus of the ER fluids before and after the stimuli of an electric field.
Conclusions
We prepared cellulose/Laponite composite particles via a facile dissolution-regeneration method, during which process the Laponite clay was added in cellulose solution and restricted in the regenerated cellulose particles by adding a pore solvent. Compared with raw cellulose, the hybrid clay composite particles showed better ER effects, including higher shear stress, higher modulus, and faster response to electric fields. The enhanced ER effect of the cellulose/Laponite composite particles compared with raw cellulose is attributed to the inserted Laponite nanosheets into cellulose and the specific morphologies of the composite particles: flake-like shape with a rough surface. It was also found that the sedimentation stability of the composite ER fluid is significantly improved because of the surface morphology and the lower density of the particles.
|
2021-03-22T17:38:13.016Z
|
2021-03-01T00:00:00.000
|
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211109771
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pes2o/s2orc
|
v3-fos-license
|
The BMP ligand Pinhead together with Admp supports the robustness of embryonic patterning
The “seesaw”-like expression of pinhead and admp establishes an alternative mechanism to ensure embryonic patterning.
INTRODUCTION
Bone morphogenetic proteins (BMPs), originally identified by their ability to induce ectopic bone formation, are multifunctional extracellular polypeptides that belong to the transforming growth factor- (TGF-) superfamily (1). Secreted BMP ligands bind as dimers to type I and type II receptors on the cell surface. The type II receptors become phosphorylated and then activate the type I receptors, which in turn phosphorylate the regulatory Smads (Smad1/5/8) (2). These phosphorylated Smads form complexes with Smad4, which then translocate into the nucleus to regulate the expression of BMP target genes (2). In zebrafish, bmp2b and bmp7a, which function as BMP heterodimers that activate Smad1/5, are initially expressed throughout the blastoderm shortly after the midblastula transition (3). BMP signaling in dorsal regions is subsequently attenuated by the BMP antagonist Chordin (Chd), and then a BMP signaling gradient forms along the dorsoventral (DV) axis and patterns tissues with high levels ventrally and low levels dorsally during late blastula stages and before the onset of gastrulation (3,4). Although many positive and negative regulators of BMP signaling have been identified during early embryonic development (4), the molecular network that generates and maintains the BMP gradient is still not well characterized.
The formation of a morphogen gradient is a dynamic process and is influenced by the kinetics of morphogen production, diffusion, and degradation. During embryonic development, the formation of a morphogen gradient is often challenged by signaling component-level fluctuations, temperature differences, size variations, and/or unequal distributions of components between daughter cells (5). Therefore, morphogen gradients should be reproducibly formed with robust stability from one embryo to the next (5). Specifically, a robust resistance of DV axis formation to perturbations has been observed in various vertebrate embryos during classic grafting and ablation experiments. When grafted to the ventral-most part of a host em-bryo, where the BMP signal is maximally activated, the Spemann-Mangold organizer of Xenopus and the embryonic shield of zebrafish retain their ability to induce a secondary body axis at the site of the graft (6,7). Furthermore, even when an amphibian blastula is bisected into dorsal and ventral halves, the dorsal half can give rise to a well-proportioned half-sized embryo (8). In addition, avian embryos can compensate for the removal of the organizer (Hensen's node) during the primitive streak stage as evidenced by the reappearance of organizer markers (9). These observations support the idea that self-regulation occurs on the dorsal side of vertebrate embryos. On the other hand, transplantation of zebrafish ventral margin cells into animal poles induces the formation of secondary tails, indicating the existence of a tail organizer (10). Therefore, both the ventral and dorsal sides of vertebrate embryos are involved in self-regulation of DV patterning, but the underlying mechanism ensuring the robust BMP activity gradient remains one of the great unsolved mysteries in developmental biology.
In zebrafish embryos, ventral BMP signaling maintains expression of the vox/vent/ved transcriptional repressors, which restrict the expression of dorsal-promoting genes, including chd (11). In Xenopus, the expression of chd is negatively regulated by BMP4 (8). Therefore, the BMP signal gradient controlled by ventral BMP ligands and their dorsally secreted antagonist Chd is theoretically unstable, where a small change in the BMP signals or Chd expression would cause severe defects in the DV body plan (12). A BMP-like protein, antidorsalizing morphogenetic protein (Admp), is uniquely expressed in and secreted by the dorsal organizer (13,14). Admp associates with Chd and facilitates Chd degradation (15). The expression of admp is repressed by BMP signals, and a depletion of the ventral BMP signals will increase admp expression, thus allowing the regeneration of a new BMP signal gradient. Therefore, Admp is an appealing candidate for ensuring the stabilization of DV patterning (8). Unexpectedly, knockdown of admp in Xenopus or zebrafish using morpholinos (MOs) only causes mild dorsalization (8,12,16,17), and the distribution of the Chd protein remains largely unchanged (17), suggesting that there are other BMP-like members that compensate for the loss of Admp function. In addition, the dorsal halves of split Xenopus embryos still retain substantial DV polarity when BMP4 and BMP7 are depleted (8), indicating that unidentified BMPlike members may function in the newly induced ventral side and are transcriptionally up-regulated to compensate for the loss of BMP ligands.
The precursor proteins of BMP family members consist of three parts: an N-terminal signal peptide that targets the protein to the secretory pathway, a prodomain that mediates proper folding, and a C-terminal mature peptide containing seven highly conserved cysteines, i.e., cysteine knots, that form intramolecular disulfide bonds (18). In addition, an Arg-X-X-Arg sequence motif in the prodomain of the precursor proteins is hydrolyzed by serine proteinases to form mature C-terminal proteins that are subsequently secreted (18). There are at least 20 structurally and functionally related BMPs, including Decapentaplegic, Screw, and Glassbottom-boat in Drosophila and BMP2/4, BMP5/6/7/8, and BMP9/10 in vertebrates. Most of these BMPs play critical roles in embryogenesis and organ morphogenesis (19)(20)(21). The characterization of previously unknown BMP members involved in embryonic development will be interesting and provide key insights into this developmental pathway.
The novel gene pinhead was originally isolated from a functional knockdown screen searching for genes involved in nervous system development and is expressed in the anterior neural plate of Xenopus neurula as a key regulator of head development (22). pinhead is located immediately upstream of admp in the genomes of various animals, ranging from arthropods to vertebrates (22,23). This genomic configuration of pinhead and admp is important for mutually exclusive expression of these genes in Ciona embryos, which lack a structure homologous to the vertebrate organizer (23). In gastrulating Xenopus embryos, pinhead is expressed in an arc around the blastopore with a distinct gap corresponding to the dorsal mesoderm, which implies a possible role in the embryonic body plan (22).
In this study, we demonstrated that Pinhead is a secreted BMPlike ligand expressed in the ventrolateral margin and has ventralizing functions in the zebrafish embryonic body plan. Similar to Admp, Pinhead was also found to promote metalloproteinase-mediated Chd degradation. Expression of pinhead was notably increased in response to the inhibition or depletion of admp and vice versa. This "seesaw"-like expression of pinhead and admp establishes a well-orchestrated alternative mechanism for the robust generation of the DV axis. This is evidenced by the normal DV polarity exhibited by pinhead or admp mutants alongside the marked dorsalization displayed when both of these genes are absent. Last, the expression of pinhead and admp is negatively regulated by BMP signaling, where this negative feedback loop between BMP signaling and pinhead/admp is important for buffering against fluctuations in dynamic BMP signaling during DV axis formation. Therefore, we propose an alternative mechanism to ensure stable axis formation that couples pinhead and admp with system control based on opposing regulation of BMP signaling and pinhead/admp expression. This work will provide important insights into the mechanisms of robustness in organisms by the self-regulating BMP activity gradient.
RESULTS
Zebrafish pinhead is a ventralizing gene expressed in the ventrolateral margin Expression of pinhead and admp occurs in a mutually exclusive manner during Ciona and Xenopus embryonic development (22,23). Admp is a BMP-like protein with important functions in the em-bryonic body plan (13,14,16). However, the developmental role of pinhead in DV patterning remains unknown. To gain insight into the functions of zebrafish pinhead [NM_205587.1, National Center for Biotechnology Information (NCBI)] during embryogenesis, we firstly characterized its expression during early embryonic development using whole-mount in situ hybridization (WISH) with an antisense probe. As shown in Fig. 1A, pinhead transcripts were undetectable before and during the sphere stage. Soon afterward, pinhead was expressed around the marginal zone but not the dorsal mesoderm, as indicated by costaining with goosecoid (gsc) (Fig. 1, A and B). During the midgastrulation stages, the expression of pinhead transcripts appeared in a DV gradient in the blastoderm margin (Fig. 1A). Abundant pinhead transcripts were consistently observed in the presomitic mesoderm, but not the axial mesoderm, at the bud stage and during somitogenesis ( Fig. 1A and fig. S1A). pinhead transcripts were no longer detectable after the segmentation stages (fig. S1B). These data indicate that zebrafish pinhead may play a role in establishing ventral cell fates during early embryonic development.
Next, the effects of pinhead overexpression on embryogenesis were assessed by injecting mRNA synthesized in vitro into one-cell stage embryos. Embryos injected with different amounts of pinhead mRNA exhibited dose-dependent ventralized phenotypes, characterized by the loss of dorsoanterior structures and the expanded ventral tissues at 24 hours post-fertilization (hpf) (Fig. 1, C and D), suggesting that Pinhead protein has ventralizing activity. In contrast to the impaired function of BMP2b by the addition of six amino acids at its C terminus in swirl mutants (24), the ventralizing activity of Pinhead was not obviously affected when a hemagglutinin (HA) epitope tag was fused to the carboxy end (Fig. 1D). Moreover, ectopic expression of Xenopus pinhead (NM_203534.1, NCBI) in zebrafish embryos generated similar ventralized phenotypes but did not result in macrocephaly (fig. S1, C and D), which had been observed in pinhead-overexpressed Xenopus embryos, suggesting an additional function of pinhead in the development of Xenopus nervous system (22).
In addition to the morphological changes, we also assessed the expression of several dorsal and ventral markers in embryos injected with 100 pg of pinhead mRNA. During the gastrula stages, injection of pinhead mRNA greatly reduced the expression of dorsal markers, including chd and gsc (Fig. 1, E and G). By contrast, the expression of the ventral markers eve1 and vent was notably expanded in response to injection of pinhead mRNA (Fig. 1, F and G). In addition, embryos injected with pinhead mRNA had a much smaller dorsal neuroectoderm (as indicated by sox3 expression), as well as an expanded ventral nonneural ectoderm (as indicated by gata2 expression) at 75% epiboly stage (Fig. 1H). At later stages, overexpression of pinhead in zebrafish embryos resulted in a slight expansion of the blood cell population within the intermediate cell mass, which is derived from the ventral mesoderm (Fig. 1I). On the basis of these observations, we conclude that Pinhead has ventralizing functions in the zebrafish embryonic body plan.
The pinhead gene encodes a functional BMP ligand Zebrafish pinhead encodes a 316-amino acid protein with a predicted hydrophobic N-terminal signal sequence (fig. S1E). Because Pinhead is a ventralizing factor and predicted to be secreted, we speculated that it is a BMP-like ligand. To address this hypothesis, we compared the sequences of Pinhead and several zebrafish BMP members, including BMP2b, BMP4, BMP7a, and Admp. Although the sequence Expression of pinhead in wild-type zebrafish embryos was analyzed by whole-mount in situ hybridization. 128-Cell and sphere stages, lateral views; 30% epiboly (ep) and shield stages, animal pole views with dorsal to the right, and dorsal views with animal pole at the top; and 75% epiboly and bud stages, lateral views with dorsal to the right, and dorsal views with animal pole at the top. In the last panel, the embryo is slightly tilted upward to expose the tail bud. of Pinhead displays little similarity to the other BMP ligands, it does contain a number of features characteristic of BMP proteins, including a consensus Arg-X-X-Arg proteolytic processing site and six characteristic cysteine residues conservatively located in the mature carboxyl terminal domain ( fig. S1F).
To examine the biochemical properties of Pinhead, we expressed Pinhead-HA protein in human embryonic kidney (HEK) 293T cells and studied the conditioned medium produced by the transfected cells (Pinhead CM). Immunoprecipitation of Pinhead CM revealed that the Pinhead protein was present in the medium ( Fig. 2A). To analyze the secretion rate of Pinhead, we treated HEK293T cells expressing Pinhead-HA with the protein synthesis inhibitor, cycloheximide (CHX), and then immunoprecipitated Pinhead proteins in conditioned medium and cell lysates at different time points, respectively. We found that about 30% of Pinhead proteins were secreted within 4 hours, and more than 90% of Pinhead proteins were present in the medium after 12 hours of CHX treatment (Fig. 2, B and C). In addition, after 8 hours of CHX treatment, we detected more than 90% of Pinhead proteins in the CM produced by the suspended cells dissociated from the gastrula embryos injected with pinhead-HA mRNA (fig. S1, G and H), suggesting that Pinhead proteins can be more effectively processed in and secreted from zebrafish embryonic cells.
To further demonstrate that Pinhead is a secreted protein in vivo, we examined whether Pinhead is secreted in zebrafish embryos by coinjecting mRNAs encoding plasma membrane-localized mCherry-CAAX protein and Pinhead-GFP protein, in which green fluorescent protein (GFP) was fused to the C-terminal end of Pinhead, into onecell stage embryos. The Pinhead-GFP fusion protein has a ventralizing activity similar to untagged Pinhead, as injection of equimolar amounts of pinhead-gfp and pinhead mRNAs resulted in similar percentages of ventralized embryos at 24 hpf (fig. S1, I and J). As expected, Pinhead-GFP protein was primarily intercellular at the shield stage (Fig. 2D). We also injected pinhead-gfp mRNA together with rhodamine-dextran into one marginal blastomere at the 16-or 32-cell stages. At later stages, the descendant cells could be indicated by rhodamine fluorescence. At the shield stage, we observed obvious GFP fluorescence at the periphery of the rhodamine-positive and rhodamine-negative cells and even the cells far away from the progeny of the injected blastomere (Fig. 2E). Immunoprecipitation of CM produced by the suspended cells from embryos injected with gfp or pinhead-gfp mRNAs showed that it was not GFP protein but Pinhead-GFP that could be detected in the medium (fig. S1K), ruling out the possibility that the high mobility of the protein observed in zebrafish embryos is due to a substantial amount of free GFP. Thus, these results suggest an efficient secretion and a long-range diffusion of Pinhead proteins in the developing embryos.
The mature form of Pinhead is one conserved cysteine residue less than other BMP ligands ( fig. S1F). Therefore, we next examined whether secreted Pinhead protein could form covalent dimers that had been proved to be essential for downstream signaling events (3). Pinhead-HA proteins were enriched from the CM by immunoprecipitations and then subsequently separated on reducing and nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), respectively. Immunoblotting analysis showed a single SDS-resistant band with an apparent molecular weight of about 70 kDa under nonreducing conditions, which migrated much more quickly under reducing conditions (Fig. 2F), implicating that most of the mature Pinhead proteins exist as disulfide-linked dimers in vivo. To deter-mine whether secreted Pinhead activates an intracellular signaling cascade, we measured the phosphorylation levels of Smad1/5/8 in Hep3B cells in the presence and absence of Pinhead CM. We found that stimulation with recombinant BMP4 or Pinhead CM substantially enhanced Smad1/5/8 phosphorylation, and combining BMP4 and Pinhead CM further promoted this phosphorylation (Fig. 2G). By contrast, Smad2 phosphorylation (p-Smad2) in Hep3B cells was induced by incubations with TGF-1 but not Pinhead CM (Fig. 2H). In addition, overexpression of pinhead in embryos had no effects on p-Smad2 expression (Fig. 2H). These data not only demonstrate that Pinhead specifically triggers the BMP pathway but also rule out the possibility that Pinhead ventralizes embryos by inhibiting Nodal signaling, which is required for the formation of the organizer and the dorsal axial structures. Moreover, Pinhead binds to and signals through BMP receptors, as Pinhead CM-induced Smad1/5/8 phosphorylation was totally abolished in the presence of the selective BMP type I receptor inhibitor dorsomorphin or DMH1, and overexpressed Pinhead was coimmunoprecipitated with BMP type I receptors ALK2, ALK3, ALK6, and ALK8 ( Fig. 2, I and J). We were not expecting to find an association between Pinhead and TGF- type I receptor ALK5 (Fig. 2J). However, this Pinhead-ALK5 association may not have biological significance, as Pinhead proteins did not induce phosphoryl ation of Smad2 in Hep3B cells and zebrafish embryos (Fig. 2H).
In zebrafish embryos, injection of 100 pg of pinhead-HA mRNA promoted phosphorylation of Smad1/5/8 during gastrulation (Fig. 2K). Pinhead protein efficiently coimmunoprecipitated with the BMP antagonist Chd and Noggin1 (Fig. 2, L and M). The pinhead overexpressioninduced DV defects in the shield-stage embryos, such as the reduction in gsc expression and the expansion of eve1 expression, were eliminated by coinjecting 10 pg of chd mRNA (Fig. 2, N and O). Consistent with these observations, at 24 hpf, injection of chd mRNA well rescued the ventralized morphology in Pinhead-overexpressing embryos ( fig. S1L). Together, these findings indicate that Pinhead is a functional BMP ligand during zebrafish embryo development.
The pinhead and admp genes repress one another and compensatorily function in DV patterning
To examine the in vivo functions of pinhead, we generated a pinhead mutant by targeting exon 1 with the CRISPR-Cas9 system. The mutant was named ph49, as there was a 49-base pair (bp) deletion that led to the loss of the translational start site ( fig. S2A). We further generated the maternal-zygotic mutant by incrossing homozygous pinhead zygotic mutants. In situ hybridization experiments revealed an obvious decrease in pinhead transcripts in the ph49 mutants, providing further evidence that this mutant is a null allele of the pinhead gene ( fig. S2B). Unexpectedly, ph49 embryos had normal morphologies at the end of gastrulation and at 24 hpf (fig. S2, C and D). In addition, we found no DV pattern defects in ph49 mutants for typically expressed dorsal and ventral genes (fig. S2, E and F). Previous studies indicate that a compensatory network may be activated to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown (25). Therefore, knockdown experiments were performed using an antisense MO (ph MO) that interfered with translation by targeting the pinhead sequence and efficiently blocking the production of the Pinhead-GFP fusion protein in embryos ( fig. S2G). However, injection of 5 ng of ph MO into wild-type embryos did not result in any obvious DV defects ( fig. S2, H and I). Therefore, the loss of pinhead does Pinhead levels in cell lysate were examined by Western blot as a positive control. Underlying data can be found in data file S1. (B and C) HEK293T cells were transfected with Pinhead-HA plasmids. Twenty-four hours later, cells were treated with CHX (20 g/ml) for the indicated times. Then, the CM and CHX-treated cells were harvested for immunoblotting (B). Pinhead-HA protein levels were quantified and normalized to tubulin (mean ± SD, three independent biological repeats; C). Underlying data can be found in data file S1. (D and E) Pinhead-GFP fusion proteins were efficiently secreted from zebrafish embryonic cells. In (D), 50 pg of pinhead-GFP mRNA and 50 pg of mCherry-CAAX mRNA were coinjected into embryos at the one-cell stage. In (E), 10 pg of pinhead-GFP mRNA together with rhodamine-dextran was injected into one marginal blastomere at the 16-or 32-cell stage. All embryos were imaged using a Nikon A1R+ confocal microscope at the shield stage. Scale bar, 10 m. (F) Pinhead-HA proteins were enriched from the CM by immunoprecipitation and then subsequently separated on reducing and nonreducing SDS-PAGE. Underlying data can be found in data file S1. (G and H) Hep3B cells were treated with Pinhead CM alone or together with BMP4 (G) or TGF-1 (H) for 1 hour and then harvested for Western blots with the indicated antibodies. The expression of -actin was analyzed as a loading control. In (H), wild-type embryos treated with 25 M SB431542 (SB) from the 16-cell stage and embryos injected with 100 pg of gfp or pinhead mRNA at the one-cell stage were also harvested at the shield stage and subjected to immunoblotting. Underlying data can be found in data file S1. (I) Hep3B cells were treated with Pinhead CM alone or together with the indicated BMP type I receptor inhibitors for 4 hours and then harvested for Western blot with the indicated antibodies. Note that Pinhead CM-induced Smad1/5/8 phosphorylation notably decreased in the presence of BMP type I receptor inhibitors. Underlying data can be found in data file S1. (J) Pinhead binds to BMP type I receptors. HEK293T cells were transfected as indicated with expression plasmids encoding Flag-tagged Pinhead and HA-tagged BMP type I receptors and harvested for immunoprecipitation with an anti-HA antibody. Underlying data can be found in data file S1. (K) Western blots of total lysates from embryos injected with 100 pg of pinhead-HA mRNA. Underlying data can be found in data file S1. (L and M) Extracellular Pinhead interacts with Chd (L) and Noggin (M). CM were prepared from HEK293T cells transfected with indicated plasmids. Immunoprecipitation assays were performed using an anti-Flag antibody. Underlying data can be found in data file S1. (N) Overexpression of chd rescues Pinhead-induced DV defects. Embryos were injected with 100 pg of pinhead mRNA alone or together with 10 pg of chd mRNA at the one-cell stage and collected at the shield stage for in situ hybridization. (O) Expression levels of gsc and eve1 were analyzed at the shield stage by real-time qPCR. Error bars indicated SD. *P < 0.05; **P < 0.01, Student's t test. NS, not significant. not disturb the formation of the DV axis, and an additional signal may be present in the embryo to compensate for the lack of pinhead.
The genes pinhead and the BMP ligand-encoding admp exist in tandem in the genomes of various animals, including zebrafish. These two genes have diametrically opposed expression patterns in the trunk epidermis in gastrulating Ciona embryos (23). We speculate that admp is an ideal candidate for buffering the loss of pinhead. In support of this hypothesis, contrary to the narrowed expression of admp in pinhead-deficient Ciona embryos (23), we found admp expression to be up-regulated in zebrafish ph49 mutants at the 30% epiboly and shield stages (Fig. 3A). To exclude the possibility that the increase in admp expression was merely an adaptation for gene loss, we injected 5 ng of ph MO into wild-type embryos. We found that admp expression also greatly increased in the pinhead morphants (Fig. 3B), suggesting that admp expression is repressed by pinhead. We also generated a null allele of admp with an 11-bp deletion in exon 1 (ad11) (fig. S3, A and B). Mild dorsalization phenotypes were observed in knockdown experiments with admp MO in Xenopus and zebrafish (8,12,16,17). By contrast, the morphology and DV polarity were not affected in ad11 maternal-zygotic mutants compared to the wild-type control (fig. S3, C to F). It had been reported that admp morphants exhibited a notable enlargement of gsc expression domain and an evident diminution of eve1 expression (14). Unexpectedly, we did not observe any marked changes in the expression of dorsal-ventral markers in embryos injected with 3 ng of admp MO (fig. S3, G and H), which had previously been used (14). This inconsistency may be due to the different experimental conditions between the studies. pinhead expression was evidently expanded in ad11 mutants and admp morphants (Fig. 3, C and D). These results reveal that pinhead and admp are expressed in a "seesaw"-like fashion through opposing transcriptional regulation in the embryonic body plan.
As shown in Fig. 3E, coimmunoprecipitation experiments revealed a steady binding of secreted Pinhead and Bamp1a, a Xolloid-related metalloproteinase that plays a pivotal role in proteolytic cleavage of Chd in zebrafish (26). The association of Pinhead with Bamp1a led us to examine whether Pinhead regulates BMP1a-mediated Chd degradation. Compared with the effects of the corresponding untagged proteins, overexpression of Admp-HA or Chd-Flag in wildtype embryos caused similar or slightly alleviated DV polarity defects at 24 hpf ( fig. S4, A to D), suggesting that the addition of C-terminal epitopes has no obvious impact on their activities. Then, BMP1a, Chd, Admp, and Pinhead proteins were prepared by collecting the corresponding CM produced by transfected HEK293T cells. When BMP1a was coincubated with Chd, we detected a decrease in Chd protein, where adding Admp further facilitated this cleavage (Fig. 3F), suggesting that the secreted BMP1a functions well in our biochemical system. Pinhead was then coincubated with BMP1a and Chd in vitro, which promoted a reduction in Chd levels (Fig. 3G). These observations demonstrate that, similar to Admp, Pinhead promotes metalloproteinase-mediated Chd degradation.
To further confirm the roles of pinhead and admp in the formation of DV polarity in zebrafish embryos, we deleted the pinhead gene in the ad11 mutants using the CRISPR-Cas9 system. One mutant was obtained with an identical 49-bp deletion in the pinhead gene in the ad11 background. The ph49 +/− ;ad11 −/− embryos develop normally and are viable and fertile, but ph49 −/− ;ad11 −/− embryos began to die 36 hpf, with a few surviving up to adulthood. Homozygous pinhead and admp double-mutant embryos were generated by crossing the surviving adults. Most of the ph49;ad11 double mutants had an ovoid shape at the bud stage and a clearly shortened posterior trunk and reduced yolk extension at 24 hpf, all of which are characteristic of dorsalization (Fig. 3, H and I). Furthermore, although injection of pinhead MO or admp MO into wild-type embryos did not lead to observable DV polarity defects at 24 hpf, coinjection of these MOs generated a dorsalized phenotype very similar to that of ph49;ad11 double mutants ( fig. S5A), excluding the potential CRISPR-Cas9 off-target effects.
The dorsalization phenotypes in ph49;ad11 double mutants were further confirmed by the expression of several dorsal and ventral markers. As shown in Fig. 3J, we observed a marked expansion in the dorsal markers chd and gsc in ph49;ad11 embryos, while the expression of these markers remained unchanged in the ph49 and ad11 single mutants compared to the wild-type embryos. Meanwhile, the expression of the ventral marker eve1 was nearly abolished in ph49;ad11 embryos ( Fig. 3K). Genetic deletion of these two genes consistently caused enlargement of dorsal-related tissues, including the prechordal plate (indicated by gsc) and notochord (indicated by ntl) (fig. S5B). There was also a large decrease in the blood cells located in the intermediate cell mass in ph49/ad11 mutants ( fig. S5C). It has been reported that dorsalized embryos exhibit a slightly widened adaxial domain and expanded somite due to loss of swirl/bmp2b (11,24). However, ph49/ad11 mutants showed reduced presomitic mesoderm (indicated by papc) at the bud stage ( fig. S5D), which might reflect a role of pinhead and admp in somitogenesis, as pinhead transcripts were highly enriched in the presomitic mesoderm at the end of gastrulation (Fig. 1A). In addition, at the shield stage, phosphorylation of Smad proteins and the BMP gradient decreased in the ph49/ad11 embryos (Fig. 3, L and M). Consistent with previous reports that bmp expression is maintained through autoregulatory feedback loops (24,27), we observed that, compared to the wild-type embryos at the shield stage, ph49;ad11 double mutants displayed reduced expression of bmp2b, bmp4, and bmp7a, which were not obviously changed in ph49 and ad11 single mutants ( fig. S5E). In addition, by knockdown of admp in ph49 mutants or injection of pinhead MO into ad11 embryos, we observed similar dorsalization phenotypes, including the changes in the expression of dorsal and ventral markers ( fig. S6, A to D), the repression of Smad1/5/8 phosphorylation ( fig. S6, E and F), and the destruction of the BMP activity gradient ( fig. S6, G and H). These results further confirm that pinhead and admp function together to regulate the formation of the BMP activity gradient and DV patterning in early zebrafish embryos.
It is well established that Admp functions in DV axis formation by enhancing Chd degradation (12,15). Moreover, Admp overexpression could enhance the ventralized phenotype of din homozygous mutants, a mutant allele of chd (28), suggesting a role for Admp in BMP signal activation through a Chd-independent manner. It has been proved that Admp has BMP-like activity and signals via the ALK2 receptor (8). Given that Pinhead has similar functional properties to Admp, we asked whether Pinhead functions as a secreted scaffold aiding in Chd degradation and a ligand involved in activating BMP signaling during zebrafish DV patterning. We first examined the expression levels of endogenous Chd in shield-stage ph49 and ad11 mutants by Western blot analysis using a previously validated antibody (17). We found that the endogenous Chd protein heavily accumulated in ph49;ad11 double mutants compared to wild-type or singlemutant embryos (fig. S7A). In contrast, the expression of Chd S7B). To avoid the influence of changes in endogenous Chd expression induced by DV defects, we further examined the expression of exogenous Chd-HA protein in ph49;ad11 double mutants and wild-type embryos. All the embryos were injected with the same amount of chd-HA mRNA (50 pg) at the one-cell stage. Western blot results showed that the expression level of Chd-HA was up-or down-regulated upon depletion or overexpression of pinhead/admp ( fig. S7, C and D). These results indicate that Pinhead can facilitate Chd degradation in vivo.
We next tested whether Pinhead also has a role in DV patterning when chd and bmp2b are depleted. As previously reported, injection of bmp2b MO into wild-type embryos caused a severe dorsalized morphology at 24 hpf, while the interference with chd function by MO generated a clear ventralized phenotype ( fig. S7E) (29,30). As expected, double depletion of chd and bmp2b gave a nearly normal morphology ( fig. S7E). Injection of 100 pg of pinhead mRNA into chd/bmp2b-depleted embryos led to a ventralized phenotype, which was slightly serious in embryos injected with the same amount of admp mRNA ( fig. S7E). Thus, similar to Admp, Pinhead might also regulate the establishment of DV regionalization via its BMP-like activity.
On the basis of these results, overexpression of either pinhead or admp would be expected to compensate for the loss of these two genes. Injection of 5 pg of bmp2b mRNA into the ph49;ad11 mutant embryos efficiently reversed the dorsalization morphologies ( fig. S7F). Injection of either 100 pg of pinhead mRNA or 50 pg of admp mRNA also considerably alleviated the DV defects in the ph49;ad11 mutants ( fig. S7F). Thus, the consistency in the molecular nature and the opposite transcriptional regulation of Pinhead and Admp provides an important compensatory mechanism by which to maintain stability of axial patterning when one of these genes is disrupted.
BMP signaling negatively regulates pinhead and admp expression
In Ciona gastrulas, pinhead is expressed in the posterior ventral epidermal cells, while admp is expressed in the dorsal epidermis. Their mutually exclusive expression is regulated at the chromatin level by a cis-acting mechanism that is widely conserved between animals (23). When the cis-acting repression is relieved, ectopic admp expression is observed in the ventral region (23). Expression of admp greatly increased in the ph49 mutant but did not spread into the ventrolateral regions, suggesting that the microdeletion in the ph49 mutant did not alter the chromosomal conformation around the gene locus (Fig. 3, A and B). Repression of admp by BMP signaling and the BMP-like activity of Pinhead prompted us to examine whether the up-regulation in admp expression in the ph49 mutants was due to a transient decline in BMP signaling induced by Pinhead deficiency.
As shown in Fig. 4 (A and B), wild-type embryos injected with bmp2b MO or treated with the BMP inhibitor dorsomorphin or DMH1 had remarkably increased expression of admp. Conversely, injection of bmp2b mRNA led to a reduction in admp expression (Fig. 4, C and E). In addition, the expression of pinhead was negatively regulated by BMP signaling, as knockdown of bmp2b induced and high levels of bmp2b inhibited its transcription (Fig. 4, A, B, D, and E). The increased expression of admp and pinhead in the corresponding mutants was reduced to a lower level than that in wild-type embryos by injection of 10 pg of bmp2b mRNA (Fig. 4, F to H), indicating a complete compression of the compensatory expression of pinhead or admp upon BMP2b overexpression. Therefore, we conclude that BMP signals negatively regulate pinhead and admp transcription. In addition, wild-type embryos injected with 10 pg of bmp2b mRNA displayed various ventralized phenotypes ranging from mild to severe at 24 hpf ( fig. S8, A and B). Upon bmp2b mRNA injection, the ph49 and ad11 single mutants exhibited a similar ventralized morphology ( fig. S8, A and B), further suggesting that the compensatory expression of pinhead or admp can make up for the gene loss in the corresponding mutants.
On the basis of the "seesaw"-like expression patterns of pinhead and admp, we speculate that, when the expression of one is disturbed in embryos, BMP signaling will temporarily be lower and expression of the other gene will be subsequently promoted to support the self-regulation of the BMP signaling levels for the embryonic body plan. To address this issue, MOs targeting pinhead or admp were injected into Tg(BRE:EGFP) embryos, in which a GFP reporter can reveal the dynamic changes in BMP activity during embryonic development (31). In support of our hypothesis, the results of realtime quantitative polymerase chain reaction (PCR) analysis revealed an early partial loss of BMP activity in the morphants, which was dynamically compensated before the shield stage (Fig. 4I). The expression of admp in ph49 mutants or pinhead in ad11 mutants was gradually elevated compared to that in wild-type embryos (Fig. 4, J and K). The promotion of admp and pinhead expression may well be due to the temporary reduction in BMP activity in the single-mutant embryos, as coinjection of 10 pg of bmp2b mRNA notably suppressed the elevation of their expression (Fig. 4, J and K).
Smad proteins directly bind to pinhead and admp enhancers
To further investigate whether BMP/Smad signaling directly represses pinhead and admp transcription, we amplified 1336 bp of the admp and 1516 bp of the pinhead promoter regions upstream of the translation start site of each gene and fused them to GFP cDNA to create reporter constructs (named −1336-ad-P-GFP and −1516-ph-P-GFP, respectively). The upstream sequence of admp drove GFP expression on the dorsal side of shield stage embryos, recapitulating endogenous expression of admp (Fig. 5A). Next, we generated serial truncations of the admp promoter and injected them into embryos. We found that the truncated promoter containing the −633-bp upstream sequence (−633-ad-P-GFP) exhibited transcriptional activity in the dorsal region similar to the full-length promoter, while the −210-ad-P-GFP construct lost the ability to express GFP (Fig. 5A), suggesting that the region between −633 and −210 bp is an enhancer essential for admp expression. To identify potential BMP/ Smad-responsive elements in this enhancer, we injected the −633-ad-P-GFP construct (100 pg) into wild-type and ph49 mutant embryos. The expression of −633-ad-P-GFP was augmented in DMH1-treated wild-type and ph49 mutant embryos (Fig. 5B), suggesting that the enhancer responds well to BMP signals. To quantitatively analyze the transcriptional regulation of admp by BMP signal, we generated a luciferase reporter plasmid (−633-ad-P-Luc) by subcloning the −633-bp upstream sequence of admp into pGL3-Basic vector. As expected, 3-or 4.5-fold enhanced transcriptional activity of −633-ad-P-Luc was observed in pinhead defective or DMH1-treated embryos (Fig. 5C). Similarly, a BMP signal-responsive enhancer that specifically drove reporter gene expression in the ventral and lateral margin of the gastrulas was identified between −431 and −225 bp in the pinhead promoter (Fig. 5, D to F).
Smad proteins physically interact with the promoters of their target genes to regulate gene expression (18). Specifically, Smad1/5 proteins, the intracellular downstream mediators of BMP signaling, directly bind to the GC-rich elements in BMP/Smad target promoters (32). There are two and four potential Smad1/5-bound GC-rich elements in the admp and pinhead enhancers, respectively ( fig. S9, Fig. 4. Expression of pinhead and admp is repressed by BMP signaling. (A and B) The expression of admp and pinhead was analyzed at the shield stage by in situ hybridization (A) and real-time qPCR in bmp2b morphants and embryos treated with 10 M dorsomorphin or 5 M DMH1 from the 1K cell stage. Error bars indicated SD. *P < 0.05; **P < 0.01; ***P < 0. luc) showed a higher transcriptional activity than the corresponding wild-type reporter. Wild-type embryos were injected with 100 pg of indicated reporter constructs at the one-cell stage. At the shield stage, these embryos were photographed (K and M) or subjected to luciferase assays (L and N). Scale bar, 100 m. ***P < 0.001, Student's t test. (O and P) Smad binding was essential for BMP signal-mediated suppression of admp (O) and pinhead (P) transcription. Mutated reporter constructs (50 pg) were injected into wild-type and indicated mutant embryos at the one-cell stage, respectively. Wild-type embryos injected with −633-ad-MTB-GFP or −431-ph-MTB-GFP reporter were treated with or without DMH1 from the 1K cell stage. Note that these mutated reporters lost their ability to respond to BMP inhibition. Scale bar, 100 m.
A and B). To explore the ability of Smad proteins to bind to these sites, we performed electrophoretic mobility shift assays using purified Smad1 proteins and synthesized probes containing presumptive Smad-binding sites. Smad1 specifically bound to admp probe B (ad Probe B) but not to the mutated probe with the "GGCGCC" to "AAAAAA" substitutions within the putative Smad1/5-binding site (Fig. 5, G and H, and fig. S9A). Meanwhile, Smad1 also bound to pinhead probe B (ph Probe B) but not its mutant (Fig. 5, I and J, and fig. S9B). When the proper mutations were introduced into each enhancer, the mutated promoters (100 pg of each) exhibited a remarkable increase of their transcriptional activities in wild-type embryos (Fig. 5, K to N), indicating a relief of direct repression by BMP/Smad signaling. Because the mutated promoters could activate much higher expression of reporter gene than their original promoters, we reduced the injection dosage to 50 pg to confirm whether they lose the ability to respond to BMP inhibition. We found that the GFP reporter driven by the mutated promoters was similarly expressed in DMH1-treated or untreated wild-type embryos and ph49 or ad11 mutants (Fig. 5, O and P). Together, these results demonstrate that Smad1/5 binds to the pinhead and admp enhancers to repress their transcription in response to BMP signaling.
The pinhead and admp genes provide a dual protection system for the robustness of embryonic patterning Embryonic DV patterning displays substantial resistance to experimental perturbations and Admp has been proposed to aid in the self-regulation of the BMP signaling gradient and the regeneration of normal DV structures (8,12). Because pinhead and admp are expressed under feedback regulations in a "seesaw"-like fashion and have compensatory functions in DV patterning, we presumed that both of these genes might be involved in the robust stability of axial patterning through fine-tuning of BMP signaling. To investigate whether pinhead and admp contribute to the buffering of BMP activity profiles against variations in gene dosage, we introduced bmp2b MO into ph49 or ad11 mutants and ph49;ad11 embryos injected with a rescuing amount of pinhead mRNA (improved ph49;ad11 mutants). As shown in Fig. 6A, injection of 1 ng of bmp2b MO induced a mild increase of chd expression in most wildtype, ph49, and ad11 embryos and caused a much stronger expansion of this dorsal marker gene in nearly 70% of the improved ph49;ad11 mutants, where the DV defects were significantly rescued by pinhead overexpression (Fig. 6, A and B). In response to bmp2b MO injection, the expression domain of chd even extended to the ventral regions in about 20% improved ph49/ad11 mutants, suggesting a much severe dorsalized phenotype (Fig. 6, A and B). Consistent with this, injection of 1 ng of bmp2b MO caused a more severe decrease in expression of the ventral marker eve1 in improved ph49;ad11 mutants compared to the control embryos (Fig. 6, A and C). These bmp2b morphants displayed different dorsalized morphologies (C1 to C5) at 24 hpf (Fig. 6, D and E). About 80% of the improved ph49;ad11 embryos showed a normal DV morphology (Fig. 6E). Upon injection of 1 ng of bmp2b MO, above 20% of wild-type embryos and pinhead or admp single mutants showed some mild cases of dorsalization defects (C1 and C2), whereas all of the improved ph49;ad11 embryos exhibited more severely dorsalized phenotypes (C3 to C5; Fig. 6E). Furthermore, a reduced amount of injected bmp2b MO (0.3 or 0.1 ng) led to marginal dorsalized phenotypes in wild-type and single-mutant embryos, while these subdose injections resulted in hyperdorsalization in improved ph49;ad11 mutants (Fig. 6, F and G). These findings indicate that pinhead and admp cooperatively confer robust resistance to the decrease of BMP signaling during DV patterning.
Because the expression of pinhead and admp notably decreased in bmp2b overexpression embryos (Fig. 4, C to E), these two genes might also play important roles when embryos are challenged with excessive BMP activity. If this deduction is correct, reduced expression of pinhead and admp should dampen the elevated BMP activity to some extent due to impeded Chd degradation and depletion of these two genes should further stabilize BMP activity profiles during DV patterning. Consistent with these predictions, injection of 10 pg of bmp2b mRNA led to severe ventralized phenotypes in above 90% wild-type, ph49, and ad11 embryos, which were obviously alleviated in about 40% improved ph49;ad11 mutants ( Fig. 6, H to J). Moreover, our study proved that, similar to Admp, Pinhead also functions in DV patterning via its BMP-like activity ( fig. S7E). To explore whether the BMP-like activity of Pinhead and Admp is involved in the robustness of embryonic patterning, we examined the expression patterns of gsc and eve1 in chd-depleted embryos at the shield stage. We observed that, upon chd depletion, the improved ph49;ad11 mutants showed a lower rate of severe ventralized phenotype than wild-type, ph49, and ad11 embryos (fig. S10, A to C), indicating that the over-activation of BMP signaling induced by chd depletion can be appeased through genetic inactivation of both pinhead and admp. These results also imply that Pinhead and Admp could act as BMP ligands to stabilize embryonic DV patterning. Collectively, these data demonstrate that pinhead and admp serve as a dual protection system for the robustness of embryonic patterning by buffering against disturbances in the dynamic BMP signaling (Fig. 6K).
DISCUSSION
Robustness is a ubiquitous property in organisms that allows a system to maintain its functions despite external and internal perturbations. These robust biological traits are often selected through evolution and facilitate evolvability (33,34). One of the best-studied models of robustness in embryonic development is the normal production of gastrula DV patterns after experimental perturbations (5-10). However, the molecular nature of this self-regulating pattern remains one of the most challenging areas that remain to be delineated in developmental biology. It is well known that a BMP signaling gradient is established through ventral BMP signals and their dorsally expressed antagonist Chd, which forms along the DV axis to pattern tissues (4). Admp, a BMP-like protein expressed as part of the feedback regulation of the Chd/BMP system on the dorsal side of gastrulating embryos, is an appealing candidate in ensuring embryonic selfregulation (8,12). Robustness can be enhanced by an "alternative" or "fail-safe" mechanism, where multiple means achieve a specific function, and the failure of one of them can be overcome by the others (33). However, whether there is an alternative mechanism to support the substantial DV polarity remains to be determined.
A previous study revealed that zebrafish admp morphants have an almost normal distribution of Chd protein (17). Consistent with this observation, the morphology and DV polarity are not affected in the ad11 mutants generated in this present study, suggesting that the loss of Admp function may be quickly compensated for by other BMP-like members. Xenopus ONT1, an olfactomedin-class secreted protein, was demonstrated to contribute to the robust stability of axial patterning with Admp in a synergistic manner (12). The negative feedback loop between pinhead/admp and BMP signals plays an important role in buffering against fluctuations in dynamic BMP signaling during DV axis formation (left panel). Meanwhile, when the function of pinhead or admp decreased or failed, the "seesaw"-like expression of these two genes will provide a well-orchestrated alternative mechanism for embryonic self-regulation (the middle and right panels). The arrows with dashed outlines in the middle and right panels indicate the quick up-regulation of pinhead or admp expression to compensate for the genetic loss of the other gene.
However, unlike Admp protein, ONT1 has no BMP ligand activity and acts as a secreted scaffold that enhances Chd degradation by facilitating enzyme-substrate association. The attenuation of ONT1 causes an increase in Admp expression but still leads to dorsalization phenotypes in Xenopus embryos (12), suggesting that ONT1 and Admp have non-overlapping functions during DV axis formation. In this study, our data indicate that zebrafish pinhead encodes a secreted BMP ligand with ventralizing functions during zebrafish embryo development. pinhead mutants had no DV pattern defects because the enhanced expression of admp fully compensated for the gene loss. Conversely, pinhead also responds to the decrease or depletion of admp to stabilize axial formation. Therefore, the "seesaw"-like expression of pinhead and admp establishes a wellorchestrated alternative mechanism for the robust generation of the DV axis. In addition, similar to Admp, Pinhead acts as a scaffold that promotes metalloproteinase-mediated Chd degradation. Thus, this alternative mechanism is mediated by the remarkable molecular similarities between Pinhead and Admp.
In Ciona embryos, pinhead is expressed in the posterior ventral epidermal cells, and MO-mediated gene knockdown experiments revealed that pinhead functions in the DV axis formation of the trunk epidermis (23). In Xenopus embryos, pinhead is expressed in the anterior neural plate of the neurula and genetic manipulation by MO injection showed that pinhead is a key regulator of head development (22). Because of a lack of expression of pinhead in the epidermal cells and the neural plate during zebrafish embryo development, it is reasonable that we found no defects in the formation of epidermal and neural tissues in our pinhead mutants.
An important unanswered question in developmental biology is how a self-differentiating morphogenetic field is established in the developing embryos. Classic embryological studies have demonstrated that admp expression is repressed by BMP signaling, and transcriptional up-regulation of admp plays a key role in compensating for the depletion of ventrally expressed BMPs (8,28). Likewise, pinhead expression is remarkably increased in embryos injected with bmp2b MO or treated with BMP inhibitors, while it notably decreased in embryos overexpressing bmp2b. Although a rescuing amount of pinhead mRNA had been introduced into pinhead and admp double mutants, these embryos were more fragile to disturbances in the dynamic BMP signaling gradient, indicating that the opposing transcriptional regulation between pinhead/admp and ventral BMP signals serves as a negative feedback mechanism and is responsible for the robust pattern formation. On the basis of these observations, we hypothesize a new framework, where the alternative mechanism is coupled with system feedback controls to ensure embryonic selfregulation. This working hypothesis is further reinforced by the identification of functional Smad1/5 binding elements in pinhead and admp enhancers. However, it has been reported that admp expression is decreased in pinhead morphants, and pinhead expression is suppressed in admp-depleted Ciona embryos during DV axis formation of the trunk epidermis (23). Furthermore, BMP signaling has no effect on admp expression but is required for pinhead expression in Ciona embryos (23). Therefore, it is possible that the effects of BMP signaling on pinhead and admp expression may be context dependent.
Expression of admp has been observed in the dorsal organizer (13,14). ONT1, which has been proposed to stabilize axial formation by restricting Chd activity, is also expressed on the dorsal side (12). In addition to the ventral and lateral region, bmp2b is also expressed in the dorsal organizer, where organizer-derived BMP2b represses chd transcription and helps control the Chd gradient during gastrulation of zebrafish embryos (17). It will be interesting to investigate whether organizer-specific bmp2b functions in the robust stability of DV patterning. However, our study showed that the expression of bmp2b in both the ventrolateral region and the dorsal organizer was not affected by single depletion of pinhead or admp, suggesting that bmp2b may not be involved in the compensatory mechanism for the robustness of embryonic patterning.
Our study revealed that pinhead is expressed in the ventrolateral margin of zebrafish gastrulas. The mutually exclusive expression and shared functions of pinhead and admp suggest that Pinhead can diffuse to the dorsal side as an extracellular signaling molecule, which is indirectly confirmed by the compensatory effects of pinhead upregulation in admp mutants. We observed an efficient secretion and a long-range diffusion of Pinhead-GFP fusion proteins in the developing embryos. In embryos, a subset of extracellular secreted factors, such as xolloid-related, twisted gastrulation, and crossveinless 2, function ventrally to promote BMP signaling through a variety of ways. The expression of these genes is positively regulated by BMPs (4,11), ruling out any potential compensatory roles they may have buffering morphogen profiles against variations. Meanwhile, BAMBI and Sizzled, secreted feedback BMP antagonists, are expressed on the ventral side as part of the BMP synexpression group and shape the BMP signaling gradient (35)(36)(37)(38). These BMP inhibitors may play additional roles ensuring reproducible DV patterns in the face of natural fluctuations (4). However, considering normal epidermal DV patterning occurs in BMP4/7-depleted dorsal halves of split Xenopus embryos and the formation of extra tails when cells from the ventral margin are transplanted into the animal pole of host zebrafish embryos (8,10), an additional BMP-like member is likely up-regulated in the ventral region to compensate for deficiencies in BMP activity. Whether expression of pinhead is induced in the bisected embryos or the grafts to preserve the ventral identity remains to be determined.
In summary, this present study suggests that pinhead and admp serve as an alternative mechanism of embryonic self-regulation, where the functions of these two genes can be restored by modular feedbacks when one component fails. It is important to note that this alternative mechanism is coupled with system control on the basis of the opposing regulation of BMP signaling and pinhead/admp expression for coping with environmental perturbations. Pinhead is the only known BMP member expressed in the ventrolateral region that is suppressed by BMP signaling. However, no expressed sequence homologous to pinhead has been found in birds and mammals (22,23). The identification of other BMP members with functions overlapping with Admp in animals that have lost pinhead gene over the course of evolution will be important for increasing our understanding of the molecular mechanism underlying self-regulative DV patterning.
Zebrafish strains
Embryos and adult fish were raised and maintained under standard laboratory conditions. Wild-type embryos were obtained from natural matings of Tubingen zebrafish. Studies in this manuscript involving zebrafish embryo collection and analyses were in full compliance with the Institutional Animal Care and Use Committee at the Institute of Zoology, Chinese Academy of Sciences (permission number IOZ-13048).
Confirmed founders were crossed to wild-type animals to raise F1 carriers for each mutant. Homozygous mutants were obtained by incrossing F1 fish carrying mutated genomic DNA. ad11 homozygous mutant embryos were injected with Cas9 mRNA and pinhead gRNA to generate germline mutants for pinhead in an ad11 mutant background. F1 animals were obtained by crossing the founders and ad11 homozygous mutants. Homozygous ph49;ad11 double mutants were obtained from the offspring of the ph49 −/− ;ad11 −/− adults.
Constructs
Zebrafish pinhead was cloned into pCS2(+) vectors containing a C-terminal HA or FLAG tag for eukaryotic expression. For Pinhead-GFP, the sequence encoding GFP protein was inserted downstream from the pinhead coding sequence with an "EFLQDIIDGSPGLE" linker separating the fluorescent protein and the Pinhead protein. C-terminal epitope-tagged Admp, Chd, Noggin1, and Bmp1a were cloned in pCS2-Flag, pCS2-HA, or pCS2-Myc vectors. All the resulting constructs were confirmed by sequence analysis.
Cell lines and transfections
HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum in a 37°C humidified incubator with 5% CO 2 . Transfection was performed using Lipofectamine 2000 (11668019, Invitrogen) according to the manufacturer's instructions. Once the HEK293T cells transfected with constructs expressing secreted proteins, such as Pinhead, Chd, and BMP1a, reached 80% confluency, they were washed three times with phosphate-buffered saline (PBS) and cultured in serum-free DMEM for 24 hours. CM was then collected from each sample and centrifuged at 3000g for 5 min, filtered through a 0.22-m filter, and concentrated to 10% of the original volume using Centriplus concentrators (Amicon).
RNA probe synthesis and WISH
Digoxigenin-uridine triphosphate-labeled and fluorescein-labeled antisense RNA probes were transcribed using the MEGAscript Kit (Ambion) according to the manufacturer's instructions. WISH was performed according to previously published methods (39). For double in situ hybridization, anti-digoxigenin-peroxidase (POD) (11633716001, Roche) and anti-fluorescein-POD (11426346910, Roche) were used as primary antibodies to detect digoxigenin-labeled pinhead probe and fluorescein-labeled gsc probe, respectively.
Embryonic treatment
To block BMP signaling, embryos were treated with 10 M dorsomorphin (P5499, Sigma) or 5 M DMH1 (D8946, Sigma) at the 1K-cell stage in the dark, and then were collected at the shield stage for WISH.
Immunoprecipitation and Western blot analysis
For immunoprecipitation assays, HEK293T cells were transfected with the indicated plasmids. Cells were harvested 48 hours after transfection and lysed with TNE lysis buffer [10 mM tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, and 0.5% Nonidet P-40] containing a protease inhibitor cocktail. Immunoprecipitation assays were performed on lysates and collected CM as previously described (39).
Immunofluorescence analysis
Embryos were collected at the shield stage, fixed in 4% paraformaldehyde overnight, washed with PBS containing 0.1% Tween 20 for 30 min, blocked with 1% bovine serum albumin for 1 hour at room temperature, and then incubated with anti-phospho-Smad1/5/8 (1:200; 9511, Cell Signaling Technology) for 24 hours at 4°C. After washing with PBS three times for 5 min each, the samples were incubated with a donkey anti-rabbit secondary antibody conjugated to DyLight 594 (1:500; 711-585-152, Jackson ImmunoResearch) overnight at 4°C. All immunofluorescence images were captured using a Nikon A1R+ confocal microscope with the same settings for all samples within each experiment.
Prokaryotic expression and protein purification
An in-frame insertion of the cDNA of the full human Smad1 gene with a glutathione S-transferase (GST) tag into the plasmid pGEX-4 T-1 was performed, and the resulting recombinant plasmid was transformed into Escherichia coli strain BL21. GST-tagged Smad1 protein expression was induced using 1 mM isopropyl--dthiogalactopyranoside, and the resulting protein was purified with glutathione Sepharose 4B beads (GE Healthcare) according to the manufacturer's instructions. Protein was digested by thrombin and dialyzed against buffer I [10 mM tris-HCl (pH 7.5), 1 mM dithiothreitol (DTT), and 0.2 M NaCl] at 4°C overnight. Proteins were brought to a final concentration of 200 g/ml.
Electrophoretic mobility shift assays
Electrophoretic mobility shift assays were performed as follows. Mixtures of purified Smad1 (10 ng) protein and 0.5 ng of carboxyfluorescein (FAM)-labeled probes were incubated at room temperature for 30 min in a 10-l reaction volume. The reaction buffer contained 25% glycerol, 50 mM KCl, 0.5 mM EDTA, 10 mM DTT, and 5 mM tris-HCl and had a pH of 8.0. The mixtures were resolved on a 6% nondenaturing polyacrylamide gel (60:1 acrylamideto-bisacrylamide ratio) containing 5% glycerol in 0.5× tris-borate EDTA buffer. The gels were visualized using a Typhoon FLA9500 Scanner.
Dual luciferase reporter assays
For detection and quantification of pinhead or admp promoter activity in zebrafish embryos, the luciferase reporter construct DNA was mixed with Renilla luciferase reporter DNA in a ratio of 10:1. Wild-type and mutant embryos were injected with 100 pg of the DNA mixture at the one-cell stage. To inhibit BMP signal transduction, embryos were treated with or without 5 M DMH1 at the 1K-cell stage and then lysed with passive lysis buffer at the shield stage for detecting luciferase activities. Each luciferase reporter assay was performed in triplicate, and the data represent the mean ± SD of three independent biological repeats after normalization to Renilla activity.
Statistical analysis
Student's t tests (two-tailed, unequal variance) were performed to analyze all datasets (Microsoft Excel software). At a minimum, experiments were performed in triplicate. All the group values are expressed as the mean ± SD. Results were considered statistically significant at P < 0.05.
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/5/12/eaau6455/DC1 Fig. S1. Pinhead is a secreted BMP-like ligand. Fig. S2. Axial formation is normal in pinhead homozygous mutants. Fig. S3. DV polarity is not affected in admp homozygous mutants. Fig. S4. The activities of Admp and Chd are not obviously affected by the addition of C-terminal epitopes. Fig. S5. Simultaneous deletion of pinhead and admp causes defects in dorsal-and ventralrelated tissues. Fig. S6. Double inactivation of pinhead and admp induces severe dorsalization in embryos. Fig. S7. pinhead and admp have similar function to rescue the DV defects in ph49;ad11 mutants. Fig. S8. Overexpression of BMP2b in wild-type, ph49 mutant, or ad11 mutant embryos leads to similar ventralized phenotypes. Fig. S9. Potential Smad1/5-bound GC-rich elements in admp and pinhead enhancers. Fig. S10. pinhead and admp stabilize embryonic patterning in chd morphants. Data file S1. The uncropped blots for the Westerns and immunoprecipitations.
View/request a protocol for this paper from Bio-protocol.
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2020-01-02T21:11:23.010Z
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2019-12-01T00:00:00.000
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v3-fos-license
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A Comparative Review of UNCRC and Indian Legislation from the Child Mental Health Perspective
Background: The United Nations Convention on the Rights of the Child (UNCRC) is the most comprehensive set of standards promoting and protecting children’s interests. It can be utilized to create appropriate policies and legislation that enshrine the values identified in the UNCRC. In India, children have been considered only in the context of their family and were welfare recipients in the past, but more legislation has been enacted to protect and promote the child’s rights. A comparative review will help identify how the new legislation enacted after India ratified the UNCRC directly or indirectly addressed children’s mental health. Methodology: Legislation enacted after 1992 with the search term “child” was identified in the Indian national portal for legislation. These were compared against specific articles of the UNCRC identified to have a direct or indirect bearing on children’s mental health. Results: The review revealed that only 11 of the 32 legislation enacted after 1992 address different aspects of children’s mental health. Only three refer to the UNCRC in their preamble or content. Six of the 11 legislation addressed Article 24, while Article 32 and Article 34 were addressed in only one legislation each. Notably, most of the legislation is focused on child protection, while very few address the participation component of the guiding principles. Discussion: The UNCRC is a valuable guide to creating a legal framework to support child rights. This review highlights the need to consider children’s mental health as a fundamental right and incorporate the principles into future Indian legislation.
T he youth population of a country is considered its demographic dividend due to the economic payoffs of the working-age population entering the workforce.The potential of this valuable resource can be realized only if it is nurtured, protected, and allowed to develop to its fullest potential by providing good health, quality education, and decent employment. 1 Youth development is influenced by their immediate surroundings (microenvironment) and more prominent societal factors (macroenvironment).The macroenvironment, among other things, includes the legal frameworks and policies of a country, which directly influences young people's health and well-being.
Mental health is an essential component of well-being.Evidence suggests that childhood mental health problems are associated with more significant risks of school absenteeism, suicide, and self-harm in the short term. 2,3Further evidence suggests that in the long term, childhood psychological issues are associated with lower educational attainment, employment prospects, social mobility, marriage stability, and other psychological characteristics that enable social engagement compared to physical health problems. 4ow mental health has been viewed in children has undergone a cultural change, which has also been reflected in government policies.A review 5 summarized how a child's needs have been viewed over the years.Specifically on mental health, the authors mention that care, protection or treatment of his or her physical or mental health, to a periodic review of the treatment provided to the child and all other circumstances relevant to his or her placement."Holistically, Article 31 recognizes the right to rest and leisure.In contrast, Article 32 prevents exploitation that might harm a child's mental and social development, and Article 33 aims to protect children from the use of illicit drugs.
Translating the UNCRC into practical steps requires governments to use it as a guide to policymaking.India ratified the UNCRC on December 11, 1992.Over the years, in India, children have been acknowledged and afforded fundamental rights (see Box 1), and several there was initially a developmental nihilism with society not taking an active interest in setting goals for children as their development was considered to be due to immutable biological or social factors.Children's mental health needs in the past have been considered an optional or discretionary good.There are concerns of over medicalization of the mental health of children now.Most countries now consider a child's mental health and well-being as a right.
The history of the development of child rights internationally is relatively recent. 6In the aftermath of World War I, children were finally recognized as individuals in their own right rather than extensions of adults.This led to the League of Nations ratifying the Geneva Declaration in 1924, which, in its preamble, mentions, "Mankind owes to the child the best that it has to give."Though not a bill of rights but more a list of adults' obligations toward children, the declaration was the first international document that addressed children in their own rights.Following this, several international treaties and conventions addressed various aspects of child rights, including protection from exploitation and a right to education.In 1989, the UN General Assembly adopted the International Convention on the Rights of the Child(UNCRC), which set the minimum standards for protecting children's rights. 7The UNCRC has become the most widely ratified international document, with 196 countries ratifying it.
The UNCRC has 54 articles broadly classified under the four headings of principles, provisions, protection, and participation.The guiding principles of the UNCRC include the child's best interests, respect for the child's views, nondiscrimination, and survival and development.These rights are classified under the headings of right to survival, protection, participation, and development.The various rights are shown in Figure 1.While the right to health has been considered fundamental, children are especially vulnerable as they depend on adults to make decisions on their behalf.
Although all four headings mentioned above are relevant to the mental health, well-being, and development of children, some articles of the UNCRC specifically address these aspects.Article 19 mentions "appropriate measures to protect the child from all forms of physical or mental violence, injury or abuse, neglect or negligent treatment, maltreatment or exploitation."Article 23 states: "a mentally or physically disabled child should enjoy a full and decent life…preventive health care and of medical, psychological and functional treatment of disabled children."Furthermore, Article 24 deals with "the highest attainable standard of health and to the facilities for the treatment of illness and rehabilitation of health." Article 25 addresses the right of the "child who has been placed by the competent authorities for the purposes of Constitutional Rights Afforded to Children in the Indian Constitution.
Review Article
laws have been created or amended to address the needs of children to protect their interests directly.We aimed to review the overlap of the UNCRC and the legislative policies in India directed toward children.Our focus would be restricted to legislations implemented at the central governmental level and will mention aspects of child mental health.
Method
First, both the authors familiarized themselves with all the articles of the UNCRC.Following this, a keyword search for "Child" with limits of Central Acts was conducted in the national portal for Indian legislation-www.indiacode.nic.in-inMay 2023.The results were restricted to Acts that were active and enacted after December 1992 (when India ratified the UNCRC).Following this, both authors extracted the full text of the legislation and independently reviewed it for information related to children's mental health or UNCRC.Consensus between the authors determined any disagreement in the selection of legislation.Once the legislation was selected, the information from the legislation was mapped onto the various articles of the UNCRC.For this review, we restricted the mapping exercise to a few Articles (Articles 19, 23, 24, 25, 29, 31, 32, 33, and 34) of the UNCRC that directly address children's mental health and well-being.A comparative analysis assessed how India's legislation protects and addresses children's rights, especially regarding their mental health.
Results
A search with the keyword resulted in 98 Central Acts on the website.Of these, 32 were enacted after December 1992.Of these, 11 (shown in Table 1) were selected based on whether mental or emotional health was mentioned in the legislation or addressed factors directly related to children's mental health.Only three pieces of legislation-the Commissions for Protection of Child Rights Act, 2005 8 (not included in the review), the Protection of Children from Sexual Offences (POCSO) Act 2012], 9 and the Juvenile Justice (Care and Protection of Children) (JJ) Act 2015 10 -cited the UNCRC in their preamble.
The definition of child varied across the different Central Acts.The Protection of Women from Domestic Violence Act 2005, 11 the POCSO Act 2012, 9 the JJ Act 2015, and the Mental Healthcare Act (MHCA), 2017 12 define a child as any person below the age of 18 years.The Prohibition of Child Marriage Act, 2006 13 identifies a child as a male who has not completed 21 years of age and a female who has not completed 18 years of age.The Right of Children to Free and Compulsory Education (RTE) Act, 2009 14 addresses children as individuals between the ages of 6 and 14 years.
Four legislations directly address protecting children from all kinds of violence, negligence, or exploitation (Article 19).The Protection of Women from Domestic Violence Act 2005 includes abuse or neglect directed toward a child (including adopted, foster, or stepchild) in the family and the aggrieved person.This Act also provides a legal recourse to protect the child from harm by restricting access through protection or custody orders.The RTE Act and the JJ Act categorically prohibit the use of physical disciplining and mental harassment in the academic setting and within childcare institutions.The JJ Act also prescribes that the JJ Board can deny a child bail if there is concern that the child would be exposed to moral, physical, or psychological danger and determines the punishment to be dealt to an individual for cruelty to a child.The MHCA sets a higher standard to ensure the safety of minors in mental health institutions and for specific treatments such as modified electro-convulsive therapy.
Article 23 protects the interests and rights of mentally and physically disabled children.The Manipur Panchayati Raj Act 1994 15 emphasizes the role of the local governance (Gram and Zilla Panchayats) to ensure that social welfare programs are created and implemented for "handicapped and mentally retarded persons."The Rights of Persons with Disabilities (PWD) Act 2016 16 takes a rights-based and participatory approach to support women and children with various disabilities.The JJ Act 2015 prescribes that if children with disabilities are harmed, the accused should bear twice the penalty for the offense committed.
Article 24 of the UNCRC gives the right to the highest attainable standard of health for children, and access to mental health care for children is enshrined in the MHCA.The Manipur Panchayati Raj Act 1994, the Manipur Municipalities Act 1994, 17 the New Delhi Municipal Council Act 1994, 18 and the Cantonments Act 2006 19 emphasize the need for the local governments to create and maintain child welfare clinics.One of the discretionary requirements is the construction and maintenance of children's homes for the vulnerable and disabled.The JJ Act identifies mental health care with behavior modification, counseling, and psychiatric support as integral to reforming children in conflict with the law.Article 25 is addressed in the JJ Act and the MHCA with respect to the treatment of mental health conditions of children in custodial settings.The care of children who need inpatient mental health services from childcare institutions is addressed in the JJ Act and the MHCA.
The development of a child's abilities and talents (Article 29) is the focus of the RTE Act, focusing on providing an opportunity (free and compulsory education) and encouraging an all-round evaluation and development of the child.The JJ Act sponsors families and institutions that cannot provide for the children's development and makes it an essential part of the rehabilitation and reintegration services.The PWD Act makes it the duty of the educational institutions to identify such children, provide them appropriate support, and monitor participation and progress of every student with disabilities with a focus on inclusion.This is also reflected in the Transgender Persons (Protection of Rights) Act 2019, 20 which focuses on inclusive educational institutions for gender-diverse individuals.
Article 31 is addressed in the Manipur Panchayati Raj Act 1994, where the responsibility of creating opportunities to participate in sports and games lies with the Zilla Parishad.As part of the rehabilitation and reintegration of children, the JJ Act recommends the provision of sports and cultural activities.The Cantonments Act 2006 and the JJ Act prescribe penalties for individuals who attempt to provide any alcohol or intoxicating substance to children, thereby supporting Article 33 of the UNCRC.
Of all the Articles of the UNCRC, Article 32 (right to be protected from • Section 12-Bail to a person who is apparently a child alleged to be in conflict with the law-… Provided that such person shall not be so released if there appears reasonable grounds for believing that the release is likely to … or expose the said person moral, physical or psychological danger … • Section 75-Punishment for cruelty to child-Whoever, having the actual charge of, or control over, a child, assaults, abandons, abuses, exposes or wilfully neglects the child or causes or procures the child to be assaulted, abandoned, abused, exposed or neglected in a manner likely to cause such child unnecessary mental or physical suffering, shall be punishable
•
Section 82-Corporal punishment-Any person in-charge of or employed in a child care institution, who subjects a child to corporal punishment with the aim of disciplining the child, shall be liable…
•
The Mental Healthcare Act, 2017 • Section 87-Admission of minor-A minor may be admitted to a mental health establishment only after following the procedure laid down in this section.
• Section 95-Prohibited procedures-electro-convulsive therapy for minors; if in the opinion of psychiatrist in charge of a minor's treatment, electro-convulsive therapy is required, then, such treatment shall be done with the informed consent of the guardian and prior permission of the concerned Board.• Section 85-Offences committed on disabled children-Whoever commits any of the offences referred to in this Chapter on any child who is disabled (as defined in the PDA ACT) as so certified by a medical practitioner, then, such person shall be liable to twice the penalty provided for such offence.• Section 18 (g)-Orders regarding child found to be in conflict with law-direct the child to be sent to a special home, for such period, not exceeding three years, as it thinks fit, for providing reformative services including education, skill development, counselling, behaviour modification therapy, and psychiatric support during the period of stay in the special home • Section 92-Placement of a child suffering from disease requiring prolonged medical treatment in an approved place-… Board, is found to be suffering from a disease requiring prolonged medical treatment or physical or mental complaint that will respond to treatment, the Committee or the Board, as the case may be, may send the child to any place recognised as a fit facility…
•
Section 93-Transfer of a child who is mentally ill or addicted to alcohol or other drugs-… that any child kept in a special home or an observation home or a Children's Home or in an institution in pursuance of the provisions of this Act, is a mentally ill person or addicted to alcohol or other drugs which lead to behavioural changes in a person, the Committee or the Board, may order removal of such child to a psychiatric hospital or psychiatric nursing home…
•
The Mental Healthcare Act, 2017 • Section 65 (2; 22; iv)-Functions of the Zilla Parishad-Encourage sports and games and construction of rural stadia
•
The Juvenile Justice (Care and Protection of Children) Act, 2015 • Section 53 (1, vii)-Rehabilitation and re-integration services in institutions registered under this Act-recreational activities including sports and cultural activities Article 32-the right of a child to be protected from economic exploitation and from performing any work that is likely to be hazardous or to interfere with the child's education, or to be harmful to the child's health or physical, mental, spiritual, moral, or social development
•
The Juvenile Justice (Care and Protection of Children) Act, 2015 • Section 76-Employment of child for begging-Whoever employs or uses any child for the purpose of begging or causes any child to beg shall be punishable…
•
Section 79-Exploitation of a child employee-Notwithstanding anything contained in any law for the time being in force, whoever ostensibly engages a child and keeps him in bondage for the purpose of employment or withholds his earnings or uses such earning for his own purposes shall be punishable… Article 33-to protect children from the illicit use of narcotic drugs and psychotropic substances
•
The Cantonments Act, 2006 • Section 285-Unauthorized sale of spirituous liquor or intoxicating drug-fine and/or imprisonment for military personnel offering or attempting to barter, sell or supply any spirituous liquor or intoxicating drug for the use of minor child
•
The Juvenile Justice (Care and Protection of Children) Act, 2015 • Section 77-Penalty for giving intoxicating liquor or narcotic drug or psychotropic substance to a child-Whoever gives, or causes to be given, to any child any intoxicating liquor or any narcotic drug or tobacco products or psychotropic substance, except on the order of a duly qualified medical practitioner, shall be punishable Article 34-to protect a child from all forms of sexual exploitation and sexual abuse
•
The Protection of Children from Sexual Offences Act, 2012 • Section 3, Section 5, Section 7 and Section 9-Defines Penetrative sexual assault, aggravated penetrative sexual assault, Sexual assault and Aggravated sexual assault respectively.
• Section 13-Defines use of child for pornographic purposes and section 14 the punishment for using child for pornographic purposes exploitation) and Article 34 (protection of children from all forms of sexual exploitation and abuse) are represented in only one legislation each.In the description of the Children in Need of Care and Protection, the JJ Act protects a child from either begging or exploitation and identifies it as a punishable offense.Similarly, various sections of the POCSO Act define the different kinds of sexual assaults and the associated punishments.Additionally, a separate section addresses the punishment for using children for pornographic purposes.It is essential to recognize that the POCSO Act defines aggravated penetrative and aggravated sexual assaults as something "that causes the child to become mentally ill."
Discussion
This comparative review highlights the interplay between the international standards (as established by the UNCRC) and the legislation in India, specifically focusing on child mental health.One key finding is that nearly a third (11 of 32) of the laws enacted from 1992 that address some aspect of children have any direct or indirect reference to their mental health.One pattern that does emerge is that even though the initial few laws were focused on protecting the well-being of children, the latter laws (e.g., the JJ Act, MHCA) specifically take a rights-based approach to children's mental health, as has been discussed in a discussion paper by UNICEF. 21This is also witnessed in the choice of words that were used to describe children with disabilities, such as "handicapped and mentally retarded" being used in earlier laws and persons with disabilities in the latter ones.Mental health is a fundamental right, as stated in the preamble of the World Health Organization.Health is defined as a positive concept-"the complete state of physical, emotional and social wellbeing". 22Even though Article 31 of the UNCRC discusses the need for rest and leisure, only two Acts categorically provide a legal mandate to implement this.Mental health as a positive concept has also been acknowledged in the Sustainable Developmental Goals (SDG) 3 of Good Health and Wellbeing.This is further clarified under the target SDG 3.4-"to reduce by one-third premature mortality from non-communicable diseases through prevention and treatment and promote mental health and well-being"-and SDG 3.5-"strengthen the prevention and treatment of substance abuse, including narcotic drug abuse and harmful use of alcohol". 23The impact on children's mental health problems cannot be overstated, as evidenced by the large-scale population-based study in Britain, which showed that mental health problems in childhood affect their economic abilities, marriage stability, and overall social mobility. 4ur review using systematic search terms and a focus on mental health revealed that only 11 of the Acts implemented at the center have aligned themselves with the UNCRC.In comparison, when Article 24 alone was considered broadly in a review, only four countries in the Eurozone were noted to have enshrined in their legislation a specific legal disposition for universal health care for all children, irrespective of their legal status. 24Several barriers have been identified to providing appropriate health care for children in India of all ages.These include structural issues such as poverty, economic inequality, illiteracy, misinformation, inadequate allocation of resources, and programmatic issues such as the implementation of programs. 25he principles of the UNCRC include the right to participation, protection, development, and survival.While the right to participation is a part of the UNCRC, only the JJ Act mentions participation, even when not under the heading of mental health care.The MHCA and the JJ Act take on a more paternalistic role in the involvement of children in their care, for example, having the parents/guardians of minors make decisions about the child's treatment.There is a description of child welfare activities and fostering sports, games, and cultural activities in the laws that focus on children's holistic development.One of the review's key findings was that a special emphasis was placed on promoting health and development among children with disabilities.
Considering the perspective of addressing the social determinants of mental health, 26 the laws reviewed here show a significant initiative of the government to keep children safe from some psychosocial stressors.In almost every scenario where children are present-either in the presence of caregivers and academic institutions or under the government's care-the laws protect them from physical, sexual, or emotional harm.The protection of children from drugs and intoxicants is also mentioned in the laws.
This article highlights where the law has not updated the current knowledge.For example, the POCSO Act mentions the effects of aggravated sexual assault on the victim's mental health without acknowledging the effects of other forms of victimization.For example, bullying (including eve teasing), stalking (including cyber-stalking), verbal violence, and discrimination toward females and gender-diverse individuals also have a significant association with children developing mental health issues. 27These non-contact forms of sexual harassment are implied to be subsumed under the mental health effects of sexual harassment.
As seen in the "Results" section, there is a wide variation in the definition of children's age that the specific laws address.While this might be appropriate for some laws, such as the RTE, that address early childhood education, it may criminalize typical adolescent phenomena such as consensual sexual exploration. 28This may lead to confusion in implementing a specific policy that provides protection and services to children.The laws are also silent regarding consent for medical procedures and engagement in research commensurate with the child's developmental abilities.This is important as both clinical and research work with children about conditions that specifically affect children or have their onset in childhood are addressed differently than those emerging later in life.
One of the major limitations of this study is that no rules of implementation, policies, or state laws were reviewed.These could have given more information about how the UNCRC has been implemented in India.Furthermore, how the UNCRC is interpreted in case law gives the current trends in its implementation.This was also not explored as a part of this review.This work's future directions should include these legal and policy framework components to understand the actual implementation of UNCRC.This would also help understand the impact of the legislation on the overall mental health and well-being of children.Some legislation is harmonized with the United Nations Commission on the Rights of Persons with Disabilities (UNCRPD).This review did not pursue the UNCRC and the UNCRPD intersection, which could have highlighted the overlap between these related standards.This review also highlights the need for clinicians to be aware of the changing trends in how the law interprets child rights in the context of mental health and in advocating for children's rights under the UNCRC framework.
This article paves the path for furthering knowledge by assessing the impact of legislation on children's mental health outcomes.Furthermore, improving age definitions ensures avoiding confusion and consistency in protecting their rights.The authors would caution that while the UNCRC provides useful guidance for enacting child-focused legislation, it must always be considered in the context of the prevailing local conditions.This is especially true in a country like India, with a strong and extended family structure to support children.This is an evolving and modifiable phenomenon, as evidenced by India's legislative history.Thus, policy advocacy with a participatory model comprising all stakeholders, including youth, families, community leaders, and mental health professionals, would be the logical way to influence the development of appropriate child-focused mental health policies.Creating specific guidance for mental health professionals on evaluating and engaging young people in decision-making will ensure the appropriate implementation of the UNCRC at all levels of care.
Conclusion
"Mankind owes to the child the best that it has to give" cannot be stated without ensuring that children have access to the highest level of mental health-focusing on their growth and development and addressing their mental health concerns.
India has made significant progress in implementing the UNCRC at the legislative level.Still, the impact is not yet felt by the persons implementing/delivering mental health-related interventions such as health prevention, promotion, and treatment of mental health conditions.At the clinical level, care remains paternalistic.Implementing the value of respecting a child's view would include active participation in their care through discussions and delivering positive mental health interventions in a participatory model with youth from different communities.
Furthermore, this article also highlights the need for all stakeholders including health professionals and policy makers to work collaboratively to create and implement policies that aid children's mental health.This will enable us to realize the greater goal of the UNCRC, which envisions a world where children are valued, protected, and empowered to participate in society as active and responsible citizens.
FIGURE 1 .
FIGURE 1.Rights of the Children as Mentioned in the UNCRC.
Constitution • Article 21A-Right to education for children between 6 and 14 years: "The State shall provide free and compulsory education to all children of the age of six to fourteen years in such manner as the State may, by law, determine."• Article 24-Children below the age of 14 years shall not work in any hazardous employment: "No child below the age of fourteen years shall be employed to work in any factory or mine or engaged in any other hazardous employment."• Article 39(e)-Children should not be abused: "… and the tender age of children are not abused and that citizens are not forced by economic necessity to enter avocations unsuited to their age or strength."• Article 39(f)-Children are to be afforded opportunities and facilities to develop: "that children are given opportunities and facilities to develop in a healthy manner and in conditions of freedom and dignity and that childhood and youth are protected against exploitation and against moral and material abandonment."• Article 45-State should provide early childhood education: "The State shall endeavour to provide … for free and compulsory education for all children until they complete the age of fourteen years."
Article 23 -
mentally or physically disabled child should enjoy a full and decent life, in conditions which ensure dignity, promote self-reliance, and facilitate the child's active participation in the community • The Manipur Panchayati Raj Act, 1994 • Section 35 (20)-Role of the Gram Panchayat-Social welfare including the welfare of the handicapped and mentally retarded • Section 65 (2; 17; i)-Functions of the Zilla Parishad-Promotion of social welfare program and activities with an emphasis on handicapped and mentally retarded persons.• The Juvenile Justice (Care and Protection of Children) Act, 2015
• 2016 • Section 4 -•••
The Rights of Persons with Disabilities Act, Women and children with disabilities-… ensure that all children with disabilities shall have right on an equal basis to freely express their views on all matters affecting them and provide them appropriate support keeping in view their age and disability•Section 24 (2, b)-Social Security-facilities for persons including children with disabilities who have no family or have been abandoned or are without shelter or livelihood Article 24-enjoyment of the highest attainable standard of health and to facilities for the treatment of illness and rehabilitation of health • The Manipur Panchayati Raj Act, 1994 • Section 35 (19)-Role of the Gram Panchayat-Participation and implementation of women and child welfare programs, promotion of school health and nutrition programs • Section 65 (2; 14; iii)-Functions of the Zilla Parishad-Maternity and child health service activities • The Manipur Municipalities Act, 1994 • Section 39 (r)-Discretionary functions of the municipality-housing and maintaining destitute, orphans, and cripples and child welfare clinics • The New Delhi Municipal Council Act, 1994 Section 11 (j)-Obligatory functions of the council-the establishment and maintenance of … and Child welfare centers … Section 12 (l)-Discretionary functions of the council-the construction and maintenance of-children's homes, houses for the deaf and dumb and for disabled and handicapped children Section 62-Duties of the Board-so far as the funds at its disposal permit to make reasonable provisions within the cantonment for-establishing and maintaining child welfare centers • Section 63-Discretionary functions of the board-the construction and maintenance of-children's homes, houses for the deaf and dumb and for disabled and handicapped children • The Juvenile Justice (Care and Protection of Children) Act, 2015
TABLE 1 .
UNCRC-Indian Legislation ComparisonTable.Definition of domestic violence-economic abuse includes deprivation of all or any economic or financial resources … for the aggrieved person or her children; disposal of household effects which may be reasonably required by the aggrieved person or her children Custody orders-if the Magistrate is of the opinion that any visit of the respondent may be harmful to the interest of the child or children may refuse to allow such visit.
• Section 18-protection orders-... Magistrate may pass a protection order prohibiting respondent from ... entering the school (if the person aggrieved is a child) • Section 21-
•
Section 19-Power of Children's Court-Provided that the reformative services including educational services, skill development, alternative therapy such as counselling, behaviour modification therapy, and psychiatric support shall be provided to the child during the period of his stay in the place of safety • Section 37-Orders passed regarding a child in need of care and protection-directions to persons or institutions or facilities in whose care the child is placed, regarding care, protection and rehabilitation of the child, including … services such as medical attention, psychiatric and psychological support including need-based counselling, occupational therapy or behaviour modification therapy, skill training, legal aid, educational services, and other developmental activities, as required, …
•
Section 104-Persons in Custodial institutions-If it appears to the person in-charge of a State run custodial institution (including beggars homes, orphanages, women's protection homes and children homes) that any resident of the institution has, or is likely to have, a mental illness, then, he shall take such resident of the institution to the nearest mental health establishment run or funded by the appropriate Government for assessment and treatment, as necessary Section 3-every child of the age of six to fourteen years including a child referred to in clause e (children with disabilities) shall have the right to free and compulsory education in a neighborhood school till completion of his/ Rehabilitation and re-integration services in institutions registered under this Actappropriate education, including supplementary education, special education, and appropriate education for children with special needs: Provided that for children between the age of six to fourteen years, the provisions of the Right of Children to Free and Compulsory Education Act, 2009 (35 of 2009) shall apply; skill development; Occupational therapy and life skill education Section 16 (iv, vi, vii)-Duty of educational institutions-provide necessary support individualised or otherwise in environments that maximise academic and social development consistent with the goal of full inclusion; detect specific learning disabilities in children at the earliest and take suitable pedagogical and other measures to overcome them; monitor participation, progress in terms of attainment levels and completion of education in respect of every student with disability • Section 31-Free education for children with benchmark disabilities-Notwithstanding anything contained in the Rights of Children to Free and Compulsory Education Act, 2009 (35 of 2009), every child with benchmark disability between the age of six to eighteen years shall have the right to free education in a neighbourhood school, or in a special school, of his choice; … that every child with benchmark disability has access to free education in an appropriate environment till he attains the age of eighteen years Obligation of educational institutions to provide inclusive education to transgender persons-… shall provide inclusive education and opportunities for sports, recreation and leisure activities to transgender persons without discrimination on an equal basis with others
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2024-02-13T16:03:47.585Z
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2024-02-11T00:00:00.000
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133237963
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pes2o/s2orc
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v3-fos-license
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Monitoring The Land Accretion Development at Coastal Area of Blanakan, Subang Indonesia
A land accretion is formed by deposition in estuaries. Recently, a land development in Subang coastal area has raised an increase. Beside its potential, coastal areas are also threatened with damage including abrasion, accretion, loss of mangrove forests, and sea water intrusion. One of the coastal areas that have been arising in very extensive land is Blanakan coastal in Subang Regency. This study aims to monitor the development of a land accretion that have been arise during the period of 1990 to 2015 and also to examine the use of a land accretion and analyze the impact of a land accretion to the social and economic conditions in the Blanakan Coastal Areas. The method used in this research was descriptive quantitative method. In this research, The Landsat imageries were overlaid came from 1990, 2000, 2010, and 2015 to determine the development of a land accretion. Based on the results of Landsat imagery overlaid over the period 1990-2015. Overall, during the period 1990-2015, accreted land formed was an area of 782.9 hectares and abrasion area of 73.3 hectares with changes in the most far reaching 1580.3 m. The use of land accretion in the Blanakan Coastal mostly used for a fishpond with a key commodity is Milkfish and Bago shrimps. The impact of land accretion to the social and economic conditions was reflected through the five indicators such as livelihoods, income, education, health, and ownership of assets.
Introduction
Coastland is an area of encounter between land and sea, in which a mutual relation still takes place1. As an archipelago state with the coastline reaching 99.093 km, the potential of coastline area in Indonesia is very vast and advantageous for various economic activities2. One of the provinces possessing potential along the coastline being currently developed is the province of West Java 3.The length of coastline in the province of West Java extending in the northern part from Cirebon district to Bekasi district is approximately 365 km long and in the southern part from Ciamis district to Sukabumi district is around 355 km.
Subang district is geographically located in the northern part of the province of West Java. Of all existing sub-districts, there are four coastal districts, namely Blanakan, Sukasari, LegonKulon, and Pusakanagara sub-districts. The length of coastline in the coast of Subang district reaches the number of 68 km 4. The utilization of coastal areas to sustain people's economic activities frequently causes destructions, such as abrasion, accretion, seawater intrusion, and the decreasing number of mangrove forests and the damage of coral reefs 5, 6. Several beaches in Subang district have been objected to abrasion as in Muara Karang -Legon Wetan until Pondok Bali, as well as along the area of Patimban. Sedimentation happened in the estuary indicated with deltas throughout the river as the aftermath of land accretion along the coastline. BPLHD reported that the furthest abrasion occurred in Indramayu sub-district of 48.57 km, whereas the longest accretion ensued along Subang Beach reaching the length of 50.44 km7.
Land accretion is a land formed by sedimentation at river mouth. In Subang district, the widest land accretion was formed in Blanakan sub-district with the average of 686.53 m, while the land accretion formed in Pusakanagara sub-district had the average 159.32 -538.50 m 7,8. Up to the present time, the land accretion in Blanakan coast is only used as an embankment. However, the utilization has not been maximized because it is hampered by weather or high tides that it causes crop failure. The measurement of the width of the land accretion was lastly conducted by BPLHD West Java in 2004 which mentioned that the land accretion in Subang district was 6000 Ha in 2002. Furthermore, there is no government agency that conducts the land accretion measurement. The measurement is usually performed by companies having business in the area9. Based on the facts mentioned, the land accretion measurement is required so it can be utilized optimally to bolster the community's economic needs and local revenue. Moreover, this research will analyze the usage of land accretion by the community. The effects of the existence of the land accretion on social and economic condition of people in Blanakan Coast will also be identified.
Methods
The method used in this research is the descriptive quantitative method. The population in this study was Blanakan sub-district. The sample in this study was a village with land accretion consisting of 7 villages, such as Cilamaya Girang, Rawameneng, Jayamukti, Blanakan, Langensari, Muara, and Tanjungtiga. As for the number of inhabitant sample was counted using purposive sampling with Slovin formulary; that is of 99 people.
The data recollection technique in this research was study of literature, study of documentation, interview, and field observation. The data processing technique was divided into 2; the first one was Landsat imagery interpretation covering RBG transformation, image enhancement, and cropping. Meanwhile the second one was social data processing performed by conducting interviews, tabulation, and creating tables and graphics.
The data analysis technique in this study was separated into 2, which were to analyze the development of the land accretion using perpendicular grid to measure the distance of development of the land accretion throughout 1990 -2015. The illustration of the usage of perpendicular grid can be seen in picture 2. As for the utilization of the land accretion area and the effects of existence of the land accretion on socioeconomic condition was conducted using cross-tabulations with parameters of income, livelihood, education, and ownership of assets/facilities. table 1 and table 2. The most significant accretion is seen in the coastal area in the western of Ciasem River, covering CilamayaGirang, Rawameneng, Jayamukti, Blanakan, Langensari, and Muara villages. The area of the land accretion forms throughout 1990 -2015 is 739.6 Ha. Besides, abrasion happened during 1990-2000 was 18.6 Ha wide. Meanwhile, at the eastern side of Ciasem River, there is only Tanjungtiga village and the most dominant dynamic was the abrasion throughout 1990-2010 that was 51,9 Ha in. Apart from abrasion, in the period of 2010-2015, land accretion is also formed but its area is not as enormous as it is in the coastal area in the western part of Ciasem River, of 47.2 Ha. Averagely, throughout 25 years, there has been land accretion formed with the total in width 786.8 Ha; roughly 31.5 Ha of land accretion is formed each year.
The land accretion formed in Blanakan coast is caused by over sediment runoff from Ciasem River which is then pushed by the stream towards the western part, proven by the wind rose resulted in data recordings from 1996 until 2010 in northern Subang beach which indicated dominant wind facing to west and southwest. Moreover, because the effects of wind, the sedimentation happening in the study areas in Blanakan coast is also influenced by the fact that the western side of Subang district coast is more slope compared to the coast in the eastern part. The sediment is then trapped in Blanakan subdistrict bay 7. Sedimentation is also influenced by the changing of coastline shape. Moreover, the coastal water is also affected by the dynamics of interaction between water inputs from the sea and freshwater from the river 10.
The western part of Blanakan Coast (bordered by Ciasem River) is an area with the widest area of land accretion. This is because its morphology which is shaped as a bay (protruded inside) and slope. This protruded inside and slope shape is what causes sediment from Patimban Beach, Pamanukan is easily trapped in the area and formed a land accretion. Apart from the morphology, there are also rivers with small estuary that all sediments coming from various directions will be collected and deposited there. As for the eastern part of Blanakan, there is only Tanjungtiga village which for the period of 1990 -2010 had undergone fairly great abrasion. Its morphological shape which is not too protruded (relatively even) to the sea eases the strong sea stream and high tidal wave to move pass it, eroding the land. Furthermore, the decreasing of mangrove forests density has reduced its function as a wave retainer that the abrasion was difficult to withstand 7.
Sample
Length (
The Utilization of the Land Accretion Area in Blanakan Coast in Blanakan Coast
The existence of land accretion is utilized by coastal communities to open an embankment to provide their economic needs. All land accretion areas in Blanakan Coast is used as embankments. According to juridical law, land accretions belong to Perhutani. However, in reality, Perhutani allows the people to work on them under the circumstances that they have to care about the sustainability of ecosystems there. To run the land accretion, people need to administer licensing letters by paying around IDR 500.000 -IDR 2.000.000 to Perhutani but through village head. By paying the money, people normally acquire 2 Ha of land for each. Since as time passes the land accretion formed is getting many, the number of people interested in managing them is increased, too. Therefore, Perhutani cooperates with local community to set up an Institution of Forest Villagers/Lembaga Masyarakat Desa Hutan (LMDH) in every village. LMDH functions as a bridge between Perhutani and the society.
The Impact of Land Accretion on Social and Economic Condition of Blanakan Coast Society
The existence of land accretion has provided several impacts on social and economic condition of Blanakan Coast Society, Subang District. Accreted land helps people in the society to satisfy their primary needs. The socioeconomic condition can be identified using 5 indicators, i.e. livelihood, income, education, health, and ownership of assets. Majority of people who live in Blanakan Coast are the owners of as well as the workers at embankments with varied embankment area ranging around 1-5 Ha.
Figure 5. The embankmentarea with main and side income
People who own embankments with the area of 1-5 Ha commonly have side livelihood to meet their necessities, such as farmers, fishermen, farmers, sellers at markets, and owners of small shops. On the other hand, the owners of embankments with the area of above 5 Ha usually own rice fields as side livelihood. The wider the embankments one has, the higher their income is. The main income of < IDR 20,000,000 places first belonging to embankment owners with an area of 1-5 Ha. The most side income is found at the group of farmers whose embankments are of 1-5 Ha, as what has been discussed earlier that it is the group with most side livelihoods. Embankment farmers whose income is < IDR 20.000.000 have more schooled compared to other income groups. Found in this group are children with level of education ranging from Primary School, Junior High School, Senior High School, and University. All respondents involved in this research state that they wish their children should be educated until attending the university so they can get better professions and income compared to their parents. As for the indicator of health, all respondents state that they have had their own lavatory even though the condition is still varied, from the ones that are feasible to use and the less feasible ones. The awareness to live healthily has been decent. The last socioeconomic which is the ownership of assets, are separated into the ownership of residence, ownership of communication means, and ownership of transportations. Of all respondents, only two of them rent their house, typically newcomers who come as embankment workers. For the ownership of communication means, the lesser the farmers' income is, the more limited the communication means they have. Most of them only have televisions and cellphones. On the other hand, the group of embankment farmers whose income is >IDR 80,000,000 owns more varies. Similar to the ownership of communication means, the lesser the embankment farmers' income is, the fewer the vehicles they have. Nearly all of the respondents ride motorbikes and in the group with the income of > IDR 20,000,000, the highest ownership of transportation means belongs to motorbikes group. Meanwhile the most varied ownership of transportation means belong to the group of embankment farmers with the income of IDR 41.000.000 -IDR 60.000.000, that are bicycles, motorbikes, and cars. health. For the government of Subang Regency, this research can be considered as a recommendation on the protection of coastal areas, especially in deciding zones of land utilization such as embankments and mangrove. This is to avoid conflicts which may arise when the area is managed by local community.
|
2019-04-26T14:24:43.718Z
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2016-11-01T00:00:00.000
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125729868
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pes2o/s2orc
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v3-fos-license
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Experimental studies of laminar-turbulent transition on a body of revolution
The focus of the present paper is hydrodynamic stability and transition to turbulence on an axisymmetric body. The objective is to trace the evolution of perturbed flow close to the surface of experimental model with increase of the angle of attack starting with zero incidence. In what follows, we briefly summarize our wind-tunnel data on this topic which were obtained at low subsonic velocities through hot-wire measurements and flow visualization. As is found, in conditions of laminar boundary-layer separation and flow instability, even small variations of the body incidence may have a profound effect on the flow pattern.
Motivation
A subject of long-term studies in subsonic fluid dynamics is the spatio-temporal structure of unstable flow on bodies of revolution. An axisymmetric configuration at zero angle-of-attack of the body represents a canonical one which was examined in recent decades. As a result, a lot of research data have been obtained which clarify the mean and nonstationary flow characteristics in conditions of the hydrodynamic instability initiating the growth of velocity perturbations, vortex formation, and transition to turbulence, see e.g. [1−10].
Indeed, an idea about the axial symmetry is appropriate and really helpful at investigation of the instability phenomena. However, at a non-zero angle of attack the flow becomes three-dimensional [11] so that the determination of its quantitative characteristics both in experiments and calculations becomes much more complicated. In this context, an issue is how large the deviation of the body position from zero incidence should be to make the axisymmetric approximation to the unstable flow no longer valid. This is the point which we are interested in here most of all. Note that in the present experimental configuration we deal with laminar boundary layer separation from the body surface. One expects, similar flow behavior on bodies of revolution is typical of quiet environmental conditions and fairly low Reynolds numbers.
Experimental arrangement
The experimental runs were performed in the subsonic T-324 wind tunnel of ITAM SB RAS which is a low-turbulent closed-circuit facility. The closed test section of the tunnel is of 4-m length with a square cross section of 1 × 1 m 2 , the free-stream turbulence level making Tu ≤ 0.04%. The test model sketched in figure 1 represented an axisymmetric body, 1140-mm in length and of 100-mm midsection radius. During the experiments the model was installed at different angles of attack ranging from 0 to 20° with an accuracy of 0.1°.
Quantitative data on the flow pattern over the body of revolution were obtained through hot-wire measurements using an AN 1003 constant-temperature anemometer from A.A. Lab Systems Ltd and a one-wire probe which was calibrated in the free stream against a Pitot-static tube. The probe was positioned in the measurement region by an automated 3D traversing mechanism. The hot-wire readings were digitized by a 14-bit analog-to-digital converter and further processed on a PC in MATLAB environment. Besides, visualization of the mean near-wall flow was carried out using a mixture of a titanium dioxide powder with kerosene which was applied onto the axisymmetric body. The data were acquired at the Reynolds number Re r = U 0 r/ν ranging from 63000 to 126000 where U 0 is the oncoming flow velocity varying from 10 to 20 m/s and r is the midsection radius of the experimental model. In what follows, x is the streamwise coordinate measured from the nose of axisymmetric body and y is the radial distance from the wall.
Wind-tunnel results
Naturally, at zero angle of attack an axisymmetric flow over the model is observed. In the present experimental conditions the laminar boundary-layer separates in the aft part of the body. The meanvelocity contours plotted in figure 2(a) demonstrate a local region of flow separation which is prone to instability. The streamwise growth of velocity perturbations in the separated shear layer results in the laminar-turbulent transition. High amplitudes of the disturbances making about 10 to 15 percent of U 0 in the downstream sections of the measurement domain are typical of the near-wall turbulence, see figure 2(b). In more detail, the stability characteristics of the present axisymmetric flow were reported in [12]. In particular, the behavior of small-amplitude oscillations was found to be in a quantitative agreement with that of the instability waves at laminar boundary-layer separation in plane configurations. The axial symmetry of the mean flow breaks down when passing from zero angle of attack to a small incidence of several degrees [13]. In this case, laminar flow separation still occurs on the windward side of the body while the boundary layer is attached to the surface on the leeward side, see figure 3. Because of much different stability characteristics of the attached flow and of the separated boundary layer, a pronounced azimuthal nonuniformity of the amplifying velocity perturbations is generated. One can observe in figure 4 that at the angles of attack = 2º and 4º the amplitudes of laminar flow disturbances on the windward and leeward sides of the body in some cross sections differ by an order of magnitude. Even more distinct transformation of the unstable flow around the model occurs when the angle of attack is further increased and the local boundary layer separation is replaced with the global one. Then, in a fully 3D separated flow the reliability of hot-wire measurements with one-wire probes goes down. Instead, visualization by surface coatings, although providing qualitative data only, is helpful to obtain a panoramic pattern. An example is given in figure 5. At the angles of attack = 10º and 20º the separated flow is featured with a pair of large-scale streamwise vortices originating in the midchord ( figure 5(a)) and in the upstream ( figure 5(b)) sections of the model. Similar vortex behavior of the flow past axisymmetric bodies at incidence was observed in a number of studies, for graphic illustrations see [14].
Concluding remarks
The above wind-tunnel results illustrate a variety of flow regimes on a body of revolution at low subsonic velocities. In the present configuration of experimental model, the hydrodynamic events at small angles of attack are dominated by the shear-layer instability of a locally separated boundary layer, while at increasing the body incidence, a globally separated flow with the prevalence of largescale vortex motion is observed.
The key point of the present consideration is the profound response of the flow pattern to small variations of the angle of attack at its close-to-zero values. The effect is basically due to different stability characteristics of the separated and attached boundary layers in the aft part of the body. As a result, minor deviations of its angular position from zero incidence generate a nonuniformity of the mean flow and, hence, strong variations of the nonstationary velocity component in the azimuthal direction. In this respect, the unstable flow over the body of revolution with laminar boundary-layer separation becomes far from being axisymmetric.
|
2019-04-22T13:08:15.097Z
|
2017-10-01T00:00:00.000
|
{
"year": 2017,
"sha1": "c4bc9e359b3b23948e703c6d614ddec6940e961a",
"oa_license": null,
"oa_url": "https://doi.org/10.1088/1742-6596/894/1/012031",
"oa_status": "GOLD",
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"Physics",
"Engineering"
],
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"Physics"
]
}
|
259304892
|
pes2o/s2orc
|
v3-fos-license
|
Does loop length change after anterior cruciate ligament reconstruction with adjustable loop cortical suspension device?: Observation of the hamstring graft completely filling the femoral tunnel
Purpose The adjustable loop cortical suspension device (ALD) is a useful femoral fixation device in anterior cruciate ligament (ACL) reconstructions, but the possibility of loosening has been suggested. The purpose of this study was to evaluate the elongation of an adjustable loop and the position of the hamstring graft inside the femoral socket. Methods The subjects were 33 patients who underwent ACL reconstruction with a hamstring tendon. The graft was fixed using ALD and completely filled the femoral socket. Magnetic resonance images were taken one week and one year after the operation. The loop length, femoral socket length, and graft length inside the socket were measured and statistically compared with the clinical outcomes. Results The loop length one week after surgery was 18.9 ± 4.4 mm, and 1 year after surgery was 19.9 ± 4.5 mm (P < 0.001). The gap between the top of the graft and femoral socket was 0.9 ± 1.8 mm one week after surgery and 1.3 ± 1.7 mm one year after surgery (P = 0.259). At one week post-operation, a gap was found in nine patients (27.3%). The loop length and gap did not strongly correlate with clinical findings. Conclusion ACL reconstruction using ALD showed a gap between the graft and femoral socket at the one week post-operation mark in 27.3% of participants. One year after the surgery, there were cases where the gap increased and/or decreased, but the elongation of the loop was 1 mm on average. Our findings suggest that ALD is clinically safe to use; however, has the possibility of initial loop elongation and non-uniform changes. Level of evidence IV.
Introduction
The cortical suspensory fixation device is widely used for femoral fixation in anterior cruciate ligament (ACL) reconstructions using the hamstring tendon. Conventionally, fixed loop cortical suspension devices (FLDs) have been used, and beneficial biomechanical and clinical results have been reported [8,11,17,22]. However, there are some disadvantages, such as improper graft insertion due to incorrect measurement of femoral tunnel length and drilling length, and difficulty in use in short tunnels.
Recently, a second-generation cortical suspension device has been developed. The adjustable loop cortical suspension device (ALD) has only one size and the insertion length of the graft tendon can be adjusted. The advantages are that it can be inserted into the bone socket to the maximum extent and the bungee effect [14] which is one of the factors that cause the enlargement of the tunnel is reduced. It is expected that the bungee effect will be reduced by completely filling the bone socket with the graft, which improves bone-tendon healing. Furthermore, it is easy to use even with a short tunnel, and it can be re-tensioned after fixation on the tibial side [3,4,21]. While clinical results are comparable to FLDs, biomechanical studies have warned that the adjustable loop may loosen and lengthen under cyclic loading [1,3].
There are many studies of biomechanics about ALD, but most of these are in vitro evaluations of ALDs alone or on the knees of cadaveric and animal studies. To date, the in vivo condition of a tendon graft completely filling the bone tunnel using ALD remains unclear. Therefore, the purpose of this study was to evaluate the loop length changes and the position of the graft which is completely filled in the femoral socket after ACL reconstruction with ALD by magnetic resonance imaging (MRI) and compare them with clinical outcomes. Since previous biomechanical studies have shown that the ALD tends to become loosened with cyclic loading, we hypothesize that the loop would exhibit elongation in vivo, and the tendon graft would shift with it. However, the results were not related to clinical results.
Patient recruitment
This study was a retrospective study. The subjects were 33 patients who underwent ACL reconstruction using an ALD from January 2016 to January 2018 at our hospital and related facilities. Inclusion criteria were primary anatomical single-bundle ACL reconstructions, with hamstring autograft. In all cases, the tendon was fixed to the femur using an ALD, and the button was in contact with the femur on a plain radiograph immediately after the operation. Those cases using an FLD (3 cases), double-bundle reconstruction (5 cases), bone patellar tendon-bone graft (BTB) (1 case) and quadriceps tendonbone graft (2 cases), and revision ACL reconstructions (2 cases) were excluded. Table 1 shows the details of the patients. All patients provided informed consent. This retrospective study was approved by the institutional review board.
Surgery procedure
All operations were performed by senior coauthors and the procedure was the same in all cases. The semitendinosus tendon (ST) was harvested and quadrupled, for a graft diameter of 7-9 mm and a minimum length of 55 mm. When the thickness was not sufficient, the gracilis tendon was also harvested, combined with the ST to make a six-fold graft, and pretensioned. The femoral bone tunnel was drilled using an outside-in technique with an Antero-Lateral Entry Femoral Aimer (Smith & Nephew Endoscopy, Andover, MA) inserted with a guide pin into the anatomical centre of the ACL attachment behind the 'resident's ridge' . Using a Flip Cutter (Arthrex, Naples, FL) of the same diameter as the graft, 12-20-mm femoral socket was prepared according to the prepared graft length. For the tibial tunnel, a guide pin was inserted into the centre of the ACL attachment of the tibial stump using a Director Tibial Guide (Smith & Nephew Endoscopy, Andover, MA), and overdrilled with the same diameter as the tendon graft. The femoral side was fixed with TightRope RT (Arthrex, Naples, FL). After graft passage and confirming the button was flipped by intraoperative fluoroscopy, the graft tendon was completely inserted in the socket. After applying preconditioning for 60 s at 100 N and repetitive knee flexion-extension to fully remove the creep, the graft was fixed at 20°of knee flexion with a Double Spike Plate System (Smith & Nephew Endoscopy, Andover, MA) at 20 N by ligament tensioners (Smith & Nephew Endoscopy, Andover, MA). Meniscal resection, sutures, and cartilage procedures were added according to the arthroscopic findings.
Postoperative therapy
All patients were rehabilitated according to the rehabilitation protocol performed at our hospital. The knee joint was placed in a brace for one week after surgery. Subsequently, joint range of motion exercise was started, and the range of motion was gradually increased weekly. After six weeks, there was no restriction. Partial weight bearing was started one week after the operation, and full weight bearing was taken in three weeks. When concomitant meniscus repair was performed, weight bearing and range of motion exercise were delayed for one-two weeks. Approximately three months after the operation, the patients were allowed to start running. Jump exercise was allowed at four months and participants returned to sports eight to 12 months after the operation.
MRI Evaluation
We had taken MRI with the following protocol in a past study of bone tunnel enlargement. One week and one year after the operation, the MRI (Philips Ingenia 3.0 T or Achieva 1.5 T, Philips Healthcare, Best, The Netherlands) examination of axial 3D proton-density-weighted imaging sequences was performed before knee range of motion exercise for all cases, and the angle of knee flexion was fixed at 10°. The following parameters were used for imaging acquisition: repetition time = 1500 ms, echo time 39 ms, field of view = 130 mm, matrix = 216 × 216, and slice thickness = 0.6 mm. Multiplanar reconstruction was then performed using the Synapse VINCENT medical imaging system (Fujifilm Medical, Tokyo, Japan). First, an axis parallel to the femoral tunnel was created and adjusted to maximize the diameter of the femoral tunnel, femoral socket, and graft inside the socket. The distance from the lateral wall of the femur to the graft tendon (loop length) (a), the distance from the femoral tunnel aperture to the femoral socket (socket length) (b), and the distance from the femoral tunnel aperture to the graft tendon in the socket (graft length inside socket) (c) were measured (Fig. 1).
The change in the ALD loop length and the change in the gap between the top of the graft and the top of the femoral socket (b-c) were calculated. The measurements were performed by a single author, who was an orthopaedic surgeon with 12-years of experience.
Clinical evaluation
Clinical results were evaluated before, and minimum two years after surgery. A Lachman test was graded as negative (≤ 2 mm), trace (3-5 mm), or positive (≥ 6 mm). Pivot-shift test was graded as equal, glide, clunk, or gross. Furthermore, Lysholm score, Tegner activity scale, and side-to-side difference measurements of a KT-1000 arthrometer (MEDmetric, San Diego, CA) performed at maximal manual forces were evaluated.
Statistical analysis
Data were expressed as mean ± standard error. Oneweek and one-year measurements were compared using a paired t-test. Spearman correlation tests were performed between the Lachman and pivot-shift tests and variations in loop length and gaps. Pearson correlation tests were performed between the Tegner activity scale scores, Lysholm scores, KT-1000 measurements, and variations in loop length and gaps. Intra-observer and interobserver reliabilities of socket length and graft length inside socket were assessed using the intraclass correlation coefficient (ICC). The measurements were performed twice with an interval at least eight weeks between measurements to minimize the memory effect. A co-author also performed measurements to assess ICC. The ICC value for the interobserver reliability was 0.62-0.83. The ICC value for the intra-observer reliability was 0.87-0.93. A post-hoc power analysis was performed using alfa = 0.05 and N (number of cases) = 33; the power was over 0.9. For all statistical evaluations SPSS version 26.0 (IBM, Tokyo, Japan) was used, and significance was assumed at P < 0.05.
Results
The loop length one week after surgery was 18.9 ± 4.4 mm, and one year after surgery was 19.9 ± 4.5 mm, and the change was 1.0 ± 1.4 mm (P < 0.001). The gap between the top of the graft and the top of the femoral socket was 0.9 ± 1.8 mm one week after surgery and 1.3 ± 1.7 mm one year after surgery, and the change was 0.4 ± 2.1 mm (P = 0.259) ( Table 2). At one week postoperatively, a gap was found in nine cases (27.3%), and a gap of 3 mm or more was found in four cases (12.1%). One year after surgery, the gap increased in 12 cases (36.4%) and decreased in seven cases (21.2%). There were four cases (12.1%) in which the gap increased by 3 mm or more and two cases (6.1%) in which the gap decreased by 3 mm or more (Fig. 2). Figure 3 shows an example of the MRI images. In cases with decreased the gap, gap was replaced by bone formation in all cases.
Regarding clinical evaluation, the range of motion of the knee joint was not different from that of the healthy side two years after surgery, and the Lachman test was negative in all cases. In the pivot-shift test, equal was recorded in 25 cases (75.8%), glide in eight cases (24.2%), and clunk and gross were not observed. The KT-1000 side-to-side difference measurement was 6.0 ± 2.1 mm preoperatively and 0.2 ± 1.1 mm postoperatively. The Tegnar activity scale was 7.0 ± 1.1 before surgery and 6.9 ± 1.1 after surgery. The Lysholm score was 82.9 ± 10.5 before surgery and 99.6 ± 1.4 after surgery ( Table 3).
Discussion
The most important finding of this study was ALD showed non-uniform deformation in vivo after ACL reconstruction. This study evaluated the elongation of an adjustable loop and the position of the hamstring graft, which completely filled in the femoral socket in ACL reconstruction by MRI evaluation. Recently, ALDs have been used because of its technical easiness, but there are no certain conclusions regarding its mechanical properties. In our study, 9 cases (27%) had a gap between the graft and the socket one week after surgery. In our procedure, after confirming the flip of the Tightrope RT button, the loop was tensioned until it did not shrink further, and the graft was fully filled in the bone socket. The presence of a gap between the graft and the socket at one week after the operation indicates that the adjustable loop had already lengthened even after the early postoperative period. Choi et al. reported the presence of a gap between the graft and the femoral socket on MRI images taken the day after an ACL reconstruction using, as in the present study, an ALD [6, 7]. They pointed out early postoperative gaps, and it is possible that the loop loosened. In a biomechanical cyclic loading test, there is a risk of adjustable loop plastic deformation. Therefore, we performed preconditioning of 100 N to fix the graft tendon; however, there was limited time to apply tension and the creep may not completely disappear. This may be the cause of the gaps within our procedures. In some studies, to avoid this phenomenon, re-tensioning to tighten the loop further after fixing the graft on the tibial side is recommended [7,13]. However, this procedure may increase the initial tension applied at tibial fixation, and which may result overconstrained knee. Therefore, we did not KT-1000 side-to-side difference (mm) 6.0 ± 2.1 0.2 ± 1.1 Tegner activity score 7.0 ± 1.1 6.9 ± 1.1 Lysholm score 82.9 ± 10.5 99.6 ± 1.4 include this in our procedures. It is up to the surgeon to allow a 1 mm gap or to choose a hyper-tensioned graft. In this study, at one year after the operation, the gap increased in 12 cases (36%), while the gap decreased in seven cases (21%). This suggests that the mechanical properties of an ALD may affect the dynamics of the tendon in the bone socket, even long after surgery. There have been few reports on gaps in the femoral socket with cortical suspension devices using MRI. Ahn et al. reported about 10% of cases with a tunnel-graft gap at six months after surgery in both FLD and ALD, but this gap was not related to the clinical results [2]. Choi et al. pointed out that the factor contributing to the gap increased six months after surgery. They reported that the loop loosened, possibly due to the rehabilitation load [7]. In our study, there were cases in which the gap increased one year after the operation. This is because the ALD loosened during plastic deformation by cyclic loads [1]. However, Iuchi et al. reported that the ALD showed a smaller elongation of the loop with increases in the lower force limit and with lower cyclic loading speeds, and the postoperative load was different in each case [15]. This result should be taken into consideration when applying the load up to three months after surgery, which is considered to complete the healing of the graft tendon in the bone tunnel. Load up during this period should be considered more carefully. In this study, the gap became shorter in six cases, in contrast to the report by Choi et al. [7]. When the MRI images of all six cases were evaluated, the gap appeared to be filled with bone. This phenomenon was only observed in our study. This might be due to the difference in the measurement method and accuracy. It is necessary to study further how bone formation in the socket progresses over a long period of time.
To date, several studies have reported the biomechanical properties of ALDs. Several papers have reported that the maximum displacement of loop length after cyclic loading is larger in ALD compare to FLD [3, 5, 10, 17,22]. Ahmad et al. showed that the ALD loop undergoes plastic deformation due to cyclic loading, and these devices show biomechanical inferiority and demonstrate heterogeneity of fixation properties [1]. In addition, regarding ultimate load to failure, there are many reports that ALDs have a weaker breaking strength than FLDs [9, 16,20,22]. However, there are still many unclear factors regarding the elongation of the adjustable loop in vivo. Kusano et al. evaluated the loop length change of the ALD by computed tomography after ACL reconstruction using BTB, but reported that the elongation was only 0.04 mm from one to 12 weeks after surgery [18]. This study also evaluated the change in loop length. The loop length increased by 1 mm on average from one week after surgery to one year after surgery. Since BTB and hamstring have different healing properties [19], a simple comparison cannot be made. However, none of the loops showed catastrophic elongation, and gap formation and loop length were not associated with clinical outcome. Given the technical benefits of ALDs, an average 1-mm elongation of loop may not be a concern. To date, there have been some reports that there is no significant difference between ALD and FLD in clinical results, such as Lysholm score, IKDC score, KT-1000 arthrometer laxity, Lachman test, and pivot-shift test [2,4,12,19,23].
This study has several limitations. The first is the potential for measurement accuracy. Although there was no problem in the measurements one week after the operation, there were some cases in which it was difficult to evaluate the bone socket and the tendon graft one year after the operation, depending on the degree of enlargement of the femoral tunnel and 'ligamentization' of the graft. Intra-rater and inter-rater errors were evaluated using reconstructed images from high-resolution MRI scans and were considered acceptable. Second, the initial measurement of loop length was performed one week after the operation.
There might have been loosening during this period. However, since the knee has been fixed for one week and the load has been limited, this bias was mitigated as much as possible. Third, there was no control group. Since the in vitro biomechanical superiority of an FLD is clear, it is considered that the elongation of the loop is less than that of ALD. We also did not evaluate the enlargement of the femoral tunnel, which is considered one of the advantages of ALDs. Since it is clinically relevant to evaluate femoral tunnel enlargement by ALDs, it is necessary to evaluate this in future studies.
Conclusion
ACL reconstructions using an ALD in which the hamstring tendon fully filled the femoral socket showed a gap between the top of the graft and the top of the femoral socket in 27.3% of cases one week after the operation, and one year after the surgery, there were cases where the gap increased and decreased. In this study, ALD showed non-uniform deformation in vivo, but it is clinically safe to use if it is used in consideration of the possibility of initial loop elongation.
|
2023-07-02T13:10:39.519Z
|
2023-07-01T00:00:00.000
|
{
"year": 2023,
"sha1": "329a12ca573c69f61b8108ec7568d8a19b40a797",
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|
32400951
|
pes2o/s2orc
|
v3-fos-license
|
Exploration of solar radiation data from three geo-political zones in Nigeria
In this paper, readings of solar radiation received at three meteorological sites in Nigeria were analysed. Analysis of Variance (ANOVA) statistical test was carried out on the data set to observe the significant differences on radiations for each quarter of the specified years. The data were obtained in raw form from Nigerian Meteorological Agency (NIMET), Oshodi, Lagos. In order to get a clear description and visualization of the fluctuations of the radiation data, each year were considered independently, where it was discovered that for the 3rd quarter of each year, there is a great fall in the intensity of the solar radiation to as low as 73.27 (W/m2), 101.66 (W/m2), 158.51 (W/m2) for Ibadan, Port-Harcourt and Sokoto respectively. A detailed data description is available for the averages across months for each quarter. The data can provide insights on the health implications of exposure to solar radiation and the effect of solar radiation on climate change, food production, rainfall and flood patterns.
a b s t r a c t
In this paper, readings of solar radiation received at three meteorological sites in Nigeria were analysed. Analysis of Variance (ANOVA) statistical test was carried out on the data set to observe the significant differences on radiations for each quarter of the specified years. The data were obtained in raw form from Nigerian Meteorological Agency (NIMET), Oshodi, Lagos. In order to get a clear description and visualization of the fluctuations of the radiation data, each year were considered independently, where it was discovered that for the 3rd quarter of each year, there is a great fall in the intensity of the solar radiation to as low as 73.27 (W/m 2 ), 101.66 (W/m 2 ), 158.51 (W/m 2 ) for Ibadan, Port-Harcourt and Sokoto respectively. A detailed data description is available for the averages across months for each quarter. The data can provide insights on the health implications of exposure to solar radiation and the effect of solar radiation on climate change, food production, rainfall and flood patterns. &
Data accessibility
All the data are in this data article.
Software
Microsoft Excel and Minitab 17 Statistical Software Value of the data • The energy sector of the economy can incorporate the data set and findings for the utilization of solar radiation received from the sites. • The vitality of these data set is widely recognised in the energy research community for forecasting minutely, hourly, daily and monthly solar radiations using time series tools which could also cater for volatility that exist in the data. • For educational purposes and environmental studies. See similar works .
• Findings from the data bring the awareness of the Nigerian government to the most suitable location for the establishment of both solar plants and research institutes to generate electricity. • The data can provide insights on the health implications of exposure to solar radiation.
Data
The raw data for this work were obtained from Nigerian Meteorological Agency (NIMET) Oshodi Lagos, as daily averages on solar radiation for three weather stations namely; Ibadan, Sokoto and Port-Harcourt. The readings were taken using the Gunn-Bellani Radiation Integrator measuring the radiations in millilitres (ml). However, for the sake of this research, the readings were converted to Watts per Sq. meters (1 ml to 13.153 W/m 2 ) covering from 1st of January, 2011 to 31st of December, 2015 and further transformed into monthly-quarterly averages (Table 7) for the specified years using Microsoft Excel software. Table 1a is the statistical summary of the quarterly averages for solar radiation from the 1st of January 2011 to 31st of December 2015. Meanwhile, it was observed that on an average, Sokoto receives the highest intensity of solar radiation followed by Port Harcourt and Ibadan. Furthermore, Table 1b shows that the data set from all sites exhibit negative kurtosis and skew, implying that the distributions are light-tailed and skewed to the left respectively.
From the graphs (Figs. 1-5), it was observed that Sokoto was top on the presented charts with an exception on December 2015. This validates that the closer the earth's surface is to the sun, the greater the radiations it receives, which is well applicable to the case of Sokoto ranking top among the other stations with a height of 309 m above sea level. Furthermore, the solar radiation received at Sokoto increases yearly within the 1st quarter. It has to be noted that the y-axis of the figures is the solar radiation reading for the zones measured in Watt per square meter.
Methods and materials
The summary of the location sites of the raw data are displayed in Table 2. Linear correlation is traditionally used to roughly determine the relationship between two variables. Table 3 shows the correlation matrix between the three meteorological stations. Though independent, the correlations among each station are positive and that of Sokoto-Ibadan and Ibadan-Port Harcourt are highly positively correlated. It is either the solar radiation levels are increasing or decreasing at the three sites simultaneously.
The ANOVA test carried out on the data set for all sites and the result was displayed in Tables 4-6. The results showed significant differences in the means for solar radiation received quarterly at the stations independently. The significant differences in the means as revealed from the ANOVA results led to further analysis using the Tukey's Simultaneous 95% Confidence Interval Post Hoc test. The aim is to detect the specific quarters where differences lie across the specified years.
The results revealed that for Port Harcourt, significant differences lie within all other quarters except for the 1st and 4th quarters and for the 2nd and 4th quarters yearly as seen in Fig. 6. Similarly, Fig. 7 shows that for Sokoto, the 1st and 3rd quarters, 3rd and 4th quarters and the 2nd and 3rd quarters as having significant differences. Lastly, Fig. 8 shows that for Ibadan, significance differences exists between the 1st and 3rd Quarters, 2nd and 3rd Quarters and the 3rd and 4th Quarters for these years. Minitab17 software was implemented for analysis on the solar radiation data, which produced the ANOVA results and the Post Hoc test for the stations (Table 7).
|
2018-04-03T02:07:04.351Z
|
2017-05-17T00:00:00.000
|
{
"year": 2017,
"sha1": "ca9d3236df2eae2c3564128a066e87c3dbeab6a3",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.1016/j.dib.2017.05.017",
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"Environmental Science",
"Geography"
],
"extfieldsofstudy": [
"Environmental Science",
"Medicine"
]
}
|
24984040
|
pes2o/s2orc
|
v3-fos-license
|
Conventional vs drug-eluting beads transarterial chemoembolization for hepatocellular carcinoma
Transarterial chemoembolization (TACE) is the current standard of therapy for patients with intermediate-stage hepatocellular carcinoma (HCC) according to the Barcelona Clinic Liver Cancer classification. The concept of conventional TACE (cTACE) is the selective obstruction of tumor-feeding artery by injection of chemotherapeutic agents, leading to ischemic necrosis of the target tumor via cytotoxic and ischemic effects. Drug-eluting beads (DEBs) have been imposed as novel drug-delivering agents for TACE, which allows for higher concentrations of drugs within the target tumor and lower systemic concentrations compared with cTACE. Despite the theoretical advantages of DEB-TACE, it is still controversial in clinical practice as to whether DEB-TACE is superior to cTACE in regard to overall survival and treatment response. In this review article, we summarize the clinical efficacy and safety of DEB-TACE for patients with intermediate or advanced stage HCC in comparison with cTACE.
INTRODUCTION
Hepatocellular carcinoma (HCC) is listed as the sixth most common cancer worldwide and the third most frequent cause of cancer-related mortality [1,2] . The majority of HCC cases occur stem from chronic liver disease and cirrhosis. Therefore, the selection of treatment modalities for HCC is determined by tumor size, multiplicity, and liver function status [3] . Transarterial chemoembolization (TACE) has been frequently performed and has become the first-line treatment option for large or multinodular HCC with preserved liver function, no evidence of vascular invasion or extrahepatic spread, and the absence of cancerrelated symptoms, which is defined as intermediate stage according to the Barcelona Clinic Liver Cancer (BCLC) classification [4,5] . Moreover, in clinical settings, TACE is considered the standard treatment for patients with early stage HCC who are not appropriate candidates for curative therapy [4] . The mechanism of action for conventional TACE (cTACE) is the selective obstruction of tumor-feeding arteries by injection of chemotherapeutic agents (doxorubicin or cisplatin) mixed with lipiodol [6] . This leads to ischemic necrosis of target tumors by cytotoxic and ischemic effects. There are several drawbacks of cTACE that are associated with ineffective treatment responses in many cases: (1) the liquid motility of lipiodol to reduce the effective concentrations of chemotherapeutic agents; (2) the inability to release drugs in a controlled and sustained manner; and (3) heterogeneity in the technique and treatment schedules. To reduce these drawbacks, drugeluting beads (DEBs) have been introduced as drugdelivering agents for TACE. After delivery of the beads to target tumors, the beads release chemotherapeutic drugs (doxorubicin or epirubicin) in a sustained fashion over a prolonged period of time [7,8] . Figure 1 shows the mechanism of action of DEB-TACE. Treatment with DEB-TACE allows higher concentrations of drugs within the target tumor and lower systemic concentrations compared with cTACE [9,10] . Thus, the use of DEBs can reduce drug-related adverse events such as postembolization syndrome. As DEB-TACE is widely used interchangeably with cTACE in many hospitals globally, it is necessary to assess the current status of DEB-TACE in comparison with cTACE. Thus, this article aims to evaluate the characteristics of each modality and to compare the clinical outcomes of DEB-TACE with those for cTACE.
cTACE VS DEB-TACE: CHARACTERISTICS cTACE
Typically, cTACE involves the infusion of chemotherapeutic drugs blended with lipiodol and embolic agents into the cancer-feeding artery [6] . Both single chemotherapeutic agents and combination chemotherapy have been used as part of the drug regimen in TACE. However, there is no agreement on the optimal anticancer drug(s) to be used in cTACE. Globally, the most widely used chemotherapeutic agent for TACE of HCC is doxorubicin. The dose of doxorubicin generally ranges from 30 to 75 mg/m 2 , at a maximum of 150 mg emulsified in 5 to 20 mL of lipiodol [11] .
Lipiodol is a key element in TACE due to its distinctive combination of features as a drug-carrying, tumorseeking, and embolizing agent [12] . Even though the principle is not concretely comprehended, it seems to be absorbed by a pump in the cancer cell wall and transported to the intracellular space. Then, upon hypoxia within cancer cells, this pump is disabled, such that lipiodol is retained within the cell. Lipiodol is confined to tumors when injected via the hepatic artery, and it is generally trapped in HCC for months, even up to a year, whereas it is washed out from non-tumor portions of the liver within 4 wk.
Several embolic agents, such as gelfoam, polyvinyl alcohol (PVA) particles, and tris-acryl gelatin microspheres, have been used over the past three decades in chemoembolization [12] . Among these embolic agents, gelfoam has recently emerged the most commonly used substance worldwide. The intended aim of embolization is as follows: To assist lipiodol to be sustained selectively in the tumor, to inhibit chemotherapeutic agent washout from HCC, and to cause ischemic necrosis.
A significant problem of cTACE is the great inhomo geneity of the technique and treatment schedules used in clinical centers worldwide. Two randomized controlled trials on cTACE used quite different technical approaches. Furthermore, some HCCs do not show lipiodol uptake which may result in lower effectiveness of the treatment [13] .
DEB-TACE
The most commonly used DEB, DC Beads (BTG, United Kingdom) are nonbiodegradable PVA microspheres, loaded with calibrated doxorubicin. They can release doxorubicin in a controlled and maintained mode [14] .
Through an ion-exchange mechanism, DC Beads actively sequester oppositely charged drugs. In initial in vitro studies, doxorubicin could be efficiently loaded into the DC beads up to 45 mg/mL, regardless of the size of beads [15] . Currently, a loading of 37.5 mg doxorubicin/mL beads is recommended, in consideration of a practical therapeutic dose and optimum handling characteristics. According to an animal pharmacokinetic study comparing two sizes of doxorubicin-eluting beads (100-300 µm and 700-900 µm) loaded with same amount of doxorubicin, treatment with the smaller beads (100-300 µm) elicited higher doxorubicin plasma levels [16] . This finding was caused by the increased surface area of the smaller beads, leading to a profuse release of doxorubicin.
In DEB-TACE, the extent of the liver cancer burden should be considered in planning the dose of doxorubicin. As a general rule, for patients within the Milan criteria (defined as single tumor ≤ 5 cm, or multiple tumors of up to three and < 3 cm each), a planned dose should be up to 75 mg doxorubicin loaded into one vial, including 2 mL of DC beads in each single treatment. Meanwhile, for patients beyond the Milan criteria, each treatment should involve a dose up to 150 mg loaded into two vials of DC beads [17] . Generally, the recommended size of beads is 100-300 µm for standard DEB-TACE procedures. This choice is based on the fact that small particles can be transported inside the tumor or in nearness to the tumor margin, and thus they are ideal for drug delivery or accurate embolization.
HCC
The survival benefit of cTACE has been the issue of a finite number of randomized controlled trials (RCTs) that have provided controversial results [18] . Among seven RCTs [19][20][21][22][23][24][25] all published between 1988 and 2002, only two trials showed favorable results in respect of overall survival [19,20] . However, a systematic review based on these seven RCTs showed that cTACE has been found to improve 2year survival (OR = 0.53; 95%CI: 0.320.89; P = 0.017) of patients with unresectable HCC, compared with best supportive care [26] . Subsequent sensitivity analysis in this study showed a significant survival benefit for chemoembolization with cisplatin or doxorubicin by analyzing 323 patients in four studies (OR = 0.42; 95%CI: 0.20-0.88), but not for embolization alone by assessing 215 patients in three studies (OR = 0.59; 95%CI: 0.29-1.20) [26] . In a current Cochrane review, the evidence based survival benefits of cTACE was challenged [27] . This metaanalysis involved RCTs published after 2002 and showed no solid evidence to support TACE or transarterial embolization (TAE) compared with conservative management, in patients with unresectable HCC. However, some experts have doubted such conclusions, because this review involved RCTs with inappropriate selection of patients and control arms, which likely biased the results of the analysis.
Primarily, DEB-TACE has been introduced to enhance the ability of drug-delivery to target tumor while reducing systemic toxicity and to provide a standardized embolic effect. The role of doxorubicin in embolic microspheres was evaluated in a randomized, cancer-size adjusted trial assessing DEB-TACE vs TAE with similar characteristics (BeadBlock-TAE) [28] . Although no survival benefit was reported in the study, the value of doxorubicin was favorable in the setting of TACE with microspheres, because DEB-TACE showed higher local response, less recurrence at 12 mo, and a longer time-to-progression than BeadBloc-TAE. Another trial assessed the rate of tumor necrosis after chemoembolization with epirubicinloaded beads vs TAE with unloaded microspheres (Embosphere particles), which was pathologically proved in explanted livers of HCC patients undergoing liver transplantation: Epirubicin-loaded beads TACE showed complete necrosis in 77% of lesions, while TAE showed complete necrosis in only 27% of lesions (P = 0.043) [29] .
A recently reported prospective clinical trial of DEB-TACE in a large Korean HCC population showed an overall 6mo survival rate was 97.4%, although more than half of patients had early stage HCC (BCLC-A, n = 77, 50.7%) [30] . Varela et al [7] firstly reported that systemic concentrations of doxorubicin were significantly lower in patients treated with DEB-TACE than patients treated with cTACE. This result was verified by Poon et al [14] , who performed DEB-TACE with possibly the highest dose of doxorubicin (150 mg). Both studies showed that none of treated patients exhibited doxorubicin-related systemic toxicity (alopecia, bone marrow suppression, or dyspnea) [7,14] . Despite the aforementioned theoretical advantages of DEB-TACE, previous studies comparing DEB-TACE with cTACE in HCC of intermediate stage have shown rather conflicting results. Recently reported metaanalysis showed that the two modalities represent comparable results, suggesting an absence of difference in tumor response between DEB-TACE and cTACE [31] . On the contrary, three other metaanalyses, assessing the efficacy of DEB-TACE vs cTACE in HCC patients, showed different results [32][33][34] . Huang et al [32] (seven studies, n = 700) and Xie et al [33] (six studies, n = 652) demonstrated that significantly better objective tumor response was found for DEB-TACE than for cTACE. In another meta-analysis of nine studies (866 patients) conducted in 2016, DEB-TACE presented significantly higher complete response rate and better overall survival, although similar objective tumor responses compared with cTACE. Regarding adverse events in these meta-analyses [32][33][34] , overall and severe adverse events were similar or slightly lower in patients receiving DBE-TACE than patients receiving cTACE. Tables 1 and 2 summarize the clinical outcomes and adverse events of the studies that were included in these meta-analyses comparing DEB-TACE and cTACE.
Among randomized controlled trials reported until recently, the largest trial is the PRECISION V phase2 trial assessing DEB-TACE vs cTACE in 212 patients with mostly HCC of intermediate stage [10] . The primary Figure 1 Action mechanism of drug-eluting bead-transarterial chemoembolization in hepatocellular carcinoma. Sustained release of chemo therapeutic agents from microbeads of uniform size, which embolize supplying vessels more distally, enables local concentration of cytotoxic agents to be higher within tumor. efficacy endpoint (response at 6 mo, P = 0.11) and primary safety endpoint (incidence of severe adverse events within 30 d, P = 0.86) were comparable in both two groups. After performing a post hoc comparison, the DEB-TACE group indicated a significant decrease in chemotherapeutic agent-related systemic and liver toxicity compared to the cTACE group. Furthermore, in subgroup analysis, the objective response rate and disease control rate were significantly better (P = 0.038 and P = 0.026, respectively) with DEBTACE than with cTACE in 67% of patients with more advanced disease (Child Pugh B, bilobular or recurrent disease, ECOG 1). Another RCT for evaluating the potential effect of DEBTACE on overall survival, compared to cTACE using epirubicin, showed no statistical differences between both modalities in terms of survival, treatment response, or adverse episodes [35] . However, it should be considered that the maximally used dose of doxorubicin/epirubicin was limited to only 75 mg for both procedures in this trial. Furthermore, the trial mainly recruited patients with low tumor burden (46% of patients with early HCC, only 20% patients with bilobar disease). Thus, this restricted one of the significant advantages of DEBTACE, which is the ability to use higher doxorubicin doses without rising drug-related systemic toxicity in patients with larger tumor burden as mentioned in the PRECISION V study. This trial indicated that DEB-TACE did not show better clinical outcomes, compared with cTACE in patients with relatively well preserved liver function and low tumor burden. A retrospective study by Song et al [9] reported that overall survival and treatment responses for DEB-TACE were significantly better than those for cTACE.
showed that overall survival, time to progression, and disease control rate were not significantly different between DEB-TACE and cTACE groups, even when subgrouped by BCLC stage [36] . However, the incidence of adverse events was significantly lower, particularly in HCC larger than 5 cm in BCLC-B patients receiving DEB-TACE [36] . Considering the results from these studies, there is still controversy regarding clinical outcomes. However, it seems that DEBTACE shows at least similar efficacy and less adverse events than cTACE. DEB-TACE might be favorable to cTACE for large HCC especially in patients with decreased liver function, even though there is lack of evidence that DEB-TACE is superior to cTACE in term of efficacy.
For advanced HCC (BCLC C), the role of chemoembolization has not been fully established. In accordance with the BCLC staging system, it recommends systemic treatment or palliative therapy to patients with advanced stage. In a small retrospective study comparing cTACE and sorafenib in patients with advanced HCC, overall survival in the cTACE group was higher than the sorafenib group (9.2 mo vs 7.4 mo, P = 0.377) [37] . Recently, two studies on DEB-TACE for patients with advanced HCC were reported: Kalva et al [38] [39] reported median survival of 13.5 mo (range, 8.2-18.7 mo) without severe adverse episodes. Subgroup analyses showed that the median survival of Child-Pugh class A patients was 17.8 mo (range, 9.0-26.7 mo). In comparison with median survivals of 10.7 mo and 6.5 mo for sorafenib in the SHARP and Asia-Pacific trials [40,41] , it appears that cTACE as well as DEB-TACE shows better or at least comparable efficacy in patients with advanced stage HCC, ChildPugh class A and good performance status.
A major limitation of TACE is a high rate of cancer recurrence. In two RCTs, a sustained response lasting for 3 to 6 mo was reported in only 28% to 35% of patients treated with cTACE [19,20] . Recently, several trials made an attempt to analyze the potential benefit of combined strategies with chemoembolization and other treatment options for overcoming this limitation. Several RCTs have sought to determine the benefit of an addition of sorafenib to cTACE or DEB-TACE in patients with more advanced HCC. The rationale for this concept is grounded in the demonstration that TACE causes hypoxia and induce angiogenesis by activating angiogenic factors and that the use of sorafenib could decrease angiogenesis. However, these RCTs have not proved definite improvement of clinical outcomes in combination therapy of sorafenib and chemoembolization, compared with chemoembolization alone [42,43] . Recently, trials have been conducted on combination of TACE with other molecular target agents, such as brivanib, sunitinib, and thalidomide. It is hoped that these ongoing trials will contribute to the determination of optimal combinations.
DEB-TACE
Apart from the overall comparison of clinical outcomes between conventional and DEB-TACE, it is still controversial as to whether DEB-TACE is superior to cTACE in large HCC (≥ 5 cm), which frequently suffers from incomplete response or recurrence after cTACE [44] Considering that liver damage given by DEB-TACE is less than that by cTACE, it might be assumed that DEB-TACE offers more therapeutic advantages over cTACE in large HCC. However, regarding response to procedures, complete response rates at 1 and 6 mo were lower in HCC larger than 5 cm, compared with HCC less than 2 cm or 2-5 cm in size [30] . Moreover, in a Korean retrospective study, there was no significant difference in survival between cTACE and DBE-TACE in HCC larger than 5 cm (36.3 mo vs 33.4 mo, P = 0.702) [36] . Therefore, the notion that a big tumor is more appropriate for DEB-TACE than for cTACE is not currently accepted. Paradoxically, small HCC (less than 2 cm) is sometimes difficult to achieve complete response to both cTACE and DEB-TACE, because the tumor vascularity is fine. In particular, unlike lipiodol in cTACE, the diameter of microspheres in DEB-TACE is still too wide to block peripheral hepatic arteries. Accordingly, the outcomes of small HCC (< 2 cm) treated with DEB-TACE, compared to cTACE are controversial. Indeed, the time to progression after DEB-TACE was shorter that after cTACE in HCC < 2 cm (10.3 mo vs 13.8 mo, P = 0.023), although there was no difference in overall survival between the two modalities [36] . Lastly, repeated sessions of a procedure could be another distinguishing advantage or disadvantage between cTACE and DEB-TACE. The severity of hepatic arterial damage has been compared between cTACE and DEB-TACE in a retrospective study. After a single session of cTACE or DEB-TACE, the incidence of hepatic arterial damage was significantly higher for DEB-TACE group than cTACE, with doxorubicin dose being a possible risk factor for such damage [45] .
CONCLUSION
In comparison with cTACE, DEB-TACE facilitates higher concentrations of drugs within the target tumor and lower systemic concentrations. Despite the theoretical advantages of DEB-TACE, it is still controversial in several clinical studies as to whether DEB-TACE is superior to cTACE in terms of efficacy. However, it seems that DEB TACE shows at least similar clinical outcomes and less adverse events than cTACE. In order to gain better results for these treatment modalities, selecting proper candidate patients for DEB-TACE or cTACE is needed. Moreover, further well-defined studies are required to identify combination strategies and to develop better
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2018-04-03T04:21:03.422Z
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2017-06-28T00:00:00.000
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Lariophagus distinguendus (Hymenoptera: Pteromalidae) (Förster)—Past, Present, and Future: The History of a Biological Control Method Using L. distinguendus against Different Storage Pests
Legal requirements and consumer demands for residue-free products pose a big challenge for pest control in grain stores. One possible alternative to chemical insecticides is biological pest control with the pteromalid wasp Lariophagus distinguendus against the weevils Sitophilus granarius, S. oryzae (Coleoptera: Dryophtoridae), and many other storage pest beetles. The use of this wasp as a biocontrol agent was already suggested in 1919 by Prof. Dr. Hase [1]. Despite many studies on host-finding and behavioral biology, the applied aspect was neglected until 1994. Nowadays the wasps are commercially available and can now even be reared on-site, facilitating their use tremendously. This review highlights the milestones in L. distinguendus research, gives insights in current studies, and ventures a glimpse into the future.
Introduction
Stored grain is threatened by a vast number of storage pests, mostly insects [2]. Nowadays integrated pest management strategies (IPM) have superseded the excessive application of chemical insecticides used against storage pests in the past. An important element of IPM is biological pest control, a technique that uses living organisms, so-called natural enemies, to suppress the populations of certain harmful animals or plants below the economic injury level [3]. To protect stored products from storage pests, antagonists that naturally occur in storage and which are already adapted to the storage environment [4,5] can be mass-reared (augmentative control) and released. The storage environment is especially suitable for biological pest control [6,7]. Temperature and humidity conditions are more stable than in the field. Furthermore, a storage building is a limited confined space reducing the risk of antagonists migrating from the application site [7]. One of the main storage pests in temperate climates in Europe is the granary weevil S. granarius [8]. It can be attacked by the parasitoid wasp L. distinguendus, which is a solitary ectoparasitoid with a wide host spectrum. It attacks different developmental stages of at least 17 beetle species in six families. However, four of the 17 species are described as hosts only once [9,10]. The beetles attacked are mostly storage pests belonging to the families Dryophtoridae, Chrysomelidae, Anobiidae, Bostrychidae, Curculionidae, and Ptinidae [9]. Only hosts which develop concealed in seeds, grain kernels, or cocoons are attacked [11,12]. The use of L. distinguendus as a biocontrol agent was already suggested in the early 20th century. Since then, many studies have dealt with different aspects of its biology. This review will summarize earlier and present work and will venture a glimpse on the future role of the wasp in biological pest control.
The 1950s and 1960s
It was more than 15 years later that Kashef resumed the studies on L. distinguendus. He noted that a thorough knowledge about the biology of L. distinguendus and the biology of its hosts is necessary for a successful application of the parasitoids against storage pests [15]. Therefore, he studied the morphology and biology of L. distinguendus and several of its hosts [15,[26][27][28][29][30]. He concentrated on the determination of the host stage most suitable for parasitization and the wasps' fecundity on different hosts [15,29,30]. He compared the reaction of L. distinguendus to known hosts such as S. paniceum and S. oryzae with beetles which had not been described as hosts before, such as the lesser grain borer R. dominica, the Australian spider beetle Ptinus tectus, and the smooth spider beetle Gibbium psylloides (Ptinidae) [30]. In this context he also brought up the question of "the natural host" and the idea of a separation into biological races due to a particular host preference and subsequent ecological isolation. Another focus of Kashef's work was on cues guiding the wasps to their hosts [31]. He was able to disprove the idea of sound being a host-finding cue as stated by Smirnov and Polejaeff [25,29].
Instead he showed that L. distinguendus is attracted by the odor of its different hosts. Even though Kashef's main focus was not on stored products protection, his work was the basis of many applied (and fundamental) studies that were yet to come.
The 1970 and 1980s
In the 1970s and 1980s, the first paper with a focus on an applied research approach was published by Gonen and Kugler, who looked at the biology of L. distinguendus on S. oryzae with the goal " . . . to obtain more information [ . . . ] of this wasp as a parasite of the rice weevil, especially those aspects which might influence its efficiency as a natural control agent" [19]. However, based on their sex ratio and longevity experiments, Gonen and Kugler doubted that L. distinguendus would be able to "cope with the rate of increase of the host population". They stated that the high number of male offspring in early larval host stages and the short lifespan of adult females were the main factors making this method ineffective. However, wasps used by Gonen and Kugler could choose between different host sizes, therefore producing sons on smaller hosts and daughters on larger ones. Putters and van den Assem could show that the absolute size of the host is not important in sex-regulation [32]. Wasps will always define a host as small or large compared to the other hosts available; therefore, in an environment of predominantly small hosts, L. distinguendus would still produce male and female offspring [32]. Thus, the objections of Gonen and Kugler could be disposed of. Several other studies were conducted by van den Assem [33,34] and his colleagues Bellows, Charnov, and Werren [35][36][37] and L. distinguendus was established as a model organism for general behavioral, evolutionary, and population ecology. The main focus of their work was on sex-ratio regulation and population dynamics [35][36][37]. They showed that parasitoids, with diploid females and haploid males, are able to adjust the sex ratio of their offspring to certain ecological conditions such as host size [38]. As part of their studies, two more hosts of L. distinguendus, the bean weevils Callosobruchus chinensis and C. maculatus (Chrysomelidae), were specified [39].
The Early 1990s
In the early 1990s, a working group from Korea started working with L. distinguendus. Their research was application-oriented, focusing on the ability of L. distinguendus to suppress populations of the rice weevil S. oryzae. First Yoo et al. concentrated on different developmental stages of S. oryzae and their suitability for L. distinguendus [40]. Later, a special focus of their work was on the impact of abiotic factors, namely temperature, on the functional and numerical response to different host densities and the development of the wasps [18,41]. Ryoo and his colleagues were able to rebut objections of Gonen and Kugler from the 1970s regarding the short life of adult females or the high number of cases in which two eggs per host were laid [19]. They could show that the lifespan of female wasps was long enough for a sufficient parasitization of hosts and that two eggs per host normally only occur rarely or at low host densities when the waste of eggs is unimportant [18,33,42]. Ryoo further stated that "the biological characteristics of L. distinguendus may be compatible with efficient control of rice weevil populations" [18]. However, Hong noted that the regulation of rice weevils "seems unlikely to occur under lower temperature conditions" [41]. A later study looked at the impact of a second parasitoid, Anisopteromalus calandrae (Hymenoptera: Pteromalidae), when applied simultaneously with L. distinguendus. Even though A. calandrae is considered to be dominant, the two wasps coexisted until the host population was suppressed [43].
From the Late 1990s-Present
Starting in the mid-1990s, the increased demand for pesticide-free products and environmentally safe pest-control methods intensified the studies on L. distinguendus tremendously. In the late 1990s and early 2000s, the main center of research was in Berlin/Germany at the BBA (Biological Federal Agency, Berlin, Germany) and the Applied Zoology Department at the Freie Universität Berlin. At that time one major point of interest was the identification of host searching cues used by L. distinguendus to detect suitable hosts (S. granarius) [44]. Based on the first descriptions by Thorne and Jones concerning the importance of learning in searching behavior [45], the influence of associative learning was also investigated [44,46,47]. Cues used during the different steps of host searching were investigated for different host complexes, revealing that for host finding as well as for host recognition semiochemicals present in the host's feces are used [48][49][50][51][52][53][54]. Chemical studies showed that the substances in the host's feces (S. granarius on wheat) which trigger (at least) host recognition are compounds commonly found in grain (e.g., tocopherol) and compounds from the host (cholesterol) [54]. These findings also explain why wasps not only respond to grains infested with S. granarius larvae but also to healthy wheat grains, a reaction probably due to associative learning. HIS (herbivore-induced synomones, either actively produced or released as a passive physiological reaction by attacked seeds to the feeding activity of the host) were also identified to play a role in host localization [55]. Furthermore, Ruther and Steidle found that L. distinguendus also reacts to volatiles emitted by astigmatic mites, again a cue most likely learned associatively [56]. In the context of host recognition, Ambriz et al. looked at cues used to locate Lasioderma serricorne (Coleoptera: Anobiidae), another host of L. distinguendus [57,58]. They found that extracts from host cocoons trigger host-recognition behavior and oviposition in L. distinguendus and that experience has an enhancing influence on oviposition. However, semiochemical cues are not only used for host (habitat) location but also to avoid unsuitable host habitats, e.g., habitats infested by mold [59].
Besides these basic studies, another major point of interest was the applied use of L. distinguendus. Schöller together with his colleague Prozell established the company Biologische Beratung Ltd, which was the first to produce and sell L. distinguendus commercially. Steidle and Schöller conducted the experiments on the dispersal ability of the wasps [60]. They showed that L. distinguendus is able to locate hosts up to 4 m vertically and horizontally in bulk grain. Steidle and Reinhard showed that wasps released in grain storage will be arrested by grain odors and low humidity and will remain in the storage environment [61]. Within the project "Strategies for the Regulation of Storage Pests in Storages and Factories for Organic Products", further studies concerning the application of L. distinguendus were conducted [62]. By means of studies and an intensive literature research, live parameters of the parasitoid were collected and a decision support application software for farmers and operators was developed [62]. Furthermore, a release schedule was calculated [62][63][64][65]. After the appointment of Steidle as the head of the Department of Animal Ecology at Hohenheim University, Germany, another center of L. distinguendus research was established. Steidle focused on basic research about the mechanisms of speciation with a focus on early learning and courtship behavior, using L. distinguendus as a model system [66]. It could be shown that the species L. distinguendus de facto consists of two species. While one of these two species is a generalist on many different hosts, the other apparently has a narrower host range and is unwilling to parasitize S. granarius, a fact that could have a huge impact on the application [66]. Furthermore, Steidle picked up earlier applied work looking at the dispersal ability of L. distinguendus, this time studying the dispersal of the wasps outside the commodity, applied as an empty storage treatment against residual pest populations [67]. Results indicate that host location outside the commodity is strongly influenced by factors such as light and that the number of wasps released should be higher than the number recommended for use inside the commodity. Additional research on dispersal and host finding was conducted by Adarkwah et al. who looked at the ability of L. distinguendus to reach hosts inside jute bags and bulk maize [68,69]. They showed that the wasps, depending on mesh size, are able to penetrate the bags and reduce pest infestations by 81%, while reduction was about 37% one meter inside the maize bulk. Further applied research was done by Hansen et al. who first focused on the influence of low temperature on the development and winter survival of L. distinguendus and its host S. granarius, showing that the wasps are able to develop, emerge, and produce viable offspring after being exposed to 6˝C for 15 weeks [70,71]. Since reproduction values of the wasps at low temperatures are higher than those of the weevils, L. distinguendus should be able to control granary pest populations even at low temperatures. In this context, Niedermayer et al. examined the parasitization ability of L. distinguendus compared to that of the closely related parasitoid A. calandrae under extreme temperature conditions [72]. L. distinguendus performs better at lower temperatures whereas A. calandrae performs better at warmer temperatures. Therefore, a combined application has been suggested.
Further studies concentrated on special circumstances possibly affecting the efficiency of L. distinguendus. Belda and Riudavets showed that in cases where the two pest species S. oryzae and R. dominica co-occur in the same storage, only populations of S. oryzae but not populations of R. dominica are affected by L. distinguendus [73]. Lucas and Riudavets investigated the combined use of L. distinguendus with other pest control methods such as mechanical polishing treatment of rice (kernels are rubbed and lose their pericarp layer), showing that even though the reduction of pests was enhanced, mechanical treatment also had a lethal effect on the wasps [74]. Additional studies regarding the combination of parasitoids with other control methods were conducted by Hansen. She investigated the use of entomopathogenic fungi and Bacillus thuringiensis in maize and could show that both treatments had negative effects on the efficacy of the wasps [75,76]. Schöller and Prozell looked at a combined use of diatomaceous earth and L. distinguendus. No positive effect of the combination could be shown [77].
To facilitate the application of L. distinguendus Steidle and Niedermayer developed a mass rearing device [9]. The device contains a self-sustaining system of host medium, hosts, and parasitoids and continuously releases wasps directly on site in storages over several months. Together with Schöller and Prozell from Berlin, the use of parasitoids in long-term storages is currently examined.
Status Quo and Outlook
Research has come a long way since the first observations by Hase, Ryabov and Kashef. At present L. distinguendus is produced commercially and distributed by at least 11 companies in central Europe (Internet inquiry using Google). Wasps are reared in the laboratory, shipped in units of about 40 wasps in plastic tubes, and are then released on location [78]. In storages, two to three releases per year are recommended (inundative release); for application against Ptinidae in buildings, wasps should be released monthly. The key market concentrates on rather small farms, mills and retail stores, most of which are in the organic sector. For the future an enhanced involvement in IPM in conventional farms and companies is desirable. Furthermore, the applicability of L. distinguendus in large storage facilities with long-term storage is currently being examined. To facilitate the application of the wasps, the rearing box of Steidle and Niedermayer should be completed and commercialized.
Future research should focus on the selection of strains most suitable for the different pest species as it is commonly done, e.g., for Trichogramma sp. The selection of strains should also play a role depending on the different climate conditions at the application sites.
Additionally, more research is needed concerning the application of L. distinguendus against the nuisance G. psylloides in households. Even though G. psylloides has been described as a host of L. distinguendus, parasitization was only observed under the artificial conditions of laboratory experiments. It is yet unclear how the wasps manage to reach their hosts which normally hide behind walls and in the ceiling. Currently it is recommended to drill holes in walls and ceilings to facilitate accessibility of the hosts. However, nothing is known about the distance between release points or a possibly negative influence of abiotic factors such as light or temperature gradients. To enhance the host-finding success and render the application of L. distinguendus as effective as possible, more research is necessary.
Summing up the results from recent research on the use of parasitoids for the control of stored products pests, we recommend the following guidelines: (1) When releasing wasps, their host-finding ability must be considered. Release points should be within 4 m of potential pest sites in bulk grain [60]. For application against residual pest populations in empty storage, release points should be, at most, 10 m apart [67] and cover no more than 10 m 2 [78]. The host-finding ability of hosts in bags strongly depends on the bag material. Wider mesh sizes facilitate the penetration ability of L. distinguendus [68].
(2) When L. distinguendus is applied outside the commodity, a significantly higher number of wasps should be applied compared to recommendations for the application in the grain bulk, because a certain amount of wasps does not manage to reach hosts [67,68,77]. Distractions due to light and obstacles must be considered and eliminated if necessary [67].
(3) To balance different host-finding ranges, a mixture of L. distinguendus and the closely related parasitoid A. calandrae should be applied [67].
(4) L. distinguendus strains should be selected in accordance to the pest species against which they are applied [66]. Especially strains originating from the host S. paniceum might be specialized on that host and are thus inefficient against other hosts [66].
(5) Temperature should always be considered when releasing parasitoids, especially in empty storage treatments. L. distinguendus should be released during the cold season or in colder geographic regions. During summer or in warmer areas A. calandrae is a suitable alternative [70][71][72]. (6) The combined application of L. distinguendus and other control methods, such as mechanical polishing, and the use of entomopathogenic fungi or diatomaceous earth are not recommended [74][75][76][77].
(7) When using L. distinguendus against pests in storage, wasps should be released four weeks after harvest and typically again in fall and spring [77]. To facilitate the application of the wasps, the use of a mass rearing device is recommended. After the initial set-up the device releases wasps continuously over several months [9].
Conflicts of Interest:
The authors declare no conflict of interest.
|
2016-08-24T23:09:51.855Z
|
2016-08-01T00:00:00.000
|
{
"year": 2016,
"sha1": "f459e0db789d8d1f11a1bd44b03c3847ce00d5ee",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/2075-4450/7/3/39/pdf",
"oa_status": "GOLD",
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142661880
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pes2o/s2orc
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v3-fos-license
|
The Application of Role Plays as an Effort to Improve the Speaking Ability of the First Year Students of SMPN 1 Tanah Putih
This is an action research. In doing this research is neededsome of the data is used to support the success. The data for thisresearch will be collected by giving pre-test, post-test andobservation. In order to get or to collect the data for this study, thewriter has to find the suitable instrument. The instrument used forthe speaking test as description. The students are asked to speakbased on the dialogue given by the teacher. In this speaking, theteacher will observe the students ability in grammar, pronunciation,vocabulary and fluency. In this research the writer involved in everycycle and in collecting the data. The researcher used the Observationsheet and test. The observation sheet is to know the students'speaking ability, a check list is used. The check list is done during theclassroom activities. in doing the check list, the researcher will invitecollaborators to the classroom. The class however, will be handled bythe researcher who is in charge of teaching speaking by using roleplay. The test was given to the student is in oral form. Before the testis given, the researcher gave a pre-test at the beginning of the cycleand post-test at the end of the cycle. So, in this research, the studentsare asked to role play their conversation in front of the class based onthe conversation given. The students' speaking ability between pretestand post-test in every cycle is increase. The research question wasanswered and the hypothesis is reliable. The use of role play is anaffective technique to improve the students' speaking ability of thefirst year students of SMP Negeri 1 Tanah Putih in academic year2010/2011. In conclusion, the speaking ability of the first yearstudents of SMP Negeri 1 Tanah Putih before treatment is poor. Butafter getting treatment, the students' speaking ability becomes good.It means that the speaking ability of the students is increase.
INTRODUCTON
Based on the level of Education Unit Curriculum 2006 for junior high school as used by SMPN 1 Tanah Putih, English lesson in junior high school is very important. As everybody knows, English as a foreign language has become a compulsory subject for the students. In Indonesia, the government has a decided that English is one of final examination subjects for junior high school students. It is a good decision because the human resources need to access information on science and technology that are presented in English, either in written or spoken forms. The function of English is to provide the students with the four language skills: listening, speaking, reading and writing. Speaking ability is basic requirements for junior high school students.
Speaking supposed to be practiced daily in teaching and learning processes in the classroom, but many students still communicate in Indonesian language. Some problems faced by students, those are English is a foreign language English pronunciation is not some with the written like in Indonesian language, the grammatical errors and less expressions. The students are also afraid to speak English because they lack of vocabularies.Based on those problems, the writer will use role play in teaching. The writer thinks that Role Play can improve the students' speaking ability.
Teaching English for beginners with appropriate method can motivate them. Brown and Yule (1994:217) say that there are four language skills that must be learned by the students in English learning. As mentioned, speaking is one of four language skills that need to be learned by the students because in speaking, people will be able to say what they want to express.
The goal of teaching speaking is communicative efficiency. Learners should be able to make themselves understood, using their current proficiency. They should try to avoid confusion in the message due to faulty pronunciation, grammar or vocabulary and to observe the social and cultural rules that applied in each communication situation.
To help students develop communicative efficiency in speaking, instructors can use a balanced approach that combines language input, structured output, and communicative output. Because of the problems above, Ur (1981) says that the use of role play has added a tremendous number of possibilities for communication practice. It was suggested is one of the extracts quoted in the previous unit that one way to vary the kinds of spoken interaction.Based on the theories above, the writer is interested in conducting a research about "The Application of Role Plays as an Effort to Improve the Speaking Ability of the First Year Students of SMPN 1 Tanah Putih".
METHODOLOGY
This is an action research. The action research consists of four steps in a cycle. They are planning, action, observing and reflecting. If the problem can not be solved in the first cycle, the research is continued to the next cycle with some revisions in the activities.Below is the description of each cycle of the classroom action research that was conducted by the researcher:
Figure 1. Action ResearchCycle (Kemmis , 1992)
In doing this research is needed some of the data is used to support the success. The data for this research will be collected by giving pre-test, post-test and observation. In order to get or to collect the data for this study, the writer has to find the suitable instrument. The instrument used for the speaking test as description. The students are asked to speak based on the dialogue given by the teacher. In this speaking, the teacher will observe the students ability in grammar, pronunciation, vocabulary and fluency.
In this research the writer involved in every cycle and in collecting the data. The researcher used the instrument as seen below:
Observation sheet
Is to know the students' speaking ability, a check list is used. The check list is done during the classroom activities. in doing the check list, the researcher will invite collaborators to the classroom. The class however, will be handled by the researcher who is in charge of teaching speaking by using role play.
Test
This research used a test. The test was given to the student is in oral form. Before the test is given, the researcher gave a pre-test at the beginning of the cycle and post-test at the end of the cycle. So, in this research, the students are asked to role play their conversation in front of the class based on the conversation given.
To analyzed the students speaking ability, the following formula is used: So, based on the description above, the classifications of the students' speaking ability can be presented as follows: The pre-test was carried out to determine the background ability of the students. Thespeaking test contains conversations.After the teaching learning process had run for a month, the post-test was conducted in order to know the development of the students who joined the role play techniques in an effort to improve the speaking ability. The administration was at the end of the first cycle.The items used for this speaking test was taken from the items of the previous pre-test. The result of the post-test was analyzed and used as the final data for this research.
RESEARCH FINDINGS
The students' speaking ability was increase after they were taught by using role play. The increase occurred to most of the students. The improvement occurred because the role play did in every meeting with some of reflection. The improvement of the students' speaking ability also occurred in some proficient list (pronunciation, grammar, vocabulary, fluency and expression).
The result of research is 75.69-38.05 = 37.64. it shows that the result of the students' speaking ability is increase. So the writer can conclude that the students' speaking ability is satisfactory. The data can be seen in the following table :
|
2018-12-15T04:59:33.977Z
|
2013-04-16T00:00:00.000
|
{
"year": 2013,
"sha1": "679d173ba30361a6ffd4f3f984b7aab0585672da",
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244490059
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pes2o/s2orc
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v3-fos-license
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Passive Recharge Burst Spinal Cord Stimulation Provides Sustainable Improvements in Pain and Psychosocial Function: 2-year Results From the TRIUMPH Study
This study assessed long-term safety and effectiveness of spinal cord stimulation using a passive recharge burst stimulation design for chronic intractable pain in the trunk and/or limbs. Significant improvements in pain, physical, mental, and emotional functioning observed after 6 months of treatment were maintained at 24 months.
C hronic pain of moderate-to-severe intensity affects as many as 20% of the population. 1 Of these, pain is disabling for approximately 12%, 2 limiting them from work productivity or participating in life roles. 3,4 Chronic pain is often emotionally distressing; resulting in a strong co-occurrence of mood issues such as depression and anxiety. 2,5 Health-related quality of life ratings among chronic pain patients have been reported as 28% lower than that of the general population. 6 Spinal cord stimulation (SCS) is a valuable pain management option for patients with intractable neuropathic pain of the trunk and/or limbs. 7,8 A commonly-used metric for assessing therapeutic response is to calculate the percentage of pre-implant baseline pain that is relieved with treatment, using a visual analog scale (VAS) or numeric rating scale (NRS). Pain ratings captured in tightly controlled studies may be inconsistent with those captured in studies with real-world observational designs (e.g., [9][10][11] ). The International Association for the Study of Pain released a revised definition of pain in 2020 saying, in part, that, ''Pain is always a personal experience that is influenced to varying degrees by biological, psychological, and social factors. . .. Although pain usually serves an adaptive role, it may have adverse effects on function and social and psychological well-being.'' 12 Because the pain experience is multi-factorial, so should be its assessment. The IMMPACT group recommends the incorporation of multiple nonpain measures such as physical and emotional functioning in pain studies, 13 and this approach is endorsed by the International Neuromodulation Society. 14 Passive recharge burst (BurstDR; Abbott, Plano, TX) SCS has emerged in the past decade as an important innovation. 15,16 This stimulation design is characterized by a fivepulse train with internal frequency of 500Hz delivered at 40Hz, with a 1 ms pulse width, utilizing a passive recharge pattern. Several randomized controlled trials have demonstrated superiority of this waveform to conventional tonic SCS. [16][17][18][19][20] The mechanisms of action of passive recharge burst (hereafter referred to as burst) are different than that of tonic, as demonstrated in animal models. [21][22][23][24] Burst firing patterns occur naturally in the medial thalamic and intra-laminar nuclei and this complex is believed to potentiate anterior cingulate cortex (ACC) neuronal activity. 25 Brain imaging studies in humans have confirmed that, unlike tonic stimulation, burst stimulation modulates the medial pain pathway in the brain that projects to the ACC and anterior insula. These regions are believed to process the emotional and affective aspects of pain. Burst stimulation may therefore exert a greater effect by not only modulating the lateral and the descending pain-inhibitory pathways, similar to tonic SCS, but also the medial aspect of the spinothalamic tract. [26][27][28][29] Focusing on pain intensity for the assessment and care of chronic pain patients results in incomplete goal setting, incorrect patient selection for treatment, and limits our understanding of therapy response and effectiveness. 30 A systematic literature review of burst SCS clinical outcomes identified that a wide range of nonpain measures have been employed in the assessment of burst SCS outcomes, and together, strongly point to holistic improvement. 31 One year results from the prospective TRIUMPH study corroborated these findings, in particular pointing to improvements in mental health and reductions in the use of opioid medications. 32 Herein we report 2-year outcomes from the TRIUMPH study.
MATERIALS AND METHODS
TRIUMPH (clinicaltrials.gov registration NCT03082261) was a prospective, post-market, single-arm study that enrolled 269 participants across 22 sites in the United States, Canada, and Europe. The purpose of TRIUMPH was to assess long-term safety and effectiveness of SCS for chronic pain in the trunk and/or limbs using a burst enabled spinal cord stimulation (B-SCS) system. The study began enrolling in 2016 and the final follow up was completed in August 2020. The study was conducted in compliance with principles of Good Clinical Practice, the Declaration of Helsinki, and present regional local laws and regulations. Study oversight was provided by local institutional review boards or ethics committees, and subjects gave their written informed consent before any study activities.
Centers were instructed to approach all eligible patients and ask for their interest in participating in the study. Patients (!18 years of age) with chronic, intractable pain of the trunk, and/or lower limbs, recommended by a physician for SCS therapy, were recruited for this study. Patients had to pass a psychological screening according to the standard of care of individual sites. Eligible patients had a baseline score on the Numeric Rating Scale (NRS) !6 over the past 24 hours for average pain specific to the area(s) of chronic pain being treated with SCS. Patients with an existing SCS system, who previously failed SCS, were planning to have a different neurostimulation system or drug pump implanted, or had a primary diagnosis of peripheral vascular disease, angina pectoris, or chronic migraine were excluded from the study.
Design and Intervention
Subject demographics and medical histories were captured at baseline. Subjects completed questionnaires regarding pain intensity (NRS), patient-reported pain relief (PRPR; a stated percentage of improvement in pain), quality of life (EQ-5D), 33 39 at baseline and at follow-up intervals (6, 12, 18, and 24 months post-permanent implant).
Before receiving a permanent SCS implant, participants underwent a trial evaluation period. Following a successful trial evaluation (defined as !50% patient reported pain relief and a willingness to have a permanent implant), a permanent SCS system was implanted. The implant was activated and programmed with both tonic and burst settings. Tonic stimulation works by emitting electric pulses delivered at a consistent frequency, pulse width, and amplitude. This waveform is dependent on paresthesia to cover painful areas and can therefore not be used at subperception levels. Burst is a waveform that delivers groups of pulses at a high frequency and at amplitudes much lower than tonic stimulation. During the interburst interval passive repolarization occurs before the next burst. This stimulation design does not use paresthesia to mask the pain sensation. Subjects were instructed in the use of their patient programmer and determined the schedule and intensity of their treatment, using tonic, burst, or a combination of waveforms. Participants with an unsuccessful trial were withdrawn from the study.
Activity level and usage of pain-related medication (including analgesics, non-steroidal anti-inflammatory drugs [NSAIDs], opioids, anticonvulsants, muscle relaxants, and psychotropic medication) were assessed at baseline and follow-up. In addition, subjects were asked about their satisfaction with treatment, overall change in their health state (Patient Global Impression of Change [PGIC]), and waveform preference at follow up. Device-related information such as programming parameters, battery consumption, and recharging activities were also collected at followup. Adverse events were captured throughout the study.
Data Analysis
Statistical analysis was completed using SAS version 9.3 (SAS Institute Inc., Cary, NC). Data are presented as means AE stanstandard deviation (SD), and proportions. Two-tailed paired t test was used for continuous variables. All statistical analyses were performed with significance accepted at P < 0.05. No adjustment for multiplicity has been made. Imputation methods were used as appropriate to account for missing data. The datasets generated during and/or analyzed during the present study are not publicly available but are available from the corresponding author on reasonable request.
RESULTS
This report includes data from 128 subjects ( Figure 1). These subjects underwent a 3-to 30-day trial period utilizing burst stimulation and an external trial stimulator, were implanted with a permanent implantable pulse generator (IPG), used burst stimulation at least sometimes, and completed the 24month assessments. Three subjects in this cohort had a tonic salvage trial after the initial burst trial failed. Twelve patients who were trialed with an all-in-one procedure (or ''on-the-table'' trial) and one patient who only used tonic stimulation throughout the follow-up period were excluded from analysis.
Average age was 58.4 AE 13.2 years and 65.6% were female. The average (SD) duration of chronic pain was 9.8 AE 8.1 years. Pain started after an accident (motor vehicle or other) in 35.1% of subjects, due to a medical condition in 29.7%, and following surgery in 14.1%. Radiculopathy and persistent spinal pain syndrome (formerly failed back surgery syndrome), diagnosed separately or combined with another chronic pain condition, were the most frequent pain diagnoses in the study. Only 12 subjects (9%) had a diagnosis that did not include one of these two indications; they were diagnosed with complex regional pain syndrome or intervertebral disc disorder. Most subjects categorized as ''Other" had nonsurgical back pain (e.g., spinal stenosis, spondylosis) ( Table 1).
Percutaneous Octrode leads were implanted in 64.4% of subjects, and a paddle lead was used in 36.6%. A nonrechargeable IPG was implanted in 82.8% of subjects. At 6 months, in participants with a rechargeable IPG, 82.6% (19/23) charged their devices on a weekly basis or less frequently and this increased to nearly all subjects (21/22) at 24 months. Greater than 21% (5/23) recharged once every 3 weeks, or less often at 6 months and this proportion remained stable until 24 months.
At each study visit, !86% of participants reported using burst stimulation. Of these, !70% used it intermittently with a duty cycle of 30 seconds on and 90 seconds off. The majority of subjects preferred burst stimulation at all follow-up timepoints, ranging from 90.2% of subjects at 6 months to 85.0% at 24 months. About half of subjects at each timepoint indicated that burst was the only waveform they used to control their pain.
At baseline, 78.1% of subjects rated that pain had a major impact in their life. At 24 months, this proportion decreased to 27.3%. With regards to the subject's activity level, the percentage reporting being moderately or very active increased from 32% at baseline to 53.1% at 24 months. The proportion of subjects who reported that they were ''moderately better," ''better," or ''a great deal better" on the PGIC was 77.3% at 24 months. Fewer than 5% of subjects did not report a change in their condition with treatment. Figure 3 presents the proportions of these three analyses at all timepoints. Subjects decreased their chronic pain medication intake for all categories with 38% reducing psychotropic medication and muscle relaxants, 46% reducing analgesic, anti-convulsant and NSAIDs, and 48% reducing opioid medications at 24 months (Table 2).
There were statistically significant (P < 0.001) improvements across all evaluated psychosocial domains at all followup timepoints. Most notably, catastrophizing measured on PCS decreased to normal population levels and were sustained at all timepoints. 34 At 24 months, 79% (34/43) were no longer clinically catastrophizing and 61% (38/62) were no longer clinically depressed. 36,40 The improvements observed at 6 and 12 months were maintained through 24 months (Figure 4). Similarly, health-related quality of life as per the EQ-5D improved across all domains. At 24 months, the EQ-5D index score was within one SD of the population norm ( Figure 5) and 64% (82/128) met the clinically important change. 41 Subject satisfaction was high with 84.4% rating themselves as 'satisfied' or 'very satisfied' with treatment at 24 months. At all timepoints, >85% of subjects expressed that they would be willing to do the procedure again for the same results, and >90% would recommend the treatment to a family member with the same condition ( Figure 6). No unanticipated adverse events have been reported. Among the 269 enrolled subjects, there were a total of 53 events in 41 subjects (15.2%). Twenty-one events were considered serious and were reported in 18 subjects (6.7%). The most common serious event was infection (n ¼ 5; 1.9%). There were also 32 nonserious events in 27 subjects. Most common nonserious events were changes in stimulation or reduced pain relief due to lead failure (n ¼ 4) or lead migration (n ¼ 4). No event occurred at a rate higher than 3%. There were no neurological injuries.
DISCUSSION
We report here that significant improvements in physical, mental, and emotional functioning can be sustained through 24 months of treatment with B-SCS. In particular, painrelated catastrophizing and depression improved with treatment. Given that psychological distress portends poor treatment outcomes, 42,43 the capacity to shift pain-related beliefs and behaviors toward more positive outlooks suggests that burst SCS, through its unique mechanism of action, is a therapy suitable to manage patients thought of as difficult to treat and to salvage nonresponders.
This study enrolled patients who were broadly representative of the SCS population. 44 This report follows those who had standard multi-day trials and who used burst at least some of the time after their permanent implant, thus giving insight into usage patterns in the real world. In addition to pain descriptors, a range of non-pain measures were collected, in alignment with best-practice recommendations. 13,14 Subjects used and preferred burst over tonic stimulation. When using burst stimulation, patients most often employed a cycling pattern (commonly 30 seconds on and 90 seconds off), whereas tonic stimulation was more often used continuously. Good clinical outcomes have been reported to be maintained while cycling burst stimulation, a setting that substantially reduces the overall electrical dosage and battery consumption. 45,46 Similar results have been achieved with lower-energy on:off cycling patterns such as 30:180 or 30:360 seconds. [45][46][47] A minority of subjects in this study (<20%) used rechargeable devices based on physician choice. Data on subjects' patterns of recharging their IPG batteries indicated that recharging fit within their lives, either as part of a normal routine or upon the device becoming depleted. Recharging was usually accomplished weekly within a few hours and with a minimum of complications. This is considerably less than the daily recharge burden that has been reported for devices using high-energy stimulation designs. 10 Pain intensity (on NRS) was statistically significant reduced at all timepoints. The PRPR was 60% and this showed maintenance of the 12-month cohort's PRPR of 59%. 32 A recent single-center retrospective review of 174 SCS patients indicated that PRPRs are strongly correlated with calculated percentage pain reductions, and that the former are consistently higher. 48 Similar trends have been observed across a variety of pain populations. [49][50][51] Thus, stated vs. calculated pain reduction percentages may not be equivalent; stated percentages, as a momentary measure in the context of current background pain, may be the more personally salient descriptor. In addition, >50% of the subjects in this report who initially said that pain had a major impact in their lives improved by at least one category, whereas nearly half of opioid users maintained their dosage reduction through 24 months. Taken together, pain has a significantly smaller footprint in patients' lives with B-SCS.
Many SCS studies have reported on reductions in opioid usage given the crisis of misuse, addiction, and overdose in the US and other countries. 32,52,53 However, chronic pain is usually addressed with multiple therapies including socalled ''rational'' polypharmacy, indicating medications across different classes. Starting at 6 months and continuing to 24 months, many patients saw a reduction in antidepressants, anticonvulsants, NSAIDs, and other analgesics. This is a substantial finding in many ways, including financial and cognitive side-effect improvements in these subjects.
In addition to successful reduction in pain intensity, all psychometric data including anxiety, depression, kinesiophobia, catastrophizing, sleep, and physical function showed statistically significant improvements. The most impactful effect of the therapy was observed on catastrophizing and depression. Interestingly, a recent multivariate regression analysis found that, after controlling for other variables, these two psychological factors were found to be significantly associated with chronic, severe low back pain and disability. This highlights their importance in persistent pain when negative beliefs about pain may become solidified, in contrast to kinesiophobia or anxiety which may be more relevant during the earlier stages leading up to chronic pain. 54 Additionally, quality of life in chronic patients is strongly associated with catastrophizing and depression, more than with pain intensity. 55,56 We observed improvements across all EQ-5D domains. Subjects reported that they were significantly improved as compared to before the implant, and rates of satisfaction with therapy were high. The magnitude and nature of these outcomes were consistent with previous reports 20, 32 and were maintained through 24 months.
Limitations and caveats of this study have been previously discussed. 32 Briefly, this study was not designed to compare tonic and burst groups and, due to its real-world nature, programming and stimulation configurations were according to subject and physician preference. Twelve subjects who underwent an on-the-table trial (4% of enrollments) were excluded from current analysis. As burst is a subperception modality, paresthesia mapping was performed with tonic stimulation in these patients. The FDA recently issued a letter emphasizing the importance of a trial stimulation period before implant. 57 Medical device reports of SCS devices for pain show that failure to achieve or maintain adequate pain control is the most common reported problem, occurring in 28% of cases. Moreover, most failed trials occur in spite of sufficient paresthesia over the painful area with trial stimulation. 58
CONCLUSION
This study shows that subjects treated with B-SCS show no decrease in therapeutic effect over time; they improve baseline to 6 months and consecutively remain at a similar level in terms of pain, quality of life, and psychological profile up to 2 years after permanent implant. At the end of the study, subjects were highly satisfied with the therapy across the multiple symptom domains of chronic pain, feeling better, more active, while taking less pain and adjuvant medication. This provides converging evidence across multiple assessment tools that B-SCS can address the intensity of pain while also improving biopsychosocial issues.
Key Points
The objective of this study was to assess long-term safety and effectiveness of spinal cord stimulation using a passive recharge burst stimulation design for chronic intractable pain in the trunk and/or limbs. Significant improvements in pain, physical, mental, and emotional functioning observed at 6 months of treatment were maintained at 2 years after implant. Participants reported a significant decrease in the impact of pain on life and in the amount of medication consumed across all drug classes. Burst spinal cord stimulation treatment had high patient satisfaction: more than 85% of subjects expressed that they would be willing to do the procedure again and >90% would recommend the treatment. The magnitude and nature of these outcomes were consistent with previous reports and maintained through 24 months.
|
2021-11-24T06:18:14.627Z
|
2021-11-22T00:00:00.000
|
{
"year": 2021,
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269934547
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pes2o/s2orc
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v3-fos-license
|
Genetic Modifications in Bacteria for the Degradation of Synthetic Polymers: A Review
Synthetic polymers, commonly known as plastics, are currently present in all aspects of our lives. Although they are useful, they present the problem of what to do with them after their lifespan. There are currently mechanical and chemical methods to treat plastics, but these are methods that, among other disadvantages, can be expensive in terms of energy or produce polluting gases. A more environmentally friendly alternative is recycling, although this practice is not widespread. Based on the practice of the so-called circular economy, many studies are focused on the biodegradation of these polymers by enzymes. Using enzymes is a harmless method that can also generate substances with high added value. Novel and enhanced plastic-degrading enzymes have been obtained by modifying the amino acid sequence of existing ones, especially on their active site, using a wide variety of genetic approaches. Currently, many studies focus on the common aim of achieving strains with greater hydrolytic activity toward a different range of plastic polymers. Although in most cases the depolymerization rate is improved, more research is required to develop effective biodegradation strategies for plastic recycling or upcycling. This review focuses on a compilation and discussion of the most important research outcomes carried out on microbial biotechnology to degrade and recycle plastics.
Introduction 1.Definition and Classification of Plastics
Plastics are a type of synthetic polymeric material based on carbon and hydrogen and with high molecular weight [1].Industrial-scale plastic production began in the 20th Century, and their use has increased over time [2,3].Plastics can be classified according to different criteria.For example, depending on the composition of their backbone, they can be homochain polymers (when their backbone is made up exclusively of carbon) or heterochain polymers (if there are other elements, like oxygen and nitrogen, present in their backbone) [4].Depending on the monomers that they are made of, they can be classified as homopolymers if they only have one monomer or copolymers if they have two or more different monomers [1].Depending on their thermomechanical properties, they can be thermosets if, during their fabrication, covalent bonds are established between polymer chains, giving them high resistance and a shape that cannot be modified thermically; thermoplastics, if no covalent bonds are established between polymer chains so they can be fused and reshaped; or elastomers if they have a high elasticity [5].Depending on the presence of benzene rings in their backbone, they can be aromatic if they have benzene rings or aliphatic if they do not have benzene rings [6].Depending on the raw material they are made from, they can be petrochemical (the raw material is petroleum) or biobased (the raw material is biomass), and depending on their degradability by organisms, they can be biodegradable or non-biodegradable.
Advantages and Disadvantages of Plastics
In general, plastics are lightweight, long-lasting, inert and cheap and easy to produce.Their diversity is such that there are plastics with the ideal characteristics for almost any application.Furthermore, the use of mixtures and alloys, as well as additives, can change the properties of the material and adjust them as desired [1].As plastics are remarkably diverse and can have vastly different properties, their uses are just as varied.They are used to make, among many others, fibers and textiles, toys, packaging, healthcare instruments, such as syringes and implants, and construction materials for insulation, pipes and cable coatings [1].Even though plastics are generally cheap and easy to produce and have good mechanical properties, the field of engineering requires especially resistant materials for very specific applications.Though they are more expensive, the so-called "engineering plastics" are used for that purpose as they boast a high performance [1].Other plastics, known as commodity plastics, are found in everyday items.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 2 of 23 they have benzene rings or aliphatic if they do not have benzene rings [6].Depending on the raw material they are made from, they can be petrochemical (the raw material is petroleum) or biobased (the raw material is biomass), and depending on their degradability by organisms, they can be biodegradable or non-biodegradable.
Advantages and Disadvantages of Plastics
In general, plastics are lightweight, long-lasting, inert and cheap and easy to produce.Their diversity is such that there are plastics with the ideal characteristics for almost any application.Furthermore, the use of mixtures and alloys, as well as additives, can change the properties of the material and adjust them as desired [1].As plastics are remarkably diverse and can have vastly different properties, their uses are just as varied.They are used to make, among many others, fibers and textiles, toys, packaging, healthcare instruments, such as syringes and implants, and construction materials for insulation, pipes and cable coatings [1].Even though plastics are generally cheap and easy to produce and have good mechanical properties, the field of engineering requires especially resistant materials for very specific applications.Though they are more expensive, the so-called "engineering plastics" are used for that purpose as they boast a high performance [1].Other plastics, known as commodity plastics, are found in everyday items.
The most used commodity plastics are poly(ethylene terephthalate) (PET), highdensity polyethylene (HDPE), poly(vinylchloride) (PVC), low-density polyethylene (LDPE), polypropylene (PP) and polystyrene (PS) [2,7,8].Products made with these plastics can be identified according to the ASTM International Resin Identification Coding System (RIC).The "Others" category includes all other plastics, as well as products made with a mixture of two or more plastics [9].The global annual production of plastics exceeded 400 million metric tonnes (400 Mt) in 2022, 362.3 of which were new petrochemical plastics.Commodity plastics make up the following percentages (see Figure 1): PET 6.2%, HDPE 12.2%, PVC 12.7%, LDPE 14.1%, PP 18.9% and PS 5.2% [10].Out of the different uses, most plastic is destined for packaging.In 2019, 142 Mt (31%) of plastics were used in packaging [3,7].Between 1950 and 2022, more than 10 billion metric tonnes (10.000Mt) of plastics were produced [2,10,11].Despite their useful properties, the use of plastics has two main disadvantages, which have to do with the beginning and the end of their lifetime: the first one is the fact that most plastics are petrochemical, and the second one is the amount of non-biodegradable waste generated.Regarding their origin, the reserves of petroleum are limited as petroleum is a non-renewable resource, and given the production rate in 2020, it is Despite their useful properties, the use of plastics has two main disadvantages, which have to do with the beginning and the end of their lifetime: the first one is the fact that most plastics are petrochemical, and the second one is the amount of non-biodegradable waste generated.Regarding their origin, the reserves of petroleum are limited as petroleum is a non-renewable resource, and given the production rate in 2020, it is estimated that reserves will last for 50 years [12].For the purposes of this article, the waste generated is the most relevant problem.
Bioplastics as an Alternative to Petroleum-Based and Non-Biodegradable Plastics
The most recent assessments estimate that out of the 9200 Mt of plastics produced until 2017, 6500 Mt (70%) have become waste.Of this waste, 5000 Mt (54%) ended up in landfills or were released into the environment [13].This plastic waste has reached virtually every corner of the planet.The five so-called garbage patches are notorious for accumulating 250,000 tons of plastic [7], but plastics are also found elsewhere.They have been found in rivers, lakes, drinking water and table salt and even in extremely remote places, such as the seabed and both polar regions.They have reached such areas mostly in the form of micro-and nanoplastics, whose size allows them to travel much further and even enter organisms' cells [14].Due to their composition and structure, plastics can remain in the environment for thousands of years [15], and their effects on living beings' health, while a complicated matter that is still under study, are undoubtedly severely negative.This has been researched especially in marine ecosystems, where they can cause harm to many different organisms [8,16,17], but they are also apparent in terrestrial ecosystems [18] and human health [19,20].Moreover, the additives used to modify the properties of plastics can also have toxic effects on organisms [21], and plastics can adsorb and accumulate heavy metals, persistent organic contaminants and even pathogens [22].
Due to their harmful effects on the environment, plastics, like any other type of waste, have to be managed appropriately.Recycling rates are overall very low, with only 600 Mt (6.5%) of plastics produced worldwide until 2017 having been recycled and more than 5000 Mt of them ending up in landfills or being released into the environment [13].Landfilling plastic waste, though simple, is not an adequate waste management strategy, as landfills can leach microplastics [23,24] as well as other contaminants [25].Moreover, plastic waste in the environment does not remain permanently unchanged, as it slowly undergoes physicochemical changes that partially break it down.These changes include photodegradation, hydrolysis and thermo-oxidation.However, this transformation results mostly in the fragmentation of plastic rather than its mineralization, generating smaller plastic fragments (micro-and nanoplastics) that still remain in the environment.In order for plastics to be mineralized and, therefore, completely removed from the environment, biological degradation is required [26].This biological degradation can be performed by different organisms but, even if plastics end up mineralized by the action of these organisms, the degradation rates are so slow and the plastic permanence is so high that most plastics are considered non-biodegradable.Given the problems derived from the properties of the most used plastics, a new type of material has received a lot of attention: bioplastics.
Bioplastics are a type of plastic that differs from conventional plastic in at least one of two ways: the raw material they are made from or their biodegradability.Bioplastics can either be made from biomass-biobased plastics-or be biodegradable or have both properties [27].A particular type of bioplastics is poly(hydroxyalkanoates) (PHAs).They are naturally produced by some microorganisms, mostly bacteria, for energy storage [28] and are both biobased (as they are produced by an organism that can be grown with renewable feedstock) [29] and biodegradable (as they can be easily enzymatically degraded) [30].Just like plastics in general, bioplastics are very varied, so grouping them in the bioplastic category is not particularly informative of their properties, especially without specifying which criteria make them such.Therefore, when discussing individual plastics, this work will specify if they are petrochemical or biobased and biodegradable or non-biodegradable (Figure 2).The main benefits of using bioplastics are summarized in the fact that biobased plastics do not involve the use of petroleum, and biodegradable plastics can be fully mineralized by organisms.However, bioplastic production still has an environmental impact when the whole life cycle is considered [31], and regarding biodegradable plastics, this property does not mean that they can be released into the environment without consequences [32].
biodegradable plastics, this property does not mean that they can be released into the environment without consequences [32].Biodegradable plastics can remain in the environment for long periods of time [33][34][35][36] and carry potentially toxic substances [37].They can even act as a reservoir of microbes with antibiotic-resistance genes [38], so their waste should be managed, too [39].In the case of biodegradable plastics, the preferred waste management strategy is carrying out their degradation by organisms.There are international standards developed to clearly define the conditions and timescale of biodegradation [40][41][42][43][44][45][46], but their use is not mandatory, so the criteria to determine the biodegradability of plastics can be quite arbitrary.However, plastic is generally considered biodegradable if there are organisms that can mineralize it in a reasonable amount of time in controlled conditions, which must always be specified.
Biodegradation of Plastic Polymers
The last few decades have seen an increasing interest in both biodegradable plastics and organisms that can degrade plastics.As new species and strains are discovered and genetic engineering is used to improve the enzymes involved in biodegradation, plastics that were previously considered non-biodegradable can now be degraded by one organism or another and to a greater or lesser extent.Therefore, the categories of biodegradable and non-biodegradable plastics are not fixed and can change over time, but in this work, we will use the usual classification.
Plastic-degrading microorganisms include bacteria, fungi and microalgae [47].This review focuses on bacterial strains.Although genetic engineering approaches have been performed for decades, only a few bacteria have been modified to improve their capability of degrading these synthetic plastics or to grant them this characteristic by modifying their metabolism.More specifically, the modifications performed consist of the heterologous expression of enzymes that come from other organisms, mutating/changing specific amino acids of the enzymes and the addition of domains or other protein structures to the enzymes.Different techniques have been used in order to carry out these modifications, Biodegradable plastics can remain in the environment for long periods of time [33][34][35][36] and carry potentially toxic substances [37].They can even act as a reservoir of microbes with antibiotic-resistance genes [38], so their waste should be managed, too [39].In the case of biodegradable plastics, the preferred waste management strategy is carrying out their degradation by organisms.There are international standards developed to clearly define the conditions and timescale of biodegradation [40][41][42][43][44][45][46], but their use is not mandatory, so the criteria to determine the biodegradability of plastics can be quite arbitrary.However, plastic is generally considered biodegradable if there are organisms that can mineralize it in a reasonable amount of time in controlled conditions, which must always be specified.
Biodegradation of Plastic Polymers
The last few decades have seen an increasing interest in both biodegradable plastics and organisms that can degrade plastics.As new species and strains are discovered and genetic engineering is used to improve the enzymes involved in biodegradation, plastics that were previously considered non-biodegradable can now be degraded by one organism or another and to a greater or lesser extent.Therefore, the categories of biodegradable and non-biodegradable plastics are not fixed and can change over time, but in this work, we will use the usual classification.
Plastic-degrading microorganisms include bacteria, fungi and microalgae [47].This review focuses on bacterial strains.Although genetic engineering approaches have been performed for decades, only a few bacteria have been modified to improve their capability of degrading these synthetic plastics or to grant them this characteristic by modifying their metabolism.More specifically, the modifications performed consist of the heterologous expression of enzymes that come from other organisms, mutating/changing specific amino acids of the enzymes and the addition of domains or other protein structures to the enzymes.Different techniques have been used in order to carry out these modifications, such as metagenomic analysis, directed and non-directed mutagenesis and the use of fusion proteins or chimeras.
This review is a compilation of the studies in which bacteria have been genetically modified, whether to grant them or enhance their ability to degrade plastics or simply to perform genetic modifications and will only mention the polymers for which modified bacteria have been obtained.Table 1 shows the modified bacterial strains that have been developed for the degradation of plastic polymers according to research published in the scientific literature (i.e., PubMed, Scopus, etc.).Since PET is one of the most produced plastics worldwide and its degradation is challenging, a special focus is put in this review in relation to the microbial biodegradation strategies developed specifically for this polymer (summarized in Table 2).
Biodegradation of PET
PET is one of the most produced petrochemical synthetic polymers, accounting for around 6.2% of global plastic production in 2022 [10], and its market size surpasses other highly produced plastics, such as HDPE, PVC or PP [74].The main reason why PET is so produced is because its molecular structure offers a lot of versatility, making it essential in our daily lives.PET is a long semi-aromatic thermoplastic polyester chain produced from ethylene glycol (EG) and terephthalic acid (TPA).Its production has two steps.First, the union of two EG molecules and one TPA molecule by esterification generates an intermediate molecule, bis(2-hydroxyethyl) terephthalate (BHET).Secondly, BHET along with catalysts, such as Sb 2 O 3 or Sb(OAc) 3 , is subjected to a process of polymerization, creating the long chain through ester bonds (Figure 3A) [5,75].When it comes to the manufacturing process, amorphous and semi-crystalline PET are produced depending on the thermal processing undergone during the polymerization.The main difference between these two types lies in the intrinsic viscosity and molecular weight.In the case of the amorphous polymer, the long polyester chains are randomly set out, resulting in a more flexible plastic.On the other hand, semi-crystalline materials are formed by amorphous domains and chains arranged in an orderly way, making the material more resistant and less ductile (Figure 3B).These differences in its molecular structure make it both chemically and thermally stable.This is what makes PET a strong and durable compound, ideal for a wide variety of applications, such as synthetic fibers for the textile industry, water bottles and packaging [5,76].between these two types lies in the intrinsic viscosity and molecular weight.In the case of the amorphous polymer, the long polyester chains are randomly set out, resulting in a more flexible plastic.On the other hand, semi-crystalline materials are formed by amorphous domains and chains arranged in an orderly way, making the material more resistant and less ductile (Figure 3B).These differences in its molecular structure make it both chemically and thermally stable.This is what makes PET a strong and durable compound, ideal for a wide variety of applications, such as synthetic fibers for the textile industry, water bottles and packaging [5,76].Despite the advantages provided by PET, it also presents the serious drawback of what to do with it after its useful life.Most PET comes to an end accumulated in landfills.It is estimated that it takes around 300 years to decompose, degrading over time because of solar radiation and heat, among other factors, and releasing harmful chemical compounds to the environment.The estimate increases if its degradation is not accelerated by heat or solar radiation, reaching 2500 years or more.PET is so difficult to degrade due to its physicochemical properties, which make it resistant to decomposition by water and organic and inorganic compounds [73,76].
For this reason, there are a series of methods, which involve mechanical, chemical and biological methods, to recycle and reuse this polymer, producing fibers and fabrics.The mechanical methods are the most widespread, but they are also very expensive, while in the case of chemical methods, the process can be harmful to the environment [70,78].
In contrast with the previously mentioned methods, biological methods are on the rise because they do not damage the environment.These methods are still in development and are based on the use of cells as factories of enzymes able to break the bonds of PET, releasing the monomers of which it is composed.After that, those different monomers can be subjected to a valorization process to generate high value-added molecules, such as polyhydroxyalkanoates, vanillic acid, gallic acid, lycopene, glycolic acid, pyrogallol, catechol and muconic acid, which can be used as flavors, cosmetics, sanitizers and animal feed and in pharmacy, among other uses [78,79].
Enzymes Involved in the PET Degradation Pathway
The reason why PET can be degraded by enzymes is because a lot of them are unspecific regarding their substrates.The current literature offers many names given to these enzymes, such as PET hydrolases, PET esterases, PET cutinases, PET depolymerases or PETases.PET esterases, PET cutinases and PET depolymerases are hydrolytic enzymes that are able to break the ester bonds of biological molecules, like suberin and cutin, and due to their unspecificity, it turns out that they can break those of PET as well.On the other hand, PETases are hydrolytic enzymes more specific to PET [80,81].Due to the Despite the advantages provided by PET, it also presents the serious drawback of what to do with it after its useful life.Most PET comes to an end accumulated in landfills.It is estimated that it takes around 300 years to decompose, degrading over time because of solar radiation and heat, among other factors, and releasing harmful chemical compounds to the environment.The estimate increases if its degradation is not accelerated by heat or solar radiation, reaching 2500 years or more.PET is so difficult to degrade due to its physicochemical properties, which make it resistant to decomposition by water and organic and inorganic compounds [73,76].
For this reason, there are a series of methods, which involve mechanical, chemical and biological methods, to recycle and reuse this polymer, producing fibers and fabrics.The mechanical methods are the most widespread, but they are also very expensive, while in the case of chemical methods, the process can be harmful to the environment [70,78].
In contrast with the previously mentioned methods, biological methods are on the rise because they do not damage the environment.These methods are still in development and are based on the use of cells as factories of enzymes able to break the bonds of PET, releasing the monomers of which it is composed.After that, those different monomers can be subjected to a valorization process to generate high value-added molecules, such as polyhydroxyalkanoates, vanillic acid, gallic acid, lycopene, glycolic acid, pyrogallol, catechol and muconic acid, which can be used as flavors, cosmetics, sanitizers and animal feed and in pharmacy, among other uses [78,79].
Enzymes Involved in the PET Degradation Pathway
The reason why PET can be degraded by enzymes is because a lot of them are unspecific regarding their substrates.The current literature offers many names given to these enzymes, such as PET hydrolases, PET esterases, PET cutinases, PET depolymerases or PETases.PET esterases, PET cutinases and PET depolymerases are hydrolytic enzymes that are able to break the ester bonds of biological molecules, like suberin and cutin, and due to their unspecificity, it turns out that they can break those of PET as well.On the other hand, PETases are hydrolytic enzymes more specific to PET [80,81].Due to the specificity, there are small differences between the different types of enzymes, such as PET cutinases, which can hydrolyze the ester bonds of aliphatic and aromatic molecules, while PETases can only hydrolyze bonds of aromatic molecules [54,65,69].To simplify the following explanation, we will refer to all of them as PETases unless it is otherwise stated, with the understanding that PETases are enzymes that hydrolyze PET.
The hydrolyzation pathway of PET is summarized in Figure 4.In most cases, PETases are capable by themselves of firstly depolymerizing the PET chain and secondly degrading its monomers-mono(2-hydroxyethyl) terephthalate (MHET) and BHET-to finally form TPA and ethylene glycol.PETases can directly produce MHET and TPA, or In contrast, if BHET is generated, they can degrade it to MHET [5,78].However, there are some PETases that are not able to carry out both steps by themselves, as is the case with PE-H (Pseudomonas aestusnigri) and IsPETase (Ideonella sakaiensis).Both can only depolymerize PET because they cannot break the ester bond of MHET, which accumulates [82].In contrast, there are enzymes capable of degrading only MHET (MHETases).IsMHETase is an example, being able to degrade MHET to finally produce ethylene glycol and TPA.Although there are enzymes that do not carry out both degradation actions by themselves, they can complement each other.IsMHETase and IsPETase were found in I. sakaiensis 201-F6 when this strain was discovered in a medium whose only carbon source was PET [81].Recently, a new category of enzymes has been discovered, BHETases, which specifically catalyze the transition from BHET to MHET.Two BHETases have been characterized, ChryBHETase from Chryseobacterium sp. and BsEst from Bacillus subtilis [83].
As a consequence of the cooperation between PETases and MHETases or the action of only PETases, PET is hydrolyzed, and TPA and ethylene glycol are released, and they can be metabolized (Figure 4).TPA presents a biochemical pathway to finally be degraded to succinyl-CoA.Regarding ethylene glycol, it can be metabolized to obtain acetyl-CoA [78].Both succinyl-CoA and acetyl-CoA are incorporated into the tricarboxylic acid cycle (TCA) to obtain energy for the bacteria and, in certain cases, some products of interest.For example, species I. sakaiensis and Geobacter sulfurreducens when cocultivated can degrade PET to ethylene glycol and generate electricity [84].In another study, a Rhodococcus josii strain PET metabolized the PET hydrolysate and synthesized lycopene, which is used in medicine for cancer treatments [85].One example of a modified bacteria is Escherichia coli modified to degrade PET and produce vanillin, present in cosmetic and food industries [70].The cleavage of the ester bond happens inside the enzyme's active site, and the ability of different enzymes to hydrolyze PET comes as a consequence of a nucleophilic similarity produced by three amino acids conserved in PETases, Ser-His-Asp.In contrast, the adjacent sequences to these amino acids are different among enzymes, varying their selectivity to substrates [50,63].
There are two proposed classifications for PETases in the literature.One of them is based on their sequence, while the other one is based on their protein structure.According to the sequence-based classification, PETases can be classified as type I and type II enzymes [5].Type I enzymes have one C-terminal disulfide bridge, and type II enzymes have two, with one of them being close to the active site.This additional disulfide bridge provides type II enzymes more thermal stability and plays a key role in the hydrolytic action [5,63,86].Another characteristic that differentiates both types is the amino acids present in the active site.Type I enzymes, such as LCC and Cut190, which will be mentioned later, have His159 and Phe/Tyr238 residues, and type II enzymes present Trp and Ser in the same locations.In addition, type II enzymes are subdivided into type IIa if they have Phe or Tyr residues instead of Ser238 or type IIb if they maintain Ser, as is the case with IsPETase.As an exception, type I enzymes may not have the disulfide bridge.This is the case with PET27 and PET30 from Aequorivita sp.CIP111184 and Kaistella jeonii, respectively.According to the structure-based classification, PETases can have three different structures, with IsPETase, Fusarium oxysporum cutinase and Chloroflexus sp.MS-G cutinase representing Structures 1, 2 and 3, respectively [87].For example, IsPETase presents an α/β-hydrolase fold and a core made up of eight β-strands and six α-helices, as well as a highly polarized surface [65].some PETases that are not able to carry out both steps by themselves, as is the case with PE-H (Pseudomonas aestusnigri) and IsPETase (Ideonella sakaiensis).Both can only depolymerize PET because they cannot break the ester bond of MHET, which accumulates [82].In contrast, there are enzymes capable of degrading only MHET (MHETases).
IsMHETase is an example, being able to degrade MHET to finally produce ethylene glycol and TPA.Although there are enzymes that do not carry out both degradation actions by themselves, they can complement each other.IsMHETase and IsPETase were found in I. sakaiensis 201-F6 when this strain was discovered in a medium whose only carbon source was PET [81].Recently, a new category of enzymes has been discovered, BHETases, which specifically catalyze the transition from BHET to MHET.Two BHETases have been characterized, ChryBHETase from Chryseobacterium sp. and BsEst from Bacillus subtilis [83].
Modifications of Bacteria and Enzymes to Improve PET Degradation
Since the discovery of PET-degrading enzymes in the last century, efforts have been made to improve their degradation rate, either by searching for new enzymes and heterologously expressing them in other microorganisms or by modifying the existing ones.It was not until 2016 that the first PETase (IsPETase) was discovered in I. sakaiensis 201-F6, a specific enzyme that allowed the bacteria to use PET as its major carbon and energy source.The experiments were carried out at 30 • C and pH 7, and this enzyme was compared with three other enzymes with hydrolytic activity toward PET [81].IsPETase has an α/β hydrolase fold like cutinases but with a larger active site [65].After this discovery, modifications of this IsPETase have been carried out in order to obtain a higher degradation rate.The importance of the amino acids of the active site or close to it is emphasized, as will be seen afterward with cutinases.In addition, the presence of one amino acid or another may affect the enzyme-substrate interaction affinity and, therefore, the degradation of the polymer [86].
However, before IsPETase was discovered, different genetic engineering methods had already been carried out to increase the degradation speed of esterases and cutinases [55].Site-directed mutagenesis was used to specifically modify the sequence close to the active site of a cutinase from Fusarium solani pisi.Mutations were carried out to create a bigger space in the active site to facilitate the entrance of the polymeric chain.The modifications that created a more hydrophobic binding site resulted in the greatest PET degradation compared with the native enzyme.PET was degraded at a rate of 12 mg/h/mg of modified cutinase at pH 8 and 50 • C, while the native enzyme degraded 0.05 mg/h/mg of the enzyme at pH 7.0 and 55 • C.
Continuing with the idea of increasing hydrophobicity, in 2011, two amino acids were removed from the active site of the cutinase Tfu_0883 from Thermobifida fusca, expanding the space and improving its capacity to hydrolyze polyesters due to better PET adsorption.In the same experimental conditions, after 4 h, the native enzyme reached a spectral value (k/s) of 0.1, while the modified enzyme reached almost 0.2.Stretching the time, at 48 h, the values were around 0.15 and 0.3, respectively [56].In addition, the amino acids of surface regions outside the active site turned out to have a direct action in PET hydrolysis, as mentioned in Herrero-Acero et al. [57].In this study, the cutinase Thc_Cut2 from Thermobifida cellulosilytica DSM44535 was modified by exchanging amino acids in the surface region.It was shown that exchanging a surface positive residue, like arginine, for a non-charged one, like asparagine and serine, makes a modified enzyme with a higher affinity for PET, according to their k cat /K m values.In this case, the modifications produced a variation in the hydrolytic capacity of the enzymes, but the hydrophobicity did not change at any time.It is speculated that these changes in the amino acid sequence may decrease the size of the active site, stabilizing the region.
A similar idea is mentioned in Kawai et al. [58], where CUT190 cutinase from Saccharomonospora viridis AHK190 was triple-mutated to Ser226Pro/Arg228Ser/Thr262Lys.It was previously known that in its native state, it is able to degrade different polymers, such as PCL, PBSA, PBS and PHB, among others.However, these modifications resulted in an enzyme with more activity and higher thermostability.A possible explanation for this result was that a polar amino acid with a positive charge, such as arginine, close to the binding site, could interact with negative charges that may be present in the substrate molecules [61].For this reason, arginine was replaced by serine.In addition, it was concluded that the addition of Ca 2+ to the medium provides greater thermostability to CUT190 [58].In another study, Han et al. [61] obtained an enzyme with a mutation near the substrate binding site that facilitated the union with the substrate and increased its thermostability to 5 • C higher than the native enzyme.
In contrast, Austin et al. [65] achieved a modified IsPETase exchanging two amino acids-Ser238Phe/Trp159His-conserved in cutinases next to the active site.The modified enzyme had a smaller binding site and resulted in a slightly higher degradation rate of PET and polyethylene-2,5-furandicarboxylate (PEF), a semiaromatic polyester derived from PET. Hydrophobicity is mentioned to be relevant in the adsorption of the polymer by the active site.In the following work with IsPETase, eight modified enzymes were obtained.Three of them with modifications in their active site-Arg61Ala, Leu88Phe and Ile179Pheresulted, respectively, in higher PET degradation rates of 13.5 mg PET/µmol PETase•L −1 per day, 17.5 mg PET/µmol PETase•L −1 per day and 22.5 mg PET/µmol PETase•L −1 per day compared with the native IsPETase of 8.2 mg PET/µmol PETase•L −1 per day.In two of them, the hydrophobicity remained constant, and in the third, it increased.However, the other five modified enzymes obtained lower results than the native one.In three of these, the hydrophobicity increased, and only in one mutant, it decreased [66].Therefore, according to these studies, not only the hydrophobicity in the active site can influence PET depolymerization, but the type of amino acid that is modified can also affect it.
Focusing on another parameter, in 2019, it was tested to see if a more thermostable IsPETase was able to degrade PET at a faster rate.Native IsPETase has a Tm of 48.81 • C, while the obtained triple-mutated IsPETase-Ser121Glu/Asp186His/Arg280Ala-had a Tm of 57.62 • C.This mutant released 14 times more monomers at 40 • C than the native enzyme, obtaining an enzymatic activity of 120.9 µM and 8.7 µM, respectively [67].
In addition, there are genetic engineering works based on metagenomics, such as in Sulaiman et al. [54], where they cloned a leaf-branch compost cutinase (LC-cutinase or LCC), whose highest identity was with a Thermomonospora curvata lipase with 59.7%, and degradation of PET was achieved under the conditions of 50 • C and pH 8 at a rate of 12 mg/h/mg of the enzyme.This result is much greater when compared to T. fusca cutinase at 55 • C and pH 7, which consumes 0.05 mg of PET/h/mg of the enzyme [88].
The main drawback of expressing LC-cutinase is that it forms aggregates with itself due to electrostatic interactions as a result of increasing the temperature.This produces an inhibition in the depolymerization of PET.For this reason, it was tested to see if glycosylating LC-cutinase could prevent this.The result was that glycosylated LC-cutinase began to form aggregates at 10 • C above its native protein [62].
Following the idea that glycosylation can influence enzymes' properties, another study used the previously mentioned cutinase Thc_Cut1 from Thermobifida cellulosilytica and generated two mutated cutinases without glycosylation sites.All were able to hydrolyze polyesters, such as PET, poly(butylene succinate) (PBS) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), but one of the mutants had higher PBS-degrading activity than the native enzyme [51].Metagenomics was also used by Wei et al. [59], exchanging amino acids from the structure of a cutinase obtained with this technique for others from TfCut2, a polyester hydrolase from Thermobifida fusca KW3.The best mutants were those with modifications near the binding site.The mutations Gly62Ala and G62Ala/I213Ser achieved a weight loss of 42% of PET films, almost three times more compared to the native protein.This may be due to the fact that the wild-type enzyme is inhibited by MHET, and the substitution of these amino acids may decrease the affinity of the mutants for this molecule.To avoid the problem of inhibition by MHET, Barth et al. [60] used the dual-protein system with TfCut2 and the carboxylesterase TfCa, both from T. fusca KW3.With this, TfCut2 degraded PET, and TfCa achieved the same with BHET and MHET.This dual system generated 2.4 times more TPA than using TfCut2 alone.On the other hand, Tournier et al. [69] obtained a modified LC-cutinase with a higher affinity for PET and higher thermostability.This was achieved by exchanging two amino acids, previously tested using in silico methods, with which the new enzyme presented a disulfide bridge.This bridge conferred greater strength in the bond to the substrate, and the other two modifications improved thermostability.In its native state, this enzyme depolymerized 50% of the amorphous PET, while the modified LC-cutinase reached 90% after 10 h.Regarding productivity, it produced 16.7 g of TPA per liter per hour as a consequence of degrading PET.The optimal ratio to obtain these results was 3 mg of mutated enzyme/per g of PET.
Another article based on metagenomics modified the anaerobic bacteria Clostridium thermocellum to constitutively express and secrete LC-cutinase in large quantities.After 14 days incubating anaerobically at 60 • C, more than 60% of amorphous PET was degraded.It was 50 mg in total, corresponding to more than 2.2 mg each day [68].
One interesting aspect is obtaining high-value by-products through the degradation of PET, as shown by Sadler et al. [70].Following this idea, they created the strain E. coli MG1655 RARE.This genetically modified strain is able to degrade PET, release TPA and obtain vanillin.This was made using PETases from I. sakaiensis and a de novo pathway to convert TPA to vanillin.To optimize the process, parameters such as temperature and cell permeabilization were improved, and 79% vanillin production was obtained compared to TPA.
In recent years, there has been a trend to create fusion proteins to depolymerize PET.This is the case with Zhu et al. [71].They modified E. coli with the BIND-PETase dimer.It is a dimer in which the so-called biofilm-integrated nanofiber display (BIND) binds to the curli of bacteria.Curli are fibers on the outside of the cell that are involved in forming biofilms.Through this method, PETase bound to BIND remains on the surface of the cell and degrades the polymer.It is a system that provides thermostability to PETase and makes it able to degrade 9.1% semi-crystalline PET and microplastics between 2.5 and 50 mg.
Based on the protein fusion strategy, Chen et al. [72] synthesized two modified LCcutinases.They based them on the modified LC-cutinase created by Tournier et al. [69] called LCC ICCG and previously mentioned, which had higher catalytic activity and thermal stability but little affinity for PET.These two fusion proteins, LCC ICCG -TrCBM and CfCBM-LCC ICCG , were made by adding a substrate-binding domain to LCC ICCG from Trichoderma reesei and Cellulomonas fimi, respectively.These domains were selected in silico based on the principles that domains from thermophilic organisms, domains that bind to ordered crystalline substrates and domains that are smaller than the rest of the protein are more adequate.It was estimated with the experimental results that the affinity for the polymer increased 1.4 fold in LCC ICCG -TrCBM and 1.3 fold in CfCBM-LCC ICCG , while they degraded 3.7% and 24.2% more PET, respectively.
Another recent project using this technique appears in Li et al. [73].The researchers modified the non-pathogenic, moderate halophile Vibrio natriegens with a chimera of IsPETase and IsMHETase from I. sakaiensis, with the aim of degrading PET present in seawater.PET was almost completely transformed to TPA, and it was determined that the strain would take 24 years to completely decompose 1 g/L of PET.The creation of chimeras is a novel technique that still has a long way to go in relation to the degradation of synthetic polymers.
Concluding with this polymer, numerous techniques for its depolymerization have been carried out to improve the efficiency of enzymes or modify bacteria with these PETases.Today, research is still needed to obtain more efficient methods for bacteria.This is due to the physical-chemical structure of PET and its own disposition in space, with the semicrystalline structure being more recalcitrant.The use of enzymes to treat the problem of this polymer has the advantage of being harmless to living beings and the environment, so further research should be carried out.
Biodegradation of Other Plastic Polymers
Genetic engineering approaches carried out to degrade synthetic polymers have not only focused on PET but also on other polymers, as described below.
Polyethylene (PE)
PE is a particular type of plastic that results from the polymerization of ethylene.A lot of plastics in this group are copolymers of ethylene and olefins, the latter of which result in branches in the plastic's structure.This results in a wide variety of polyethylenes with different properties [89].Multiple organisms have been discovered that can degrade different types of polyethylene [90][91][92].
In a study of genetic engineering, Gyung Yoon et al., who expressed the alkane hydroxylase gene (alkB) of Pseudomonas sp.E4 in E. coli BL21, found that it was able to degrade low-molecular-weight polyethylene (LMWPE) [53].Kong et al. bound the latex clearing protein derived from Streptomyces sp.strain K30 (LcpK30) to the anchor peptide LCI and expressed it in E. coli in order to improve its ability to degrade low-density polyethylene (LDPE) [93].
Poly(butylene Adipate-co-terephthalate) (PBAT)
PBAT is a long-chain aliphatic-aromatic copolyester formed by subunits of TPA, adipic acid and 1,4-butanediol linked by ester bonds.It is estimated that its global production was more than 360 million tons in 2018 [94].Its importance relies on the fact that, like PET, it is a plastic used in daily life due to the advantages it presents.It is easy and cheap to produce, resistant and very versatile due to its chemical structure, which gives it the property of being rigid and flexible [95].PBAT is produced by the condensation of TPA, 1,4-butanediol and adipic acid, and catalysts with tin, zinc and titanium are used [96].It is widely used in the textile industry, agriculture and packaging.It is also present in organic waste bags because its ester bonds are more easily degradable by enzymes.Its biodegradability has been demonstrated in previous studies, so it is considered a bioplastic [97].
However, its biodegradation rate is low [95].For this reason, some studies have been carried out with cutinases to better understand the hydrolysis mechanism of PBAT.Due to the presence of ester bonds in PBAT, it is possible to extrapolate the cutinases research carried out on PET to biodegrade PBAT.This happens with Yang et al. [98], where researchers studied the structure of T. fusca cutinase TfCut.This enzyme is known to degrade PBAT into its monomers.A double-mutated cutinase, TfCut-DM, was created.TfCut-DM depolymerized all the PBAT into TPA after 48 h of culture, while, at that time, the native enzyme released more 1,4-butanediol-TPA than TPA.On the other hand, genes related to anaerobic living beings, such as Clostridium botulinum, have been researched.This is the case with the esterases Cbotu_EstA and Cbotu_EstB, which are expressed heterologously in E. coli BL21-Gold(DE3).They have been shown to be able to hydrolyze PBAT.However, some differences in the amino acids of its active site make Cbotu_EstA more efficient than Cbotu_EstB.In silico representations show that the active site of the first protein is larger, which can facilitate the entry of the polymer [49].
Poly(butylene succinate) (PBS)
PBS is an aliphatic polyester formed by the condensation of succinic acid and 1,4butanediol.In the past, it was produced from compounds derived from petroleum.Nowadays, it is produced from renewable resources, such as sugar cane and corn, through bacterial fermentation [99].This is a point in its favor in terms of being produced in greater quantities, along with the physical and chemical properties it has.It has a crystalline structure that gives it rigidity and some ductility and a wide temperature range with which to work.Among other uses, it is present in the textile industry and is a possible substitute for polypropylene [100].
The same proteins that hydrolyze PET and PBAT break the ester bonds of PBS.A study on the genetic engineering of cutinases is the one of Gamerith et al. [51], mentioned above for PET.In the article, they used Thc_Cut1 cutinase and two mutants, of which one of them resulted in hydrolyzing the PBS at a higher rate, degrading 92% of the dry weight after 96 h.This confirms its potential for PBS biodegradation.
Polylactic Acid (PLA)
PLA is an aliphatic polyester formed by monomers of lactic acid.In its manufacturing, it is normally made from hydroxyl acids.It is based on bacterial fermentation, as in the case of PBS, to synthesize lactic acid.Later, lactic acid is purified, and the monomers are condensated and linked by ester bonds.PLA is considered a bioplastic that can be chemically depolymerized to lactic acid in recycling processes.PLA is rigid and transparent, and compared to other bioplastics, it has a long durability.Among its uses, it is used as food packaging, for 3D printing, in medicine and the textile industry.PLA can be degraded by cutinases and esterases, as in the case of PET, PBAT and PBS [101].
There are almost no genetic engineering studies focused on PLA, partly because the same enzymes can hydrolyze multiple polymers and partly because PLA is not as recalcitrant as others, such as PET, making PLA less interesting when it comes to developing biodegradation strategies.There is, however, a patent on a protease from Thermus sp.Rt41A that has been mutated and whose variants are meant to be used to biologically degrade PLA and recover lactic acid monomers [48].
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)
PHBV, one of the most known PHAs, is a copolymer of hydroxyl-butyrate (HB) and hydroxyl-valerate (HV).When compared to PHB, another well-studied PHA, PHBV has better flexibility and a lower melting temperature [102].The ratio between both monomers determines the plastic's properties, with a higher content of HB resulting in higher strength and a higher content of HV resulting in higher flexibility and toughness [103].PHBV's properties can be improved in different ways [104], for example, by mixing it with agroresidues, which results in green composites that have enhanced physical properties [102], or by forming small crystal nuclei and then drawing the material, resulting in fibers with high tensile strength [105].PHBV has many different uses [106].It can be used for coatings and packaging [107][108][109] as well as 3D printing [110].PHBV can also be used in the textile industry, especially when blended with poly(lactic acid) (PLA), a biobased and biodegradable plastic [111], and in cosmetics as microbeads for exfoliants [112].The medical field has also found various uses for PHBV.For example, it can be used to make biocompatible scaffolds [113,114] or slowly release drugs like insulin [115] or drugs for treating tumors [116].Each of the monomers of PHBV is synthesized in a different metabolic pathway.In one pathway, acetyl-CoA is transformed into acetoacetyl-CoA, which, in turn, is transformed into 3-hydroxybutyryl-CoA.In the other pathway, oxaloacetate is sequentially transformed into threonine, 2-ketobutyrate, propionyl-CoA, 3-ketovaleryl-CoA and R-3-Hydroxyvaleryl-CoA [117].
PHBV waste can be recycled.For example, PHBV-based face masks can be chemically recycled through hydrolysis [118].However, as PHBV is a biodegradable plastic, carrying out its biodegradation is preferable.Its biodegradability when released to the environment is slow and depends greatly on environmental conditions [119], but PHBV has been estimated to biodegrade in rivers [120], and the highest degradation is reached when in activated sludge [121] or compost [122,123].PHBV-degrading bacteria have been isolated from soil, specifically Actinomadura sp.AF-555 [124] and Streptomyces sp.MG [125], but very little is known about specific enzymes that can degrade PHBV.In an aforementioned study, the enzymes Thc_Cut1 and Thc_Cut1_koST from Thermobifida cellulosilytica DSM44535, expressed in P. pastoris, were able to degrade PHBV, though to a very limited extent [51].
Polyvinyl Acetate (PVAC)
PVAC, also known as PVAc, is a petrochemical plastic that is mostly used as an adhesive, particularly for paper and wood [126], but it has also found a use as part of graphene-derived composites [127].The monomer, vinyl acetate monomer (VAM), is synthesized through the oxidative addition of acetic acid to ethylene, and acetic acid can be, in turn, synthesized from ethylene [128].Polymerization reactions in PVAC include emulsion-, suspension-and solution polymerization [129].
It has been reported that fungi can grow on items made from PVAC, suggesting its biodegradation [130], but PVAC is generally considered to be non-biodegradable [128].Liu et al. [50] developed a chimeric lipase-cutinase (Lip-Cut), originally from Thermomyces lanuginosus and Thielavia terrestris NRRL 8126, respectively, and expressed it in P. pastoris.They studied the use of this Lip-Cut in the process of deinking waste paper and found that the synergy between both enzymes improves the ability of the cutinase to both degrade PVAC and deink paper.Also, regarding paper waste treatment, Liu et al. [131] fused the cutinase from Humicola insolens to the E. coli anchor peptide OMP25, which can be adsorbed on the polyester polymethylmethacrylate (PMMA).They expressed the fusion protein in P. pastoris and concluded that the anchor peptide enhances the cutinase's ability to degrade PVAC and the so-called stickies in waste paper recycling.
Poly(ε-caprolactone) (PCL)
PCL is a plastic well known for its use in the biomedical field [132,133], as it is biocompatible [134].It can be used in drug delivery or wound dressing [135], but it stands out as a scaffold for tissue engineering, whether on its own [136] or as part of a blend or composite material [137].Its monomer is ε-caprolactone, and its polymerization can be carried out by opening the monomer's ring followed by the addition or, alternatively, by polymerizing 6-hydroxycaproic acid [138].
PCL is a biodegradable plastic [139,140], and its degradation is especially high when in anaerobic sludge [141] and compost [142] or when performed by bacteria isolated from compost [143].Regarding engineered bacteria to degrade PCL, two examples have already been mentioned.Sulaiman et al. [54] found that LC-cutinase could degrade PCL in addition to, and better than, PET.The aforementioned CUT190 cutinase from Saccharomonospora viridis AHK190, when double-mutated and expressed in E. coli Rosetta-gami B (DE3), had an improved degradation activity of PCL, among other polymers [58].
Polyurethane (PU or PUR)
PU is a polymer synthesized by the union of a diol and a diisocyanate molecule linked by amide bonds.To accelerate the process, catalysts, such as 1,4-diazabicyclo[2.2.2]octane or dibutyltin dilaurate, can be added.Polyurethane is the eighth most consumed polymer in the world, with 18.6 MMt/yr.Its structure can be highly varied, and it has a lot of uses.Its main use is as a foaming agent, but it is also manufactured in thermal insulation processes and to protect against abrasion and corrosion, among other uses.Its drawback is that this manufacturing requires toxic substrates, making it difficult to recycle.For this reason, alternative routes to synthesize it are being sought.In addition, using biodegradation to eliminate this polymer in a harmless way is being studied [144].PU has amide bonds that are hydrolyzed by amidases, releasing the monomers.To increase the degradation of PU, some researchers improved the adsorption of an amidase from Nocardia farcinica to PU by fusing it to a polymer-binding module originally from a polyhydroxyalkanoate depolymerase obtained from Alcaligenes faecalis [52].However, there are not many studies based on the biodegradability of PU.
Conclusions
The magnitude of plastic usage and the later production of its waste, which in most cases remains in nature for decades, makes the search for alternative bioplastic materials or other compounds a matter of major importance for today's society.In addition, the biodegradation of these materials and, where possible, the use of their constitutive monomers as carbon and energy sources for the growth of microorganisms that can produce valuable compounds are important goals to achieve [145].
Despite the fact that there are a large number of studies based on genetic modifications, most of them were carried out through modifications in the amino acid sequence.New strategies have been recently tested, such as the use of chimeras or fusion proteins, among others, but these approaches are a minority.Further research in the use of genetic techniques, as well as combining different strategies, is necessary to obtain new strains or proteins with a greater degradative capacity of these synthetic polymers.
An example of a new research field is the use of transcriptional regulators, such as Garrido et al. [146] have carried out.This work tested if the FarA transcription factor in Aspergillus oryzae was directly related to the synthesis of CutL1 and HsbA, a cutinase and a hydrophobic surface binding protein that participate in the degradation of poly(butylene succinate-co-adipate) (PBSA), a biodegradable polymer.The deletion of FarA produced a mutant with minimal concentrations of cutL1 and hsbA compared with its native strain and without the ability to degrade PBSA.This study differs from the ones described in the main text, as it consists of the basic research of an organism and its genetic regulation.This does not mean that the other studies ignore the subject of gene expression.In contrast, modifying bacteria to express an enzyme requires careful consideration of the genetic elements necessary for its successful production.The difference relies on studying genetic expression in the native organism, which cannot only help us understand their genetic regulation and metabolism but also give us hints on different approaches to enhance plastic degradation and how to use the native organism instead of relying on a different host organism.
It is also relevant to point out that the research tends to focus on enhancing an enzyme's ability to degrade specific polymers, with almost no studies evaluating how the modifications performed may change specificity toward other polymers.Limiting the analysis to just a few substrates-for which the enzyme is already specific-is comprehensible, but using a wider variety of polymers could indicate if the modifications have a greater effect than expected and may be more useful.
In addition, there is little work on the tridimensional structure of the different enzymesnative or modified-that have been mentioned.Deeper research is necessary on this matter in order to better understand why changes happen regarding the affinity for the substrate or the stability, among other characteristics, according to the exchanged residues.Obtaining the structure of enzymes can be achieved not only through X-ray crystallography but also in silico by modeling.This is especially useful when proteins cannot be successfully purified and crystallized, but modeling goes even further.From molecular dynamics to substrate docking, these tools are used in some of the referenced studies to analyze enzymes' properties and enhance their activities.
Similar to plastics themselves, approaches to studying their biodegradation are diverse, and this variety often comes with a lack of consistency between studies.The conditions in which the enzymes are used can differ, and some studies do not calculate enzymatic activity and limit themselves to detecting solid plastic disintegration or monomer liberation, and the works that calculate enzymatic activity can use different units.This can sometimes make it complicated to compare their conclusions.
Overall, more research is required to develop effective, i.e., quicker, safer and more efficient, biodegradation strategies for plastics, if possible.This applies not just to the plastics that are briefly addressed in this work but also to all plastics in general.Microbial biotechnology and genetic engineering approaches, together with the current development of artificial intelligence tools that provide a new direction in the study and design of novel of enzymes, can facilitate the generation and optimization of several types of plastic-degrading enzymes and valorization processes.
Nowadays, we are getting closer to achieving this aim thanks to the latest advances in DNA sequencing, metagenomics, bioinformatics, genome mining and machine learning tools, in conjunction with new genetic engineering techniques, such as CRISPR-Cas technologies.
Figure 2 .
Figure 2. Classification of plastics according to the raw material and biodegradability.PHAs (including a major type, i.e.PHBV) constitute a special category of plastics since they are completely natural.
Figure 2 .
Figure 2. Classification of plastics according to the raw material and biodegradability.PHAs (including a major type, i.e., PHBV) constitute a special category of plastics since they are completely natural.
Figure 4 .
Figure 4.Chemical structures of PET and products related to its hydrolyzation.Each arrow represents an enzymatic reaction.The presence of multiple arrows, like between EG-acetyl-CoA
Figure 4 .
Figure 4.Chemical structures of PET and products related to its hydrolyzation.Each arrow represents an enzymatic reaction.The presence of multiple arrows, like between EG-acetyl-CoA and TPAsuccinyl-CoA, refers to the fact that there is more than one chemical reaction between one molecule and another.Adapted from [80].
Table 2 .
Heterologous expression of enzymes to degrade PET.
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2024-05-22T15:07:41.549Z
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2024-05-01T00:00:00.000
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Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1
The Hepatocyte Growth Factor (HGF)/c-Met signaling pathway regulates hepatocyte proliferation, and pathway aberrations are implicated in the invasive and metastatic behaviors of hepatocellular carcinoma (HCC). In addition to c-Met, heparin acts as a co-receptor to modulate pathway activity. Recently, anti-metastatic and anti-cancer effects of heparin have been reported. However, the role of heparin in the regulation of HGF signaling remains controversial and the effects of heparin on HGF-induced biological responses during hepatocarcinogenesis is not yet defined. In this study we determined the effects of heparin on HGF-induced activities of HCC cells and the underlying molecular mechanisms. Here, we report for the first time that heparin inhibits HGF-induced adhesion, motility and invasion of HCC cells. In addition, heparin reduced HGF-induced activation of c-Met and MAPK in a dose-dependent manner, as well as decreased transcriptional activation and expression of Early growth response factor 1 (Egr1). HGF-induced MMP-2 and MMP-9 activation, and MT1-MMP expression, also were inhibited by heparin. Stable knockdown of Egr1 caused a significant decrease in HGF-induced invasion, as well as the activation and expression of MMPs. Parallel to these findings, the overexpression of Egr1 increased the invasiveness of HCC cells. Our results suggest that Egr1 activates HGF-induced cell invasion through the regulation of MMPs in HCC cells and heparin inhibits HGF-induced cellular invasion via the downregulation of Egr1. Therefore, heparin treatment might be a therapeutic approach to inhibit invasion and metastasis of HCC, especially for patients with active HGF/c-Met signaling.
Introduction
Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and the third leading cause of cancer-related deaths worldwide [1]. The lethality of HCC is partly associated with its resistance to currently available anticancer agents and a lack of biomarkers that can detect the early stages of the disease. Although liver transplantation and surgical resection improves overall survival in patients with small, non-invasive and nonmetastatic tumors, there is still no effective treatment for patients with advanced disease [2,3].
In addition to the clinical and histopathological characteristics of advanced HCC, recent data have shown that HGF and its high affinity receptor tyrosine kinase c-Met are implicated in the development and progression of HCC [4]. Furthermore, it has been demonstrated that HCC patients with active HGF/c-Met signaling have a significantly worse prognosis and higher association with metastatic disease than those without [4][5][6][7]. Based on our current understanding of the HGF/c-Met pathway in HCC, several strategies have been proposed for inhibiting HGF and/or c-Met expression or activity at different levels in HCC management [8][9][10]. Although over-expression of c-Met was found to be involved in aggressive liver tumors and associated with poor prognosis in HCC [4,5,11], a contradictory deficiency of c-Met in hepatocytes has been reported to initiate tumorigenesis in liver [12].
Recently, the c-Met receptor tyrosine kinase was reported as a potential molecular target for the personalized treatment of HCC in patients with an active HGF/c-Met pathway [4]. HGF-induced activation of c-Met mediates cellular motility and invasion, and is important for cell proliferation, morphogenesis, angiogenesis, as well as protection from apoptosis. Acting as HGF co-receptors, heparan sulfate proteogylcans (HSPGs), heparin and dermatan sulfate (DS) also have a crucial role in the efficient activation of c-Met through ligand oligomerization [13][14][15]. Furthermore, HS and heparin facilitates c-Met signaling and mediates distinct HGF responses such as mitogenesis, cellular motility and invasion in target cells [16,17]. Heparin also has been used for the prevention or treatment of cancer-associated thrombosis, and positive effects of heparin on patient survival have been reported in several studies [18]. Recently, anticancer and anti-metastatic activities of heparin were described in animal models and in patients with metastatic disease [19]. In contrast to these results, a recent randomized trial showed no overall survival benefit for patients treated with heparin [20,21]. Despite this single apparent inconsistency, the activities and underlying molecular mechanisms of heparin and HSPGs as potential anti-cancer and anti-metastic agents warrant further investigation. Since the involvement of HGF/c-Met signaling in the invasiveness and metastasis of HCC is known, and heparin mediates HGF-induced biological responses, we hypothesized that heparin is an important regulator of the HGF-induced activities of HCC cells. To test the hypothesis, we first determined the effects of heparin on HGF-induced cellular responses of HCC cell lines. We show for the first time that heparin inhibits HGF-induced adhesion, motility and invasion of HCC cell lines. To understand the underlying molecular mechanisms of these effects, we examined c-Met and downstream signaling molecules by immunoblotting and found that heparin inhibited HGF-induced c-Met phosphorylation and the activation of downstream signaling molecules. We identified differentially expressed genes by cDNA microarray and selected a subset for validation experiments; among these was Egr1. Egr1 is a member of the zinc-finger transcription factor family that regulates wide spectrum of cellular responses from survival to apoptosis, growth to cell cycle arrest and senescence, and differentiation to transformation [22][23][24][25]. It has been reported that HGF induces Egr1 transcription in HCC cell lines, and that Egr1 regulates HGF-induced biological responses including angiogenesis, scattering, invasion and motility in a variety of cell types [10,[26][27][28]. To test the hypothesis that Egr1 is a critical mediator of inhibition of HGF signaling by heparin, we characterized the expression of Egr1 in HGF and/or heparin treated HCC cells. We found that HGF-induced activation of c-Met signaling increased the expression level of Egr1, parallel to increased adhesion, motility and invasion of HCC cell lines. Heparin, in contrast, inhibited HGF/c-Met signaling and HGFinduced cellular responses via the inhibition of Egr1 expression as well as downstream targets of Egr1 such as Membrane type 1 metalloprotease (MT1-MMP), matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9).
Heparin inhibits HGF-induced, invasion and migration of Hepatocellular Carcinoma (HCC) Cell Lines
To study the role of heparin on HGF/c-Met signaling in HCC, we first analyzed the effects of heparin and/or HGF on the migration and invasion of SK-HEP-1 HCC cells. In the absence of heparin, HGF significantly increased the motility and invasion of SK-HEP-1 cells (p,0.05). However, in the presence of heparin, HGF-induced motility and invasion were significantly reduced in a dose-dependent manner (p,0.05) ( Figure 1A, 1B and 1C). Similarly, heparin significantly inhibited (p,0.05) HGF-induced adhesion, motility and invasion by HuH-7 and Hep3B HCC cells (Figure S1A, S1B and S1C). Similar data were obtained with Mahlavu and SNU-449 HCC cells (data not shown). HGF or HGF plus heparin treatment did not significantly affect the proliferation of the HCC cell lines that we have tested (data not shown). Apoptosis was similarly not effected by HGF and/or heparin treatment up to 72 h in SK-HEP-1 cells (data not shown).
Heparin inhibits HGF-induced expression and transcriptional activity of Egr1
To identify differentially expressed genes in SK-HEP-1 cells treated with HGF and/or heparin, we used the Affymetrix GeneChip cDNA microarray. One of the genes exhibiting a significant difference was Egr1. To confirm that the expression level of Egr1 was affected by HGF and/or heparin treatment, we performed RT-PCR and immunoblot analysis in SK-HEP-1 cells. The effect of heparin on HGF-induced migration and invasion were analyzed by using modified Boyden chambers and the Roche xCELLigence System in SK-HEP-1 cells. Following serum deprivation, SK-HEP-1 cells were left untreated (0) or treated with HGF in the presence or absence of heparin, then invasion and migration assays were performed. In both panels, values are expressed as the ratio of migrating or invading cells in HGF and/or heparin-treated wells to control wells treated with heparin alone at the indicated concentrations (1A, 1B and 1C). Results are representative of three or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups. doi:10.1371/journal.pone.0042717.g001 We observed significantly higher Egr1 mRNA and protein levels in HGF-treated cells compared to untreated cells in a timedependent manner (Figure 2A and 2C). Heparin treatment led to dose-dependent decreases in the levels of Egr1 mRNA and protein ( Figure 2B and 2D). To further investigate the effects of HGF and/ or heparin on Egr1 transcription, cells were transfected with pGL2-Luc-B-Egr1 plasmid and luciferase activity was measured. As shown in Figure 2E, HGF induction transactivated the Egr1 promoter in a time-dependent manner. Furthermore, the HGFstimulated increase in the reporter activity of Egr1 was abolished by heparin treatment in a dose-dependent manner ( Figure 2F), consistent with the mRNA results.
Heparin inhibits HGF-induced activation of c-Met and ERK1/2, MMP-2 and MMP-9 If Egr1 is a target of the HGF/c-Met signaling pathway and heparin inhibits HGF-induced migration and invasion via Egr1, then heparin is likely to inhibit HGF-induced c-Met activation and downstream signaling. To assess c-Met and ERK1/2 activation by HGF in SK-HEP-1 cells, serum-deprived cells were treated with HGF for various periods and c-Met activation status was measured using phospho-Tyr1234/1235-c-Met-and phospho-Thr202/ Tyr204-ERK1/2-specific antibodies. The levels of phosphorylated c-Met and ERK1/2 increased in a time-dependent manner upon HGF treatment ( Figure 3A). Heparin inhibited HGF-activated c-Met and ERK1/2 activation in a dose dependent manner ( Figure 3B). Since previous studies have shown that Egr1 mediates MMP-2 and MMP-9 activation and MT1-MMP expression, we determined the effects of HGF and/or heparin on the levels of MMP-9 and MMP-2 activation and by gelatin zymography. Treatment with HGF increased MMP-9 and MMP-2 activation, whereas heparin suppressed these effects ( Figure 3C, 3D).
Heparin reduces binding of HGF to c-Met
To address the question of whether heparin interferes with binding of HGF to its receptor c-Met, we used surface plasmon resonance spectrometry (Biacore), which directly measures binding in real time ( Figure 4). HGF injection over the c-Met coated surface created a specific binding signal. When HGF was premixed with heparin and injected over c-Met surface, dose-dependent inhibition of the HGF/c-Met interaction was observed. When heparin alone was injected, no meaningful binding was detected. Similar data were obtained with four different heparin types. The c-Met antagonist SU11274 inhibits Egr1 expression and transcriptional activity We used SU11274, a potent and specific c-Met inhibitor, to further define the role of HGF/c-Met signaling in the regulation of Egr1 expression. Treating SK-HEP-1 cells with HGF in absence and presence of SU11274 showed that HGF-induced p-Met activation and Egr1 expression were significantly decreased by SU11274 ( Figure 5A). The HGF-stimulated increase Egr1 reporter activity was abolished by treatment with this c-Met inhibitor in a dose-dependent manner ( Figure 5B). These results further support the concept that HGF/c-Met signaling mediates Egr1 activation.
Egr1 knockdown suppresses HGF-induced invasion by attenuating MMP expression and activation
To investigate the direct involvement of Egr1 in HGF-induced cell invasion, we first transfected SK-HEP-1 cells with pCDNA 6/ TR which express that tet repressor. Stable cells were cotransfected with the tetracycline-regulated pSUPER.retro.neo GFP tet RNAi construct, which was directed against a sequence containing Egr1. Stable cells were then analyzed for downregulation of Egr1 after 24 h of tetracycline treatment. Ten-fold downregulation of Egr1 was detected by immunoblotting in pSUPER.retro.neo+GFP tet/Egr1 clones ( Figure 6A) but not in mock transfectants ( Figure 6B). Egr1 knockdown resulted in a significant decrease of HGF-induced cell migration and invasion (p,0.05) ( Figure 7A, 7B, 7C). Whereas HGF induced the activation of MMP-2 and MMP-9, and the expression of MT1-MMP, Egr1 knockdown resulted in decreased activation and/or expression of these MMPs ( Figure 7D, 7E, 7F), indicating that Egr1 is required for HGF-mediated MMP-2 and MMP-9 activation and MT1-MMP expression. Similar data were observed with stable knockdown of Egr1 in Hep3B cells ( Figure S2). To further demonstrate the critical role of Egr1 in HCC cell invasion and motility, we transiently transfected SNU-449 cells with the pCMV6-AC-GFP-EGR1 plasmid to generate Egr1 overexpres-sion. Constitutive overexpression of Egr1 ( Figure 8A) resulted in increased basal (unstimulated) motility ( Figure 8B) and an 8-fold increase in basal invasion of SNU-449-Egr1 cells ( Figure 8C). Consistent with the observed increased in invasiveness, Egr1 overexpression induced MMP-9 proteolytic activity ( Figure 8D). These cells did not express MMP-2 or MT1-MMP, thus the effects of Egr1 on their expression and/or activity could not be determined. As anticipated, treatment of SNU 449-pCMV6-AC-GFP-EGR1 cells with HGF or HGF plus heparin did not further increase Egr1 expression ( Figure 9A) or cell motility and invasion ( Figure 9B, 9C).
Discussion
HGF/c-Met signaling regulates cellular motility, invasion, and metastasis in various types of human cancer [29,30]. It has been reported that activation of HGF/c-Met signaling is associated with intrahepatic metastasis, vascular invasion, poor prognosis, and drug resistance in HCC. Therefore, the c-Met receptor tyrosine kinase is thought to be a promising target for the personalized treatment of HCC [1,30,8]. Recent studies also suggest that due to its anti-metastatic and anti-invasive activities, heparin may be a potential therapeutic for the treatment of cancer [20,21]. Although there is considerable information on the role of heparin in HGF/c-Met signaling generally, the effects of heparin on HGF/c-Met signaling in HCC are not yet known.
We show for the first time that heparin inhibits HGF-induced cell invasion, cell motility, and cell adhesion in HuH-7, Hep3B, SK-HEP-1, Mahlavu HCC cell lines. The effects of heparin on HGF-induced migration by other cell types have been reported in a few studies. Rubin et al [16] reported that heparin facilitates HGF signaling through interaction with c-Met and the N domain of HGF, and heparin treatment increases both mitogenesis and motility in HS deficient 32D/c-Met cells. Similarly, Lyon et al. [15] showed that the activity HGF was dependent on the presence of sulfated GAGs on the cell surface and that both HS and DS increased HGF induced cell motility in GAG deficient CHO pgsA-745 cells. Most, if not all HGF target cells possess abundant cell surface HS and DS proteo-and glycosaminoglycans. Thus, these studies identify an important positive role for cell surface HS and DS proteoglycans in HGF signaling, and suggest that soluble heparin may competitively antagonize the effects of these cell surface glycans, consistent with the results as we present here for HCC cells. Surface plasmon resonance results also supported the hypothesis that added soluble heparin can inhibit the interaction of HGF with c-Met.
HGF-induced motility and invasion has become an important target for the prevention and/or treatment of metastasis in HCC. The stimulatory effects of HGF on the motility and invasion of HCC cells are well documented [1,5,31,32], and activation of cell motility and invasion are also rate-limiting steps of tumor metastasis [33]. Concordantly, the activation of HGF/c-Met signaling positively correlates with tumor metastasis in HCC [1]. In addition, HGF/c-Met signaling induces epithelial-mesenchy-mal-transition (EMT), a pivotal event in the development of invasive and metastatic cancer progression in HCC cells [34]. We propose that heparin may be an effective inhibitor of motility, invasion and metastasis in this context. In support of this concept, we note that positive effects of heparin on the survival of cancer patients are documented in animal models and clinical studies [21,35]. In 2011, Akl et al. [35] conducted a systematic review on the effects of heparin on the survival of cancer patients with no therapeutic or prophylactic indication for anticoagulation and reported a potential survival benefit from heparin therapy. Moreover, the randomized controlled trials that were reviewed did not report any significant effects on bleeding, suggesting that the survival advantage of heparin might be related to its interaction with signaling molecules. Recent reports suggest that the therapeutic effects of heparin for HCC and other cancers also might be due to inhibition of cell invasion and metastasis, rather than inhibiting the growth of primary tumors [36][37][38]. Consistent with those studies, in the presence HGF we did not observe significant effects of heparin on cell proliferation; HGF treatment alone did not induce the proliferation of HCC cells that were studied up to 96 hrs. We note that although the effects of HGF on the proliferation of primary hepatocytes are well documented, the role of HGF on the proliferation of HCC cells remains controversial [39,40]. Stimulatory effects of HGF on the proliferation of some HCC cell lines have been reported [39], whereas the anti-mitotic and apoptotic effects were reported by others [40].
In order to identify genes that regulate HGF-stimulated migration and invasion and that were also sensitive to the inhibitory effects of heparin, we used cDNA microarray expression profiling. We provide evidence that Egr1, a zinc finger transcription factor known to mediate HGF-induced motility and invasion, had significantly reduced activity as a regulator of HCC cell invasion in the presence of heparin. The diverse involvement of Egr1 in the regulation of growth, differentiation, survival and stress responses has been reported in various models [22,41,42]. Egr1 protects normal cells from transformation by inducing apoptosis or growth arrest upon DNA damage, and its role in chemoresistance and radioresistance in tumor cells is well documented [43][44][45]. In breast, colon and lung cancer, fibrosarcoma and glioblastoma, Egr1 is considered a tumor suppressor gene [46][47][48], but Egr1 can act both as a tumor suppressor and as a tumor promoter, depending on the cell context [22,49].
While Egr1 is crucial for the proliferation of hepatocytes and plays an important role in liver regeneration [50], Egr1 expression level and its impact in HCC are controversial. One study reported Egr1 overexpression in HCC [51], whereas another found downregulation of Egr1 expression [52]. Recent data suggests that different biological functions of Egr1 in HCC might arise due to increased HGF/c-Met signaling [25,26]. Various studies show that Egr1 is a critical transcription factor in mediating HGFinduced expression of genes that encode regulators of cell migration, invasion, angiogenesis, malignant progression and metastasis [25][26][27]53]. We show that HGF induction of MMP-2 and MMP-9 activation, and MT1-MMP expression, is also mediated by Egr1 in HCC. The only other known mediator of HGF induced transcription of MMPs was Ets-1 [54]. Egr1 mediation of MMP activation and expression by HGF is critical in the context of HCC progression because several studies have shown that in human HCC, these MMPs are associated with metastasis and aggressive behavior [55,56]. We further provide functional evidence that Egr1 is a critical regulator of HGFinduced invasion by HCC cells: silencing Egr1 in SK-HEP-1 and Hep3B HCC cell lines decreased HGF-induced invasion and MMP secretion, and Egr1 overexpression in SNU-449 cells increased invasion and MMP levels. Our results predict that the impact of HGF-induced Egr1 activity in HCC is strongly prometastatic.
We show that heparin inhibited HGF-induced Egr1 expression and gene promoter activity in a dose-and time-dependent manner, providing a basis for inhibition of HGF-induced activation of MMP-2 and 9 through decreased MT1-MMP expression. Consistent with this mechanism, the silencing of Egr1 abolished the inhibitory effects of heparin on HCC cell invasion. Heparin inhibition was a direct consequence of decreased HGF-induced c-Met phosphorylation and pERK1/2 activation. The use of specific inhibitors of c-Met and MAPK confirmed that HGF-induced Egr1 expression and cell invasion depend on the MAPK pathway in HCC, as reported previously [22,26,57]. Consistent with these findings, the ectopic overexpression of Egr1 in HCC cells rescued cells from the inhibitory effects of heparin on HGF-induced invasion.
Together, our results define the molecular signaling pathway by which heparin inhibits the HGF-induced expression of transcription factor Egr1 and in turn, HCC cell motility and invasion ( Figure 10). These studies provide functional and mechanistic support for further preclinical and clinical investigations into the use of heparin to prevent and treat HCC metastastic progression. Figure 6. Stable vector-mediated, inducible knockdown of Egr1. To achieve an inducible knockdown of Egr1, we firstly transfected SK-HEP-1 cells with pCDNA6/TR which express tet repressor. Stable cells were selected using Blasticidin before co-transfecting SK-HEP-1 T-REx cells with tetracycline-regulated pSUPER.retro.neo+GFP tet RNAi construct, which was directed against a sequence containing Egr1. Stable cells were selected using G418. After selection, clones were analyzed for downregulation of Egr1 after 24 h of tetracycline treatment. Ten-fold down-regulation of Egr1 was detected by immunoblotting in pSUPER.retro.neo+GFP tet/Egr1 clones (6A) but not in mock transfected clones (6B). The graph compares the signal intensities obtained from Egr1 bands. doi:10.1371/journal.pone.0042717.g006 . HRP-anti-mouse/rabbit secondary antibodies were from Pierce (IL, USA). ECL detection system was from Pierce (II, USA). c-Met inhibitor SU11274 was from Calbiochem 448101 (Darmstadt, Germany). Tetracycline was purchased from Sigma C4881.
In-vitro motility and invasion assays
In-vitro motility and invasion assays were performed as described [59]. The motility and invasion abilities of SK-HEP-1, Egr1 overexpressed-SNU-449 and Egr1 knockdown-SK-HEP-1 Egr1.1.16 were measured using 8 mm pore size of Biocat Cell Enviroment control and matrigel invasion inserts, respectively (BD Bioscience, CA, USA). Briefly, %70 confluent cells were starved overnight with DMEM containing 2% FBS and cell line were treatment with tetracycline (1 mg/ml) in DMEM containing 2% FBS for 24 hrs. Untreated cells were used as a control. Lower chambers were filled with 750 ml of DMEM containing 2% FBS, to which HGF (40 ng/ml) and/or heparin and tetracycline were added at the indicated concentration. Cells were trypsinized, washed in DMEM and added to upper chambers (1610 4 cells/ insert) with heparin and tetracycline at the indicated concentrations and incubated at 37uC for 24 h. After incubation, media was removed, cells on the upper portion of each membrane were wiped with cotton swabs, whereas cells that had transversed through the membrane were fixed and stained with Diff-Quick (Siemens Healthcare Diagnostics, IL, USA). Total cell numbers on the bottom surface of the membranes were counted using brightfield microscopy. Three or more independent experiments were performed in at least triplicate. Bars represent fold difference in mean migrating or invading cell numbers. Fold differences were calculated by dividing the experimental results by the control results. Invasion and motility of cells were also monitored using a real-time cell electronic sensing RT-CES system (xCeLLigence-Roche Aplied Science [60]. For real time motility analysis, the lower chambers filled with DMEM containing 2% FBS, to which HGF (40 ng/ml) and/or heparin were added at the indicated concentrations. Measurements of cell index were taken every 30 min. For invasion studies, the membrane on the bottom of the top chamber of CIM plates were coated with 25 mL of a 0.25 mg/ ml of Matrigel (BD Biosciences) in serum free media and incubated at 37uC for 2 hours. Then 2610 4 cells were added to the upper chamber in DMEM with 2%FBS, which contained heparin and tetracycline at the indicated concentrations. Subsequent steps were performed in the same manner as described for cell migration assays.
Reverse Transcriptase PCR (RT-PCR)
SK-HEP-1 cells were cultured until 60%-70% confluence and serum starved overnight. Cells were then treated with HGF and/ or heparin at the indicated time points. Primers for Egr1 gene (F59CTCTCTGAACAACGAGAAGGTGCT39 R59AGATGGTGCTGAGGA CGAGGA) were selected with aid of PrimerQuest software (Integrated DNA Technology). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Total RNA was prepared using Trizol, and reverse-trancripted into c-DNA by using M-MuLV Reverse Transcriptase kit (MBI Fermentas, USA). Synthesized c-DNAs were used as template for PCR (Polymerase Chain Reaction) reactions. All PCR reactions were carried out using 2 ml/cDNA from the reverse transcription mix which was performed using 2 mg total RNA, for 28 cycles. Amplified products were analyzed by electrophoresis on 2% (w/v) agarose gels. Negative controls without reverse transcriptase and without DNA were included for each set of experiments. The gels were photographed using a transilluminator system (Vilber Lourmat). All RT-PCR supplies were from Fermentas, MBI (USA). Band intensities were quantified as pixels by using ImageJ software.
Transient Transfection of Egr1 expression vector
SNU-449 cells were transfected with p CMV6-AC-GFP-EGR1 vector (Origene, RG209956, USA), using Fugene HD Transfection Reagents Roche (Mannheim, Germany) according to the manufacturer's instructions. After 48 h cells were harvested and cell lysates normalized for protein concentrations and westernblotting was performed to determine the protein level of Egr1.
Gelatin-Zymography
The activities of MMP-2 and MMP-9 in conditioned medium were measured by gelatin-zymography assays. Briefly, cells were grown in serum-free medium then treated with HGF (40 ng/ml) and/or heparin (1, 10, 100 mg/ml) for 24 hrs. SK-HEP-1 Egr1.1.16 clone were treated with HGF (40 ng/ml) and tetracyclin (1 mg/ml) for 24 h and cultured supernatant was directly used for detection of secreted MMPs. Cultured cell media were prepared with SDS sample buffer without boiling or reduction, and then equal amounts of medium for each condition were loaded on 10% polyacrylamide gels containing 0.1% SDS and 1 mg/ml gelatin. After electrophoresis the gels were washed with 2.5% Triton X-100 and then incubated in 50 mM Tris-CI (pH7.0) solution containing 10 mM CaCl and 150 mM NaCI at 37uC overnight. Then, the gels were stained with 0.25% Coomassie Brilliant Blue R-250 (Bio-RAD) solution for 1 h and destained with destaining buffer (40% methanol, 20% acetic-acid) until bands became clear. Gelatinolytic activity was visualized as a transparent band against a blue background. Relative densities of MMP-2 and MMP-9 bands were measured by scanning the photographic negatives and quantified as pixels by using ImageJ software.
Cell Adhesion and Proliferation Assays
Cell proliferation rate was determined using the CyQUANT NF Cell Proliferation Assay (Invitrogen) according to the manusfacturer's protocol. Briefly, SK-HEP-1 cells were seeded in 96-well plates at a density of 1610 3 cell/ml. The next day, nearconfluent cells were starved overnight in DMEM with 2%FBS. Then cells were treated with HGF (40 ng/ml) and/or heparin (1, 10 and 100 mg/ml) for 24 and 48 hrs. Growth media was later removed, green-fluorescent CyQUANT GR dye was added to the wells and incubated for 30 min at 37uC. The fluorescence intensity of each sample was measured using a fluorescence microplate reader with excitation at 485 nm and emission detection at 530 nm (Biotek, USA). Results represent averages of triplicate samples obtained from at least two independent experiments. Proliferation and adhesion were also monitored using xCeLLigence system (Roche Aplied Science) for 72 h.
Surface Plasmon Resonance Binding Assays
Experiments were done with a Biacore T-100 instrument and CM5 sensorchips. HBS-P (10 mM HEPES, pH 7.4, 150 mM NaCl and 0.05% P-20) was used as the running buffer with 10 ul/ min flow rate. All flow cells were coated with 4000 RU protein A/ G using amine coupling method according to manufacturer's instructions (GE Healthcare). IgG-Fc was loaded (100 RU) to flow cell #1 to be used as reference surface. Fc tagged recombinant human c-Met was loaded (350 RU) to flow cell #2. All HGF and heparin injections were done for 60 seconds over both flow cells and the reference-subtracted signal (flow cell #2-#1) was provided in the figures. Raw data was exported to Prism software (v5.0, GraphPad Software, La Jolla, CA) for drawing final figures.
Statistical analysis
Data were analyzed by Mann-Withney U test. P values of #0.05 were considered statistically significant. All statistical procedures were performed using Minitap-15 software. Figure S1 Heparin inhibits HGF-induced adhesion, migration and invasion in HuH-7 and Hep3B HCC cell lines. The effect of heparin on HGF-induced migration and invasion of HuH-7 and Hep3B cell lines were analyzed by using Roche xCELLigence System. Briefly, serum deprived HuH-7 and Hep3B cell lines were left untreated or treated with HGF in the presence or absence of heparin. Adhesion, migration and invasion assays were then performed. E-PLATES, CIM-plates and Matrigel coated CIM-plates were used for real time adhesion, motility and invasion assays, respectively. In both panels, values are expressed as the ratio of adhesive (1A), migrating (1B) or invading (1C) cells in HGF and/or heparin-treated wells. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups. (TIF) Figure S2 Stable knockdown of Egr1 significantly decreases HGF-induced cell invasion in Hep3B cells. To achieve Egr1 knockdown, Hep3B cells were first transfected with the pSUPER.retro.neo+GFP tet RNAi construct. Stable cells were selected using G418 and marker-selected clones were analyzed for downregulation of Egr1 by western blotting (2A). The graph compares the signal intensities obtained from Egr1 bands. HGF-induced cell invasion in the Egr1 knockdown Hep3B cell line was examined using modified Boyden chamber assays (2B). (TIF)
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2017-09-15T23:28:45.370Z
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2012-08-13T00:00:00.000
|
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268005430
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pes2o/s2orc
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v3-fos-license
|
Possibilities of exploitation of useful metals from used Li-ion batteries
The paper presents the possibilities of recovering cobalt from used batteries. Given the evolution of the electric car market, the demand for cells of those raw materials for batteries such as cobalt, nickel, lithium will increase in the coming years. Efforts must be made by all actors involved to meet these needs in a sustainable manner. The recycling of used batteries involves the utilization of valuable elements present within them. Electric car batteries are part of the circular economy in which battery materials are recovered and reused to produce other batteries. Recycling is important because it reduces the pressure on the demand for raw materials.
Introduction
Currently, the car market is made up of two large categories of vehicles [1]: -Conventional vehicles (ICE); -Alternatively-powered vehicles (APVs) present in table 1.The evolution of new car sales in the EU is shown in Figures 1 and 2. There is a decrease in sales of conventional vehicles and an increase in alternative vehicles.Given the increase in the number of electric cars on the car market, it can be considered that batteries are placed at the center of this transition in the field of clean mobility systems [2].A very important influence on the demand for batteries is also manifested by an increase in the market demand for electric vehicles intended for passenger transport.
Battery manufacturing and recycling at the end of life has become a priority and a strategic goal for many regions of the world, especially China and the EU [2], [3].Although there are currently different types of batteries, Li-ion batteries [1], [2], [3], [4] are estimated to dominate the electric vehicle market for at least another decade.
The required materials [2], [5] Li-ion battery manufacturing are: lithium, cobalt, nickel, graphite, etc.The demand for lithium, cobalt and graphite will increase by about 500% by 2050 [1], [2] taking into account the evolution of the market.The resources of these materials are found in few regions of the world, mainly in Argentina, Chile, Bolivia and the Democratic Republic of Congo [6].Battery The Li-ion battery consists of a group of interconnected cells.A cell consists of the following (figure 3): a graphite anode with a copper collector, a cathode consisting of a metal oxide with an aluminum collector, separator and electrolyte.The chemical composition of the cathode defines the type of Li-ion battery: NCA, NMC, LMO, LFP, LTO.
Figure 3. A Li-ion battery [2]
There are currently other types of batteries / production technologies under development and testing.The largest Li-ion battery factory is Tesla Gigafactory 1 Nevada followed by four factories in China.Europe has a battery production growth rate of over 10% by 2030 [2], [7], [9], [10].
Currently, battery recycling has a number of problems: technological limitations, recycling efficiency limitations, low recycling rates, lack of material collection and recovery infrastructure.Recycling also has an impact on the environment through the emergence of other waste, high energy consumption and greenhouse gas emissions.Europe is making financial efforts to increase battery production but also to streamline their recycling process.Some of the financial resources are used for research and innovation in the production and recycling of batteries [9], [10].Battery recycling focuses mainly on the elements: cobalt, lithium, aluminum, copper, manganese.
Experimental researches
The process of recovery and recycling of used batteries involves life cycle (figure 4): -Obtaining materials (extraction, preparation, processing, refining); -Manufacture of battery components; -Manufacture of batteries; -Equipping and using batteries on electric cars; -Recycling.The paper presents the research carried out in the laboratory phase on the possibilities of capitalizing on cobalt in the form of pigment from used Li-ion batteries.
The technological flow of cobalt recovery in the form of pigment is presented in figure 5.
Li-ion batteries from electric vehicles were used for laboratory research.They were dismantled manually and the content was separated on components (cathode, housing, contacts, foils).
Figure 6 shows the aspects during the dismantling of the batteries.Figures 7 and 8 show the components of the dewed batteries as well as an average of them.
Only the battery cathode was used to recover the cobalt.Ultrasound was used for the cathode paste cleaning process on the aluminum cathode.The tests and equipment used are shown in figure 9.The recovery yield of the active paste is calculated as follows [8]: where: 0 is the initial mass of the cathode film (Al+paste foil); is the final mass (Al foil), after ultrasonication in acidic medium (acetic acid).
The results obtained are presented in figure 10.
Conclusions
Because cobalt is an non-renewable resource, its recycling is a strategic issue.The solution presented in the paper proposes the capitalization of cobalt in the form of pigment.Cobalt blue is an inorganic pigment consisting of a cobalt aluminate spinel.The presented method of recovering cobalt in the form of a pigment is a viable and environmentally friendly method.The method involves the manual disassembly of the batteries and the recovery of the cathode paste in an ecological environment.The resulting cathodic paste is used for pigment production.Cobalt side sources are very valuable.Disposable Li-ion batteries have a high cobalt content.The obtained cobalt blue is a pigment used in various industrial sectors.
Figure 1 .Figure 2 .
Figure 1.Trends over time in the EU (in units, 2017-2021) for conventional vehicles on Applied Sciences (ICAS 2023) Journal of Physics: Conference Series 2714 (2024) 012018 IOP Publishing doi:10.1088/1742-6596/2714/1/0120183 manufacturing, in addition to the consumption of poor raw materials, also requires large amounts of energy and generates carbon and waste emissions.
Figure 10 .7
Figure 10.The efficiency of the cathode paste recovery operation
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2024-02-27T17:20:51.463Z
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2024-02-01T00:00:00.000
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52089186
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pes2o/s2orc
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v3-fos-license
|
Diagnostic Potential of Systemic Eosinophil-Associated Cytokines and Growth Factors in IBD
Despite the acknowledged contribution of eosinophils to the disease pathogenesis, available data on cytokines closely related to the peripheral eosinophils in inflammatory bowel disease (IBD) are scattered. We assessed the concentrations of eosinophil-associated cytokines and growth factors in the group of 277 individuals (101 patients with Crohn's disease (CD), 77 with ulcerative colitis (UC), 16 with irritable bowel syndrome (IBS), and 83 healthy controls) and referred to IBD activity and the levels of hsCRP. As compared to IBS patients or healthy controls, patients with CD had significantly higher levels of IL5, IL8, IL12(p70), GM-CSF, and TNFα and patients with UC, the levels of eotaxin, IL4, IL5, IL8, IL12(p70), IL13, GM-CSF, and TNFα were also higher. As compared to CD patients, patients with UC had significantly higher levels of eotaxin, IL4, IL5, IL8, and IL1. In turn, the concentrations of hsCRP were significantly higher in CD than UC. Except for IL13, all cytokines and hsCRP positively correlated with CDAI. In UC, a positive correlation with MDAI was observed for hsCRP, GM-CSF, IL12(p70), and IFNγ and a negative one for IL8. The concentrations of hsCRP, GM-CSF, IFNγ, IL12(p70), and RANTES were higher in UC patients with active than inactive disease whereas those of IL8 and TNFα were significantly lower. Eotaxin, determined individually or in a panel with IFNγ and hsCRP, showed fair accuracy in differentiating CD from UC. If confirmed on a larger representation of IBS patients, IL8 might support differential diagnosis of organic and functional conditions of the bowel. GM-CSF, in turn, demonstrated to be an excellent indicator of bowel inflammation and may be taken into consideration as a noninvasive marker of mucosal healing. In summary, eosinophil-associated cytokines are elevated in IBD, more pronouncedly in UC, and may support the differential diagnosis of IBD and aid in monitoring of mucosal healing.
Introduction
Crohn's disease (CD) and ulcerative colitis (UC) are two main types of inflammatory bowel disease (IBD), a group of chronic inflammatory conditions of the bowel. IBD incidence is increasing worldwide, both in pediatric and adult populations [1,2]. The disease is characterized by the wide spectrum of intestinal and extraintestinal complications. Pathogenesis of IBD is multifactorial and still not fully elucidated. Nevertheless, the consensus is that the crucial role is played by an impaired immune response to the gut microbiota, evoked by environmental triggers and/or genetic factors [3,4]. The adaptive immunity seems to play the first fiddle in the pathogenesis of IBD [4]. However, the inflamed mucosa is infiltrated also by cytokine-rich innate immune cells [5] and their unceasing activation contributes to local and systemic inflammations [6]. IBD diagnostics and differential diagnostics pose a challenge for clinicians. IBD symptoms are nonspecific and overlapping. Establishing diagnosis requires a performance of series of laboratory, endoscopic, radiologic, and histologic tests. Those tests are expensive, often invasive, related to some risk, and poorly acceptable by patients. Biochemical markers currently used in clinical practice are insufficient and not recommended to serve as therapeutic targets [7]. Therefore, as a key part of the precision medicine strategy, the alternative, noninvasive biomarkers of IBD are intensively searched for [8,9].
Eosinophils are acidophilic multifunctional granulocytes involved in inflammation and immunity that remain outside the mainstream research on IBD. However, they are a rich source of cytotoxic proteins, pro-and anti-inflammatory cytokines, chemokines, and growth factors and are likely to contribute to both inflammatory and regenerative phases of the disease. Accordingly, peripheral eosinophils of IBD patients are primed and preactivated. They display increased responsiveness, adhesiveness, migration, and degranulation [10,11] and are characterized by upregulated secretion of their mediators, for example, eosinophilic cationic protein [12] or eotaxin [13,14]. Peripheral eosinophilia is associated with less favorable outcome, that is, higher incidence of primary sclerosing cholangitis or need for surgical intervention [15]. Interestingly, prevalence of peripheral eosinophilia significantly differs between CD and UC. It has been reported to occur more often in UC patients, both in pediatric [16] and adult [15] populations. Locally, increased number and activation of eosinophils have been repeatedly observed in areas of active inflammation. Functionally, as evidenced in animal models of colitis, eosinophils seem to exert proinflammatory and promotility effects in IBD, thus contributing to diarrhea, tissue inflammation, and destruction, as well as fibrosis and formation of strictures. Additionally, they have been suggested to contribute to tissue repair and remodeling (reviewed in [17][18][19]). However, knowledge concerning eosinophils and quiescent bowel remains inconsistent.
Despite the acknowledged contribution of eosinophils to the disease pathogenesis, available data on cytokines closely related to the development and activity of peripheral eosinophils in IBD patients are either scattered or nonexistent. Herein, we assessed cytokines and growth factors crucial for eosinophil proliferation and differentiation (GM-CSF, IL5, and IL4), for their release from bone marrow (IL5), for their survival (IL5, IL13, GM-CSF, and IL4) and priming in circulation (IL5, GM-CSF, IL4, and TNFα), and for extravasation (IL4, IFNγ, TNFα, IL8, and eotaxin) and homing into the gut (eotaxin, RANTES, IL5, IL8, IL13, GM-CSF, and TNFα) as well as responsible for inducing production and secretion of their mediators (TNFα, IFNγ, IL-5, GM-CSF, and eotaxin) (reviewed in [10,17,20]). We also evaluated IL12 as a key inductor of eosinophil apoptosis and as a mediator of antieosinophil actions of glucocorticosteroids [10]. This paper focuses on the potential of eosinophil-associated cytokines and growth factors as biomarkers in IBD, in reference to high-sensitive C-reactive protein, an inflammatory marker commonly used in IBD clinics [8]. or the coexistence of other severe systemic diseases, malignancies, liver diseases, or pregnancies were not included. The clinical activity of the disease was assessed using the Crohn's Disease Activity Index (CDAI) for CD and the Mayo Disease Activity Index (MDAI) for UC. Severity of bowel inflammation in UC patients was assessed using a Mayo endoscopic score. IBD patients were treated with 5′-aminosalicylate (5′-ASA) derivatives. Healthy controls were recruited from among students of our university and hospital staff or from among outpatients of Research, Science, and Educational Center of Dementia Diseases, Scinawa, Poland, diagnosed with mild cognitive disorders (no significant medical history) or from blood donors from the Regional Center for Blood Donation and Therapeutics in Wroclaw, Poland. Female to male ratio in study groups was as follows: 50/51 in CD, 34/43 in UC, 12/4 in IBS, and 38/45 in controls, p = 0 147. Age distribution was as follows (medians with 95% CI): 31 yrs (30-36.9) in CD, 40 yrs (33.8-45) in UC, 53 yrs in IBS, and 35 yrs in controls (p = 0 104).
Ethical Considerations.
The study protocol was accepted by the Medical Ethics Committee of Wroclaw Medical University (KB-575/2011). The study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 1983, and an informed consent was obtained from all patients.
Analytical
Methods. Blood was drawn by venipuncture, following an overnight fast, allowed to clot for 30 minutes, and centrifuged (15 minutes, 720 ×g). Resulting sera were collected, aliquoted, and kept frozen at −80°until examination. We applied a flow cytometry-based method utilizing magnetic microspheres conjugated with monoclonal antibodies (Luminex xMAP® Technology) to measure the concentrations of eotaxin, GM-CSF, IFNγ, IL4, IL5, IL8, IL12(p70), IL13, RANTES, and TNFα. Analytes were measured in duplicate using Bio-Plex Pro™ human cytokine, chemokine, and growth factor magnetic bead-based assays according to the instructions provided by the manufacturer but with a lower serum dilution factor and the Bio-Plex 200 platform with HRF (Bio-Rad, USA). Standard curves were drawn using 5-PL logistic regression, and the data were analyzed using Bio-Plex Manager 6.0 software. Data on highsensitive C-reactive protein (hsCRP), measured using an enhanced immunoturbidimetric method with the CRPex-HS CRP test (Good Biotech Corp., Taichung, Taiwan), were retrieved from our earlier study on this population (available exclusively for IBD patients).
Statistical Analysis.
Normality of data distribution was tested using the χ 2 test and homogeneity of variances using Levene's test. Data were log-transformed if necessary and presented as geometric means or medians accompanied by 95% confidence interval. Multigroup comparisons were conducted using one-way ANOVA or the Kruskal-Wallis H test. Two-group comparisons were conducted using t-test for independent samples (with Welch correction if appropriate) or the Mann-Whitney U test. Correlation analysis with the disease activity scores was conducted using the Spearman test. Frequency analysis was conducted using the χ 2 test or Fisher's exact test. Receiver operating characteristic (ROC) curve analysis was conducted to evaluate the strength of observed associations and the suitability of analytes as disease markers. A backward stepwise method of logistic regression, followed by the Hosmer and Lemeshow goodness-of-fit test and ROC analysis, was used to assess the discriminative power of combined marker evaluation. Model building was preceded by the analysis of linearity, collinearity, and interactions of candidate variables. To validate the final model, we applied the V-fold cross-validation. Z statistics was used to evaluate the significance of difference between area under ROC curves (AUCs; measure of test accuracy) obtained for training and validation sets. All calculated probabilities were two-tailed, and p values ≤ 0.05 were considered statistically significant. The statistical analysis was conducted using MedCalc statistical software version 15
Eosinophil-Associated Cytokines in Bowel Diseases.
Patients with UC had significantly higher levels of eotaxin, IL4, IL5, IL8, and IL13 than patients with CD or IBS and healthy controls. They also had higher levels of TNFα, GM-CSF, and IL12(p70) than IBS patients and healthy controls but not CD patients. The levels of RANTES and IFNγ were higher in UC patients but only in comparison to healthy controls ( Figure 1).
Patients with CD had significantly higher levels of IL5, IL8, IL12(p70), GM-CSF, and TNFα than IBS patients and healthy controls and higher levels of IL13, IFNγ, and RANTES as compared to healthy controls ( Figure 1).
Patients with IBS had significantly higher levels of IL12(p70) and IFNγ than healthy controls but lower levels of IL8 ( Figure 1).
In UC, active disease was associated with more pronouncedly elevated levels of GM-CSF (47 pg/ml (39-57) versus 23. Except for IL13, all evaluated cytokines and growth factors weakly to moderately correlated with CDAI. In turn, only IL13 and GM-CSF displayed a positive association with MDAI (Table 1).
Of the examined cytokines, GM-CSF, IL12(p70), and IFNγ positively correlated with the degree of endoscopic inflammation in UC patients (Table 1). IL8 displayed a negative correlation due to high cytokine levels in patients without inflammation. When the analysis was restricted to patients with inflamed mucosa, the association tended to be positive (p = 0 107).
The levels of hsCRP were significantly higher in active than inactive CD: 38.6 mg/l (27-65) and 1.74 mg/l (0.7-21), p = 0 002, respectively. They were also higher in active than inactive UC: 18 mg/l (5.5-22.6) and 1.75 mg/l (0.3-3.8), p < 0 001, respectively. There was a positive correlation between hsCRP and the indices of clinical activity of CD and UC as well as endoscopic inflammation in UC (Table 1).
Eosinophil-Associated Cytokines as Differential Markers in IBD.
Symptomatology of UC and CD may sometimes overlap, rendering both IBD types difficult to distinguish. Moreover, there is still a group of patients with unclassified IBD, in whom the definitive distinction between UC and CD is not possible despite of all recommended tests [21]. Of the evaluated eosinophil-associated cytokines, eotaxin 7), p = 0 035) were more pronouncedly elevated in active UC than CD. In turn, hsCRP was significantly higher in active CD than UC (26.6 mg/l (17.2-41.2) versus 8.9 mg/l (4.3-18.4), p = 0 011). We applied ROC analysis to evaluate the suitability of these cytokines and hsCRP as individual differential markers (Figure 2(a)). As depicted in Table 2, eotaxin displayed superior accuracy as a marker of active UC, followed by hsCRP as a marker of active CD.
In order to discern the independent predictors of the disease type and evaluate the power of their combined assessment, we applied logistic regression. All cytokines, the levels of which significantly differed between active UC and CD, were selected as potential explanatory variables, and active CD was coded as 0 while active UC coded as 1. Data on CRP levels were unavailable for a few patients; thus, the analysis was limited to 115 patients with active IBD (45 with UC and 70 with CD). First, a univariate regression analysis was conducted for each variable and all variables were found to significantly affect the dependent variable.
Next, candidate variables were tested for linearity, collinearity, and interactions. The model was built with eotaxin, IL13, IFNγ, and hsCRP (ratio of analyzed variables to cases was 29), and a backward stepwise method was used. Eotaxin (Table 2) and allowed for a correct classification of 72.2% of cases; however, the model's pseudo R-squared was rather low: Nagelkerke R 2 = 0.273. The accuracy of a validated model (V-fold cross-validation) was comparable ( Table 2). Lack of significant difference in accuracy between training and validation sets suggests that the predictive power of the model was not overestimated.
Eosinophil-Associated Cytokines as Negative Markers of
Mucosal Healing (MH) in UC. Data on endoscopic findings were available for UC patients, among whom 16 had no signs of active bowel inflammation (coded as 1 for the purpose of ROC analysis and logistic regression) and 37 had an ongoing inflammation (coded as 0). Of patients with active inflammation, 10 were assigned Mayo endoscopic score 1, 15 score 2, and 12 score 3.
GM-CSF, IFNγ, and IL12(p70) as well as hsCRP were significantly correlated with Mayo endoscopic subscore (Table 1). Individually, the accuracy of GM-CSF as the marker of mucosal healing was superior to those of other cytokines and hsCRP (Figure 2(b), Table 3).
Logistic regression (backward stepwise methods) was applied to discern the independent predictors of mucosal healing and to evaluate the power of potential multimarker assays. GM-CSF, IFNγ, IL12(p70), and hsCRP were tested as potential explanatory variables. However, they were found to be interrelated; GM-CSF was positively correlated with hsCRP (r = 0.56, p < 0 00001), IL12(p70) (r = 0.404, p = 0 003), and IFNγ (r = 0.035, p = 0 011). Also, hsCRP was correlated with IFNγ (r = 0.36, p = 0 014). As such, we attempted to build three separate models: one exclusively with GM-CSF, second containing IFNγ and IL12(p70) combined together, and third with hsCRP and IL12(p70) combined together. However, testing for linearity and collinearity excluded two latter combinations of variables. At the end, only a model with GM-CSF was built. Regression coefficient for GM-CSF was b = −7.7, p < 0 001; OR was 0.0005 (0-0.0335); constant = 10.33, p < 0 001. The overall model fit was χ 2 = 30.5, p < 0 00001, and the results of the Hosmer and Lemeshow test were χ 2 = 6.8, p = 0 658 indicating a good logistic regression model fit. The model predictive power was excellent (Table 3) and allowed for a correct classification of 87% of cases. The model's pseudo R-squared was high as well: Nagelkerke R 2 = 0.619. The accuracy of the V-fold cross-validated model was comparable proving the model predictive power not to be overestimated (Table 3).
Eosinophil-Associated Cytokines as Markers
Differentiating between IBD and IBS. Functional bowel disorders like IBS display a similar set of symptoms to IBD making their differential diagnosis difficult. Herein, we evaluated the eosinophilassociated cytokines as potential differential markers.
As depicted in Figure 1, IL5, IL8, IL12(p70), TNFα, and GM-CSF were significantly higher in both CD and UC than in IBS. Of these, IL8 had the highest AUC and Youden index. At optimal cut-off values, IL5 and TNFα displayed superior specificity, IL12(p70) and GM-CSF displayed superior sensitivity, and the sensitivity and specificity of IL8 was equally high (Figure 2(c), Table 4).
Logistic regression was applied to discern the independent indicators of active IBD (coded as 1 and IBS coded as 0). Candidate variables (IL5, IL8, IL12(p70), GM-CSF, and TNFα) were tested for linearity, collinearity, and interactions. IL5, IL8, and TNFα occurred to be interrelated with the following correlation coefficients: r = 0.74 for IL5 and IL8, r = 0.6 for IL5 and TNFα, and r = 0.62 for IL8 and TNFα. As such, three separate models were tested: each model included GM-CSF and IL12(p70) and either IL5, IL8, or TNFα. Of these, the model with IL8 (IL8, GM-CSF, and IL12(p70)) displayed the best fit. However, following the analysis of variable contribution to the model's predictive power, exclusively IL8 was retained. Regression coefficient for IL8 was b = 4.49, p < 0 0001; OR was 89.4 (11.8-679); constant = −13.9, p < 0 0001. The overall model fit was χ 2 = 43.3, p < 0 00001, and the results of the Hosmer and Lemeshow test were χ 2 = 5.3, p = 0 727 indicating a good logistic regression model fit. The model predictive power was excellent (Table 4) and allowed for a correct classification of 93% of cases. The model's pseudo R-squared (Nagelkerke R 2 ) was 0.502. AUC obtained after V-fold cross-validation was slightly lower, but there was no significant difference compare to AUC of a training set proving the model predictive power not to be overestimated (Table 4).
Discussion
Eosinophils are gaining an increasing attention as the cells of unique properties among leukocytes, which can damage or repair surrounding tissue and modulate the activity of immune cells [22]. Accordingly, the infiltration of lamina propria by eosinophils has been shown to be an early histological marker of UC [23] and a predictor of clinical relapse [24] as well as poor response of UC patients to medical therapy [25]. Recently, local eosinophilia has been reported to be related to the stricturing phenotype of CD [26]. Peripheral eosinophilia has been observed in IBD as well and linked to poor prognosis [15]. Peripheral eosinophils isolated from IBD patients have been characterized as primed, displaying increased responsiveness, adhesiveness, migration, and degranulation [10,11]. Still, little is known on cytokines involved in managing growth, distribution, and priming of eosinophils as well as in aiding their homing into the gut. Except for eotaxin, which has become a novel therapeutic target in UC [27], eosinophil-associated cytokines, such as GM-CSF, IL3, IL5, and IL13, have not been comprehensively analyzed in the context of IBD. Available data are scanty, mostly derived from the analysis of small cohorts, and obtained exclusively from multiplexed analyses intended for screening purposes, in which cytokines less abounded in sera frequently balance at the verge of lower detection limits of the tests, making their evaluation problematic [28][29][30]. Herein, the dilution of our samples was adjusted to enable credible analysis of these analytes. It allowed us to show that compared to healthy individuals, IBD patients had significantly higher concentrations of all evaluated eosinophil-associated cytokines and growth factors, although the elevation of eotaxin and IL4 was observed exclusively in UC patients. Differential diagnosis of UC and CD might be challenging [21]. In this respect, it is easy to perform and noninvasive cytokine assays would be welcomed to assist clinicians in the diagnostic process. In line with higher prevalence of local [31] and peripheral eosinophilia [16] in UC than CD, we found several of eosinophil-associated cytokines to be more pronouncedly elevated in UC. We evaluated suitability of eotaxin, IL13, IFNγ, and hsCRP as differential markers assessed individually as well as a multicytokine panel. Individually, eotaxin performance was fair and superior to other markers. Eotaxin is a critical chemoattractant specific for eosinophils, and an elevation of its circulating concentrations in IBD has already been reported by several groups [13,14,28,29,32]. However, previous comparative analyses of eotaxin in CD and UC have been conducted exclusively in small cohorts and yielded contradictory results [13,14,28,32]. The addition of IFNγ and hsCRP improved the discriminative power of the assay as compared to sole determination of eotaxin but not markedly. Of note, Iboshi et al. [33] found the expression of IFNγ to be more accentuated in UC than in CD.
Chronic bowel inflammation is a hallmark of IBD. The assessment of its severity is imperative for the determination of the disease activity and establishment of a therapeutic approach. It became particularly crucial in view of the recent change of priorities in IBD therapy from control of symptoms to the restriction of inflammation [1]. Mucosal healing (MH), currently assessed exclusively by endoscopy, is related to more favorable clinical outcomes and has lately become one of the therapeutic endpoints in clinical trials. However, clinical trials have shown a poor correlation between clinical symptoms and endoscopy. It is estimated that almost half of CD patients with clinical remission are still presenting with endoscopic features of active disease whereas almost 40% of patients with inactive disease in endoscopy are displaying clinical symptoms of the disease [34]. Since clinical indices are not sufficient, biomarkers, which could be applied in MH evaluation, as well as in monitoring the disease course, predicting its relapse, and recognizing flare, are intensively sought after [34][35][36]. Herein, hsCRP, GM-CSF, IFNγ, and IL12(p70) were significantly and positively correlated with the degree of bowel inflammation, expressed as Mayo endoscopic subscore. Of these, GM-CSF has emerged as a superior MH marker in UC. GM-CSF displayed high accuracy, falling within the range typical for biomarkers applied in clinical practice (85-95%). Importantly, GM-CSF accuracy remained high following its validation. Taking into account the invasiveness and a risk for complications associated with endoscopy, serum-based MH markers such as GM-CSF might help to assist in the selection of patients and limit the need for invasive examinations. GM-CSF has been the only cytokine other than eotaxin significantly elevated in sera of UC patients in Coburn et al.'s study [29]. GM-CSF controls proliferation and differentiation if eosinophils in the bone marrow provide survival signals and induce the release of their granule proteins. GM-CSF is also responsible for eosinophil priming in circulation and acts as their chemoattractant [20,37]. However, as reviewed by Han et al. [38], data available on GM-CSF functionality in the bowel are equivocal. A beneficial role has been attributed to this growth factor in the small intestine, where it might contribute to maintaining intestinal barrier function, likely through stimulating proliferation of intestinal epithelial cells (IECs). Accordingly, no difference in GM-CSF expression has been noted between inflamed and noninflamed IECs. On the other hand, colonic mucosa has been shown to overexpress GM-CSF when inflamed or cancerous and the growth factor had no effect on colonic cell proliferation. Despite unresolved issue of GM-CSF function in IBD, growth factor-based therapies have been attempted and yielded promising results. In animal models of colitis, GM-CSF application has resulted in an improvement of clinical symptoms and histological findings as well as in accelerated ulcer healing. In human CD, a therapy with recombinant GM-CSF has lessened the disease severity (reviewed in [38]). In the light of these findings, our observation on GM-CSF increase in IBD patients might represent a protective mechanism. However, healing capabilities of GM-CSF might be counteracted by concomitant elevation in the levels of GM-CSF autoantibodies. Their presence in IBD patients and the association with a more aggressive disease phenotype have been previously reported and implied in functional GM-CSF deficiency caused by antibody interference with GM-CSF binding to its receptor [39,40]. It is estimated that the endoscopy in about half of adult and 70% of pediatric patients presenting with symptoms indicative of IBD yields negative results. These patients are subsequently diagnosed with functional bowel disorders, for example, IBS, the condition which share many clinical symptoms with IBD. In this respect, a reliable but less invasive differential tool is needed [41]. From among eosinophilassociated cytokines analyzed by us, IL5, IL8, IL12(p70), TNFα, and GM-CSF were significantly higher in both CD and UC than in IBS. Of these, IL8 had superior accuracy in differentiating IBS and IBD and allowed for a correct classification of 93% of patients. IL8 is a cytokine which is the most loosely associated with eosinophils. Although secreted by these leukocytes and acting as their chemoattractant [20], IL8 is primarily responsible for neutrophil trafficking. Correspondingly, another neutrophil-associated protein, namely, calprotectin, has been claimed as having adequate sensitivity and specificity to assist in differentiating IBD from IBS. Even though IL8 herein displayed an excellent accuracy, validated using V-fold cross-validation technique, our results should be interpreted with care and treated as indicative only because of the small number of patients with IBS included in our cohort.
Conclusions
Our results obtained on a large cohort of patients showed that the concentrations of circulating eosinophil-associated cytokines are elevated in IBD and that this elevation is more accentuated in UC. Evaluated as prospective differential markers, eotaxin, individually and in a panel with IFNγ and hsCRP, showed fair accuracy in differentiating CD from UC. IL8, if confirmed on a larger representation of IBS patients, might, in turn, support differential diagnosis of organic (IBD) and functional (IBS) conditions of the bowel. GM-CSF demonstrated to be an excellent indicator of bowel inflammation and may be taken into consideration as a noninvasive marker of mucosal healing.
Data Availability
The data used to support the findings of this study are included within the article.
Ethical Approval
The study protocol was approved by the Medical Ethics Committee of Wroclaw Medical University (KB-575/2011).
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2018-08-28T09:51:17.024Z
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2018-07-26T00:00:00.000
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225097006
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pes2o/s2orc
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The Good, the Bad and the Ugly: Pitfalls and Best Practices in Automated Sound Static Analysis of Ethereum Smart Contracts
Ethereum smart contracts are distributed programs running on top of the Ethereum blockchain. Since program flaws can cause significant monetary losses and can hardly be fixed due to the immutable nature of the blockchain, there is a strong need of automated analysis tools which provide formal security guarantees. Designing such analyzers, however, proved to be challenging and error-prone. We review the existing approaches to automated, sound, static analysis of Ethereum smart contracts and highlight prevalent issues in the state of the art. Finally, we overview eThor, a recent static analysis tool that we developed following a principled design and implementation approach based on rigorous semantic foundations to overcome the problems of past works.
Introduction
Blockchain technologies are revolutionizing the distributed system landscape, providing an innovative solution to the consensus problem leveraging probabilistic guarantees and incentives. In particular, they allow for the secure execution of payments, and more in general computations, among mutually distrustful parties. While some cryptocurrencies, like Bitcoin [27] provide only a limited scripting language tailored to payments, others, like Ethereum [32], support a quasi Turing complete 1 smart contract language, allowing for advanced applications such as trading platforms [25,28], elections [26], permission management [7,11], data management systems [5,29], or auctions [13,17]. With the growing complexity of smart contracts, however, also the attack surface grows. This is particularly problematic as smart contracts control real money flows and hence constitute an attractive target for attackers. In addition, due to the immutable nature of blockchains, smart contracts cannot be modified once they are uploaded to the blockchain, which makes the effects of security vulnerabilities permanent. This is not only a theoretical threat, but a practical problem, as demonstrated by infamous hacks, such as the DAO hack [1] or the Parity hacks [2,3] which caused losses of several millions of dollars. This state of affairs calls for reliable static analysis tools which are accessible to the users of the Ethereum system, that is, developers, who need to be able to verify their contracts before uploading them to the blockchain, and users interacting with existing smart contracts, who need tool assistance to assess whether or not those contracts (which are published in human unreadable bytecode format on the blockchain) are fraudulent.
State of the art. The bug-finding tool Oyente [24] (published in 2016) pioneered the (automatic) static analysis of Ethereum smart contracts. This work highlighted, for the first time, generic types of bugs that typically affect smart contracts, and proposed a tool based on symbolic execution for the detection of contracts vulnerable to these bugs.
A particular compelling feature of Oyente is that it is a push-button tool that does not expect any interaction or deeper knowledge of the contract semantics from the user. On the downside, however, Oyente does not provide any guarantees on the reported results, being neither sound (absence of false negatives) nor complete (absence of false positives) and, thereby, yielding only a heuristic indication of contract security. Given the importance of rigorous security guarantees in this context, several approaches have been later proposed for the verification of Ethereum smart contracts. In particular, alongside tools that aim at machine-checked smart contract auditing [6,9,18,19], a line of work focused on automation in the verification process to ensure usability and broad adaption. Despite four years of intense research, however, until now only four works on such sound and fully automatic static analysis of Ethereum smart contracts have been published. Furthermore, all of these works exhibit shortcomings which ultimately undermine the security guarantees that they aim to provide.
Our contributions. Motivated by these struggles, we overview the difficulties that arise in the design of sound static analysis for Ethereum smart contracts, and the pitfalls that so far hindered the development of such analyzers. To this end, we first give a short introduction to the Ethereum platform and its native smart contract language (Section 2). Afterwards, we illustrate the challenges in automated smart contract verification by overviewing existing works in this field with emphasis on their weak spots (Section 3 and 4). We conclude the paper summarizing our experiences in the design of eThor [30], a static analyzer for EVM bytecode we recently introduced, along with a breakdown of the components we deem crucial to achieve efficiency and soundness (Section 5).
Ethereum and the EVM Bytecode Language
Ethereum is (after Bitcoin) the second most widely used cryptocurrency with a market capitalization of over 14 billion U.S. dollars 2 . Compared to Bitcoin, Ethereum stands out due its expressive scripting language, that enables the execution of arbitrary distributed programs, so called smart contracts. Ethereum smart contracts are stored on the blockchain in bytecode that is jointly executed by the network participants (also called nodes) according to the Ethereum Virtual Machine (EVM) -Ethereum's execution environment that is implemented in different clients 3 . Smart contract execution is part of Ethereum's consensus protocol: The state of the system is determined by a jointly maintained, tamper-resistant public ledger, the blockchain, that holds a sequence of transactions. Transactions do not only indicate money transfers, but can also trigger contract executions. To derive the state of the system, every node locally executes the transactions in the blockchain as specified by the EVM. For advancing the system, nodes broadcast transactions which are assembled into blocks and appended to the blockchain. While the correctness of the system is ensured by the nodes validating all blocks, fairness is established by a proof of work mechanism: Only nodes (called miners) capable of solving a computationally hard puzzle are eligible to propose a block. This adds randomness to the selection of proposers and prevents a minority from steering the evolution of the system.
Ethereum Ecosystem
The state of the Ethereum system (called global state) consists of the state of all virtual accounts and their balances in the virtual currency Ether. Smart contracts are special account entities (contract accounts) that in addition to a balance hold persistent storage and the contract code. While non-contract (so called external ) accounts can actively transfer fractions of their balance to other accounts, contract accounts are purely governed by their code: A contract account can be activated by a transaction from another account and then executes its code, possibly transferring money or stimulating other contracts. Similarly, a contract can be created on behalf of an external account or by another contract. We will in the following refer to such inter-contract interactions as internal transactions, as opposed to external transactions that originate from external accounts and are explicitly recorded on the blockchain.
EVM Bytecode
The EVM bytecode language supports designated instructions to account for the blockchain ecosystem. These encompass primitives for different flavors of money transfers, contract invocations, and contract creations. Most prominently, the CALL instruction allows for transferring money to another account while at the same time triggering code execution in case the recipient is a contract account. Other domain-specific bytecodes include instructions for accessing the blockchain environment such as the instructions SSTORE and SLOAD for reading and writing the cells of the persistent contract storage. Further the instruction set contains opcodes for accessing information on the ongoing (internal) transaction (its caller, input, or value transferred along) and for computation: The EVM is a stack-based machine supporting standard instructions for arithmetic and stack manipulation. The control flow of a contract is also subject to the stack-based architecture: conditional and unconditional jump instructions allow for resuming execution at another program position that is determined by the value on the stack. While these features would make EVM bytecode Turing-complete, to enforce termination the execution of EVM smart contracts is bounded by an upfront-specified resource called gas. Every instruction consumes gas and the execution halts with an exception when running out of gas. The gas budget is set by the initiator of the transaction who will pay a compensation to the miner of the enclosing block for the effectively consumed amount of gas when executing the transaction. Due to the low-level nature of EVM bytecode, smart contracts are generally written in high-level languages (most prominently the Solidity [4] language) and compiled to EVM bytecode.
Related Work on Automated Sound Static Analysis of Ethereum Smart Contracts
We overview the state of the art in the automated static analysis of Ethereum smart contracts. So far there have been works on four static analyzers published that come with (explicit or implicit) soundness claims: the dependency analysis tool Securify [31] for EVM bytecode, the static analyzer ZEUS [22] for Solidity, the syntax-guided Solidity analyzer NeuCheck [23], and the bytecode-based reachability analysis tool EtherTrust [14]. By implicit soundness claim, we mean that the tool claims that a positive analysis result guarantees the contract's security (i.e., absence of false negatives with respect to a specific security property). While Securify, ZEUS, and EtherTrust implement semantic-based analysis approaches, NeuCheck is purely syntax-driven. Securify supports data and control flow analysis on the EVM bytecode level. To this end, it reconstructs the control-flow graph (CFG) from the contract bytecode and transforms it into SSA-form. Based on this structured format, it models immediate data and control flow dependencies using logical predicates and establishes datalog-style logical rules for deriving transitive dependencies which are automatically computed using the enhanced datalog engine Soufflé [21]. For checking different security properties, Securify specifies patterns based on the derived predicates which shall be sufficient for either proving a property (compliance patterns) or for showing a property to be broken (violation patterns). ZEUS analyzes Solidity contracts by first translating them into the intermediate language LLVM bitcode and then using off-the-shelf model checkers to verify different security properties. In the course of the translation, ZEUS uses another intermediate layer for the Solidity language which introduces abstractions and that allows for the insertion of assumptions and assertions into the program code which express security requirements on the contract. The security properties supported by ZEUS are translated to such assertion checks, possibly in conjunction with additional property-specific contract transformations.
NeuCheck analyzes Solidity contracts by pattern matching on the contract syntax graph. To this end, it translates Solidity source code into an XML parse tree. Security properties are expressed as patterns on this parse tree and are matched by custom algorithms traversing the tree.
EtherTrust implements a reachability analysis on EVM bytecode by abstracting the bytecode execution semantics into (particular) logical implications, so called Horn clauses, over logical predicates representing the execution state of a contract. Security properties are expressed as reachability queries on logical predicates and solved by the SMT solver z3 [12]. EtherTrust was a first prototype that later evolved into the eThor analyzer, which we will discuss in Section 5.1.
All presented tools focus on generic (contract-independent) security properties for smart contracts. However, the underlying architectures allow for extending the frameworks with further properties. Foremost, the tool eThor supports general reachability properties and hence also functional properties characterized in terms of pre-and postconditions. For the soundness considerations in this paper we put the focus on the abstractions of generic security properties.
Challenges in Sound Smart Contract Verification
EVM bytecode exposes several domain-specific subtleties that turn out to be challenging for static analysis. Furthermore, characterizing relevant generic security properties for smart contracts is highly non-trivial and subject to ongoing research. We will examine both of these problems in the following.
Analysis Design
We summarize below the main challenges that arise when designing a performant and still sound analysis for Ethereum smart contracts: -Dynamic jump destinations: Jump destinations are statically unknown and computed during execution. They might be influenced by the blockchain environment as well as the contract state. As a consequence, the control flow graph of a contract is not necessarily determinable at analysis time. -Mismatch between memory and stack layout: The EVM has a (stack) word size of 256 bits while the memory (heap) is fragmented into bytes and addressed accordingly. Loading words from memory to the stack, and conversely writing stack values to memory, requires (potentially costly) conversions between these two value formats. -Exception propagation and global state revocation If an internal transaction (as, e.g., initiated by a CALL) fails, all effects of this transaction including those on the global state (e.g., writes to global storage) are reverted. However, such a failure is not propagated to the callee, who can continue execution in the original global state. Modeling calls must thus save the state before calling in order to account for global state revocation.
-Native support for low-level cryptography: The EVM supports a designated SHA3 instruction to compute the hash of some memory fraction. As a consequence, hashing finds broad adaption in Ethereum smart contracts, and the Solidity compiler bases its storage layout on a hash-based allocation scheme. -Dynamic calls: The recipient of an (inter-contract) call is specified on the stack and hence subject to prior computation. Consequently, the recipient is not necessarily derivable at analysis time, resulting in uncertainty about the behavior of the callee and the resulting effects on the environment. -Dynamic code creation: Ethereum supports the generation of new smart contracts during transaction execution: A smart contract can deploy another one at runtime. To do so, the creating smart contract reads the deployment code for the new contract from the heap. The newly created contract may hence be subject to prior computation and even to the state of the blockchain.
In order to effectively tackle these challenges, several contributions of independent interest are required, such as domain-specific abstractions (e.g., suitable over-approximations of unknown environment behavior); the preprocessing of the contract to reconstruct its control flow or call graph; (easily checkable) assumptions that restrict the analysis scope (e.g., restriction to some language fragment); and optimizations or simplifications in intermediate processing steps (e.g, contract transformations to intermediate representations). Altogether, these analysis steps enlarge the semantic gap between the original contract semantics and the analysis, making it harder to reliably ensure the soundness of the latter. In the following, we will review in more detail the tension between soundness and performance of the analysis, and how past works stumbled in this minefield.
Soundness Ensuring the soundness of the analysis requires a rigorous specification of the semantics of EVM bytecode. The original semantics was written in the Yellow Paper [32]. This paper however, from the beginning exhibited flaws [15,18,19] and underspecified several aspects of bytecode execution. The ultimate truth of smart contract semantics could therefore only be extracted from the client implementations provided by the Ethereum foundation. In the course of time, several formal specifications of the EVM semantics have been proposed by the scientific community [15,18,19], leading the Yellow paper to be replaced by an executable semantics in the K framework [18] For the high level language Solidity, despite first efforts within the scientific community [8,10,20,34,35], there exists at present no full and generally accepted formal semantics. Consequently the semantics of Solidity is only defined by its compilation to EVM bytecode. Since the compiler is subject to constant changes, Solidity constitutes a moving target.
The complexity and uncertainty about the concrete semantics made most works build on ad-hoc simplified versions of the semantics which do not cover all language features and disregard essential aspects of the EVM's execution model. ZEUS [22], for instance, defines an intermediate goto language for capturing the core of Solidity. The semantics of this language, however, is inspired by the (ad-hoc) semantic modeling used in Oyente [24], inheriting an essential flaw concerning global state revocation: In case that an internal transaction returns with an exception, the effects on the global state are not reverted as they would be in real EVM (and Solidity) executions. Since the translation to the intermediate language is part of the analysis pipeline of [22], such a semantic flaw compromises the soundness of the whole analysis.
Also Securify [31] introduces an ad-hoc formalism for EVM bytecode semantics. This is not, however, related to the dependency predicates used for the analysis, but just serves for expressing security properties. It is hence unclear to which extent the dependency predicates faithfully reflect the control flow and value dependencies induced by the EVM semantics. Assessing the correctness of this approach is difficult, since no full logical specification of the dependency analysis is provided 5 . Indeed we found empirical indication for the unsoundness of the dependency analysis in the presence of complicated control flow. Consider the example contract depicted in Figure 1. For better readability, we present the contract in the high-level language Solidity, a language inspired by JavaScript, that is centered around contracts which are used analogously to the concept of classes in object-oriented programming languages. The depicted contract Test has a global boolean field test. Global fields are reflected in the persistent storage of the contract and constitute the contract state. The public function flipper() allows every account but the one with address 0 to flip the value of the test field: For checking the restriction on the calling account, the flipper() function accesses the address of the caller using Solidity's msg.sender construct. For writing the test field, the internal function flip() is called. Internal functions are not exposed to the public, but are only accessible by the contract itself and calls to such functions are compiled to local jumps. The use of internal functions consequently substantially complicates the control flow of a contract.
We identified a correctness issue that affects both the soundness and completeness of Securify. This incorrectness becomes evident in the violation pattern that checks for unrestricted writes. An unrestricted write is a write access of the global storage that can be performed by any caller. The violation pattern states that such an unrestricted write is guaranteed to happen if there is a SSTORE instruction whose reachability does not depend on the caller of the contract. This pattern should not be matched by the Test contract since the only write access to the Test's sole variable test in function flip() is only reachable via the function flipper where it is conditioned on the caller (msg.sender). Hence not every contract can write the test variable, but the write access depends on the caller. Still Securify reports this contract to match the violation pattern, consequently proving the wrong statement that there is no dependency between writing the test field and the account calling the contract. Note that even though showing up in a violation pattern (hence technically producing false positives), the underlying issue also affects the soundness of the core dependency analysis 6 . Securify specifies a may dependency relation to capture (potential) abstract dependencies between program locations. For correctly (i.e. soundly) abstracting the dependencies in real programs, the absence of a may dependency should imply a corresponding independence in the real program. Since the may dependency relation is used in both compliance and violation patterns, without such a guarantee Securify can be neither sound nor complete. The example refutes this guarantee and thereby illustrates the importance of providing clear formal correctness (i.e. soundness) statements for the analysis.
These two examples show how the missing semantic foundations of the presented analysis approaches can lead to soundness issues in the core analysis design itself. These problems are further aggravated once additional stages are added to the analysis pipeline for increasing performance, since such additional stages are often not part of the correctness considerations.
Performance For performance reasons, it is often unavoidable to leverage wellestablished and optimized analysis frameworks or solvers. This leaves analysis designers with the challenge to transform their analysis problem into a format that is expressible and efficiently solvable within the targeted framework while preserving the semantics of the original problem.
ZEUS [22] makes use of off-the-shelf model checkers for LLVM bitcode and hence requires a transformation of smart contracts to LLVM bitcode. The authors describe this step to be a 'faithful expression-to-expression translation' that is semantics preserving, but omit a proof for this property. The paper itself later contradicts this statement: The authors report on LLVM's optimizer impacting the desired semantics. This indicates that the semantics of the LLVM bitcode translation does not coincide with the one of the intermediate language, since it would otherwise not be influenced by (semantics-preserving) optimizations.
The Securify tool [31] makes use of several preprocessing steps in order to make EVM bytecode amenable to dependency analysis: First it reconstructs the control flow graph of a contract and based on that transforms the contract to SSA form. The correctness of these steps is never discussed. Indeed we found Securify's algorithm for control flow reconstruction to be unsound: The algorithm fails when encountering jump destinations that depend on blockchain information. In such a case the control flow should be considered to be non-reconstructable since a jump to such a destination may result in a valid jump at runtime or simply fail due to a non-existing jump destination. Securify's algorithm however does not report an error on such a contract, but returns a (modified) contract that does not contain jumps. Such an unsound preprocessing step again impacts the soundness of the whole analysis tool since it breaks the assumption that the contract semantics is preserved by preprocessing.
Security Properties
The Ethereum blockchain environment opens up several new attack vectors which are not present in standard (distributed) program execution environments. This is in particular due to the contracts' interaction with the blockchain which is in general controlled by unknown parties and hence needs to be considered hostile. It is a partly still open research question what characterizes a contract that is robust in such an environment. A well-studied property in this domain is robustness against reentrancy attacks. We will focus on this property in the following to illustrate the challenges and pitfalls in proving a contract to be safe.
Reentrancy Attacks Reentrancy attacks became famous due to the DAO hack [1] in 2016 which caused a loss of over 60 Million dollars, and ultimately led to a hard fork (a change in the consensus to explicitly ignore this particular incident) of the Ethereum blockchain. The DAO was a contract implementing a crowd-funding mechanism which allowed users to invest and conditionally withdraw their invested money from the contract. An attacker managed to exploit a bug in the contract's withdraw functionality for draining the whole contract, stealing the money invested by other participants. We illustrate the main workings of this attack with a simplified example in Figure 2.
The depicted DAO contract has a global field bal which is a mapping from account addresses to the payments that they made so far. The two (publicly accessible) functions of the contract allow arbitrary entities to invest and withdraw money from the contract. If an account with address a calls the invest function, the money transferred with this invocation is registered in the bal mapping. Similar to msg.sender, Solidity provides the variable msg.value to access the value transferred with the currently executed (internal) transaction. The withdraw function when being called by a, will check the amount of money invested by a so far and in case of non-zero investments, transfer the whole amount of Ether (as recorded in bal[a]) back to a. This is done using Solidity's call construct for function invocations: it initiates a transaction to the specified address (here a) and allows for the specification of the value to be sent along (using .value()). The attack on the DAO contract can be conducted by an attacker that deploys a malicious contract Mallory to first make a small investment to the DAO contract ( 1 ) that they later withdraw ( 2 ). When the withdraw function of the DAO contract calls back to the sender (Mallory, 3 ), not only the corresponding amount of Ether is transferred, but also code of Mallory is executed. This is as in the case that not a specific (Solidity) function gets invoked with a contract call, the contract's fallback function (a function without name and arguments) is executed. Mallory implements this function to call the DAO's withdraw function ( 4 ). Since at this point the balance of Mallory in the bal mapping has not been updated yet, another value transfer to Mallory will be initiated ( 5 ). By proceeding in this way, Mallory can drain all funds of the DAO contract.
The depicted attack is an example of how standard intuitions from (sequential) programming do not apply to smart contracts: In Ethereum one needs to consider that an internal transaction hands over the control to a (partly) unknown environment that can potentially schedule arbitrary contract invocations.
Formalizing Security Properties While bug-finding tools typically make use of heuristics to detect vulnerable contracts, there have been two systematic studies that aim at giving a semantic characterization of what it means for a contract to be resistant against reentrancy attacks: The resulting security definitions are call integrity [15] and effective callback freedom [16].
Call integrity follows non-interference-style integrity definitions from the security community. It states that two runs of a contract in which the codes of the environment accounts may differ, should result in the same sequences of observable events (in this case outgoing transactions). In simpler words, another contract should not be able to influence how a secure contract spends its money. Intuitively, this property is violated by the DAO contract since an attacker contract can make the contract send out more money than in an honest invocation.
In contrast, effective callback freedom is inspired by the concept of linearizability from concurrency theory: It should be possible to mimic every (recursive) execution of a contract by a sequence of non-recursive executions. The DAO contract violates this property since the attack is only possible when making use of recursion (or callbacks respectively). After each callback-free execution, the investments mapping will be updated, so that a subsequent execution will prevent further withdraws by the same party.
While [15] shows how to over-approximate the hyperproperty call integrity by three simpler properties (the reachability property single-entrancy and two dependence properties), [16] does not indicate a way of statically verifying effective callback freedom, but proves this property to be undecidable. This leaves sound, and (efficiently) verifiable approximations an open research question. Checking Security Properties The state-of-the-art sound analyzers discussed so far do not build on prior efforts of semantically characterizing robustness against reentrancy attacks, but come up instead with own ad-hoc definitions.
Securify Securify expresses security properties of smart contracts in terms of compliance and violation patterns over data flow and control flow dependency predicates. In [31] it is stated that Securify supports the analysis of a property called 'no writes after call' (NW) which is different from (robustness against) reentrancy, but still aims at detecting bugs similar to the one in the DAO. The NW property is defined using an ad-hoc semantic formalism, and it states that for any contract execution trace, the contract storage shall not be subject to modifications after performing a CALL instruction. Intuitively, this property should exclude reentrancy attacks by preventing that the guards of problematic money transfers are updated only after performing the money transferring call. However, this criterion is not sufficient e.g., since reentrancies can also be triggered by instructions other than CALL. For proving the NW property, the compliance pattern demands that a CALL instruction may not be followed by any SSTORE instruction. We found this pattern not to be sufficient for ensuring compliance with the NW property (nor robustness against reentrancy). We will illustrate this using a variation of the DAO contract in Figure 3. This contract implements the exact same functionality as the one in Figure 2. The only difference is that the access to the balance mapping is handled via the library contract Lib. Ethereum actively supports the use of library contracts in that it provides a specific call instruction, called DELEGATECALL, that executes another contract's code in the environment of the caller. When calling Lib.write in the withdraw function, such a delegated call to the (external) library contract is executed. Executing write in the context of contract DAO will then modify DAO's storage (instead of the one of the Lib contract). In order to let the write and the get functionality access the right storage position (where DAO stores the bal mapping), these functions take as first argument the reference to the corresponding storage location. Same as the version in Figure 2, this contract is vulnerable to a reentrancy bug. Also, it violates the NW property: The storage of the contract can be changed after executing the call (when writing the bal) mapping. Still, this contract matches the compliance pattern (which should according to [31] guarantee the contract to satisfy the NW property), since it does not contain any explicit SSTORE instruction. This example illustrates how without a proven connection between a property and its approximation, the soundness of an analyzer can be undermined. This issue does not only constitute a singular case, but is a structural problem: There are counter examples for the soundness of 13 out of the 17 patterns presented in [31], as we detail out in [30].
ZEUS In [22], the property to rule out reentrancy attacks is only specified in prose as a function being vulnerable 'if it can be interrupted while in the midst of its execution, and safely re-invoked even before its previous invocations complete execution.' This definition works on the level of functions, a concept which is only present on the Solidity level, and leaves open the question what it means for a contract to be robust against reentrancy attacks. The authors distinguish between 'same-function-reentrancy' and 'cross-function-reentrancy' attacks, but do not consider cross-function reentrancy (where a function reenters another function of the same contract) in the analyzer. We found that without excluding cross-function reentrancy also single-function reentrancy cannot be prevented.
Consider the versions of the DAO contract depicted in Figure 4 that aim to prevent reentrancy using a locking mechanism. The global lock field tracks whether the withdraw function was already entered (indicated by value 1). In that case, the execution of withdraw throws an exception. Otherwise the lock is set and only released when concluding the execution of withdraw. While the two depicted contracts implement the exact same withdraw function, the first contract's function is vulnerable to a reentrancy attack, while the second one is safe. This is as the first contract implements a public switchLock() function that can be used by anyone to change the lock value. An attacker could hence mount the standard attack with the only difference that they would need to invoke the switchLock() function once before reentering to disable the reentrancy protection in line 5. Without exposing such functionality, the second contract is safe, since every reentering execution will be stopped in line 5.
This example shows that ZEUS' approach of analyzing functions in isolation to exclude 'same-function-reentrancy' is not sound. Another issue in the reentrancy checking of ZEUS is caused by the reentrancy property exceeding the scope of the analysis framework. For proving a function resistant against reentrancy attacks, ZEUS checks whether it is ever possible to reach a call when a function is recursively invoked by itself. However, the presented translation to LLVM bitcode only models non-recursive executions of a function. Consequently, the reentrancy property cannot be expressed as a policy (which could be translated to assertions in the program code), but requires to rewrite the contract under analysis to contain duplicate functions that mimic reentering function invocations. This contract transformation is not part of any soundness considerations. As a result, not only the previously discussed unsoundness due to the lacking treatment of cross-function reentrancies is missed, but it is also disregarded that Solidity's call construct is not the only way to reinvoke a function. Indeed there are several other mechanisms (e.g., direct function calls) that allow for the same functionality. Still, ZEUS classifies contracts that do not contain an explicit invocation of the call construct to be safe by default.
NeuCheck The NeuCheck tool formulates a syntactic pattern for detecting robustness against reentrancy attacks. The pattern checks for all occurrences of the call function whether they are followed by the assignment of a state variable. As discussed for Securify, the absence of explicit writes to the storage does not imply that the storage stays unchanged. Hence the example in Figure 3 would also serve as a counter example for the soundness claim of NeuCheck. Also, as discussed for ZEUS, call is not the only way of invoking another contract, what reveals another source of unsoundness in this definition. Furthermore, neither the security properties that the tool aims for are specified nor any justifications for the soundness of this syntactic analysis approach are provided.
How to Implement a Practical, Sound Static Analysis?
After exposing the problems that can arise when designing a practical, sound static analysis, we discuss how we tackled them in developing eThor and the underlying static analysis specification framework HoRSt [30]. We then present the elements we identified as essential for designing an automated sound analysis: A semantic foundation, sound abstractions, and a principled implementation.
Overview of eThor
eThor was preceded by an earlier prototype, called EtherTrust [14]. EtherTrust implemented the rules of a formal abstract semantics in Java ™ and exported them to z3 . While this design showed promising preliminary results, it turned out to be too inflexible for our purposes: Changes in the abstract semantics had to be tediously translated to Java ™ code; the non-declarative manner of specifying rules made them hard to write and review; and the lack of a proper intermediate representation made it difficult to implement custom optimizations before passing the verification task to z3 . These limitations are addressed by HoRSt [30], a dedicated high-level language for designing Horn clause based static analyses. HoRSt allows for the specification of Horn Clause based semantic rules in an declarative language and can apply different optimizations before translating them to z3 formulae. Thus, the semantics specification and the tool implementation are logically separated and systematic experiments with different versions of the semantics are possible. Additionally, optimizations can be implemented independently from specific semantics, improving the overall performance in a robust fashion.
eThor [30] combines HoRSt with an abstract EVM semantics, a parser to read EVM bytecodes, and a set of EVM-specific preprocessing steps, including the reconstruction of the control flow and the propagation of constants. It supports general reachability analysis and in particular allows for (soundly) verifying that a contract is single-entrant (following the definition in [15]).
What distinguishes eThor from prior work discussed in Section 3 is its well defined analysis specification that is supported by rigorous formal soundness proofs, as well as its principle implementation design. Prior works do not come with thorough formalization and proofs what ultimately leads to soundness issues in the analyzers, as we confirmed empirically. In contrast, eThor lives up to its theoretical soundness guarantees in an extensive evaluation while still being practical in terms of runtime and precision. In the following, we will discuss in detail the semantic foundations, modular design and implementation of eThor as well as its empirical performance evaluation.
Semantic Foundations
A formal soundness guarantee requires a formal semantics of the system under analysis. Such a semantics might be specified on paper [33] or in an executable fashion [15,18], but in any case has to be precise enough to unambiguously capture all relevant aspects of the system. While semantics defined in prose tend to be more readable, executable semantics lend themselves to automated testing or tooling (e.g., the generation of interpreters or symbolic debuggers [18]). eThor builds on the semantics presented in [15] which consists of a logical specification as well as an executable F * semantics that was rigorously tested for its compliance with the Ethereum client software.
Using a formal semantics, security properties can be precisely characterized. eThor bases its analysis for the absence of reentrancy attacks on the notion of single-entrancy [15]. Single-entrancy captures that the reentering execution of a contract should not initiate any further internal transactions. This property rules out reentrancy attacks and also contributes to the proof strategy for the more general call integrity property as detailed out in [15].
However, an executable semantics combined with precisely defined security properties alone does not yield a useful analysis tool. While these components allow experts to semi-automatically verify contracts (using frameworks such as [6,15,18,19]), automation generally requires abstractions to be feasible.
Sound Abstractions
A first step to reduce the complexity of the analysis problem, and hence to make it amenable to automation, is to over-approximate the target property. For eThor , we over-approximate the single-entrancy property by the simpler call unreachability [14] property. Call unreachability breaks down single-entrancy to a simple criterion on the execution states of a single contract, as opposed to reasoning about the structure and evolution of whole call stacks. Such overapproximations have to be proven sound -every program fulfilling the overapproximated property also has to fulfill the original property. A corresponding proof for single-entrancy is conducted in [14].
To further simplify the analysis task, the relevant parts of a contract's execution behavior need to be abstracted in a sound manner. In eThor for this purpose we devised an abstract semantics based on Horn clauses that we proved in [30] to soundly over-approximate the small step semantics in [15]. The abstract semantics simplifies and summarizes complex execution scenarios that may emerge due to the uncertain blockchain environment, as we exemplify in the following.
In the largely unknown blockchain environment it is infeasible to track constraints on all unknown values. Instead, following a standard technique in abstract interpretation, we enriched our domain of concrete computation values with a new value , signifying all possible values. This designated symbol overapproximates under-specified values while dropping constraints on them. Some computations, such as the SHA-3-computations or unaligned memory accesses in EVM, are due to their complexity over-approximated by in eThor .
Further, we abstract the initial invocation of a contract and its reentering executions as they might be scheduled by (unknown) contracts which are called during execution. In eThor we ignore the call stack up until the first execution of the analyzed contract, and assume a contract to be called in an arbitrary environment instead. Also, we only distinguish between the first execution of a contract under analysis in a call chain and a reentering execution of such a contract. In this way we collapse all reentering executions while modeling relevant storage invariants with a sophisticated domain-specific abstraction.
For an extended discussion of the abstractions used in eThor , including those for inter-contract calls, gas treatment, and memory layout, we refer to [30].
In summary, eThor provides a reliable soundness guarantee for the singleentrancy property, proving that a contract labeled secure by eThor satisfies single-entrancy. This guarantee stems from the soundness of the abstract semantics with respect to the rigorously tested small step semantics and from the proof that call unreachability (formulated in terms of the abstract semantics) soundly approximates single-entrancy. The soundness of the abstract semantics further enables the sound verification of arbitrary reachability properties that are expressed in terms of the abstract semantics. In particular this holds for functional contract properties phrased as pre-and postconditions: eThor can prove that a contract starting in a state satisfying the precondition is never able to reach a state that does not satisfy the postcondition.
Internal Horn Clause Representation
Horn Clause Transformations
Implementation Strategies
To arrive at a fast and stable analysis implementation, the analysis task is usually reduced to a known problem supported by performant and well-maintained solvers. This does not only save implementation time and helps performance, but it also adds an abstraction layer that facilitates reasoning. For HoRSt we decided to use z3 , respectively Constrained Horn Clauses over smt-lib's linear integer arithmetic fragment, as translation target. We chose z3 since it is a state-of-the-art solver for program analysis and the fragment suffices to formulate reachability properties.
Architecture In eThor , the generation of smt-lib code that models the abstract semantics of a contract is structured into separate and well-defined phases.
As can be seen in Figure 5, the input of eThor consists of a contract with reconstructed control flow. The bytecode of the contract is then parsed and constants are propagated within basic blocks. With this information, the abstract semantics (provided as a HoRSt specification) is instantiated to a set of Horn clauses which are, after several transformation steps, translated to smt-lib formulae.
Optimizations The performant analysis of real-world programs might require the usage of different optimizations, such as leveraging domain-specific knowledge in a pre-processing step. Such preprocessing may include propagation of constants [30,31], reconstruction of the control flow [30,31], computation of memory offsets [31], and pruning of irrelevant parts of the input [30,31]. As mentioned in Section 4.1, unsoundness introduced in any optimization or preprocessing step (e.g., by using an unsound decompiler) immediately affects the soundness of the whole analysis. It is hence crucial to formally reason over each step. In eThor the control flow graph reconstruction of a smart contract is realized by symbolically computing the destinations of all jump instructions based on a simplified version of the sound abstract semantics used for the later reachability analysis. Therefore, all soundness considerations from the full abstract semantics carry over to the preanalysis. Since this version of the semantics falls into the datalog solvable fragment as implemented by the Soufflé solver, we encoded this simple abstract semantics as a Soufflé program. To automate the generation of such preprocessing steps in the future we plan to extend HoRSt with Soufflé as additional compilation target.
Evaluation To ensure the correctness and perfomance of an analysis tool, it is inevitable to extensively and systematically test the tool implementation. To this end synthetic, well-understood inputs help to identify problems regarding precision, performance, and correctness early. These, however, may not be representative of the challenges that are found in real-world contracts. Data gathered from a real-world setting, on the other hand, might be of uncertain ground truth or too complex to give guidance in early stages of the development. In our experience, an automated test suite with corpus of synthetic and real-world inputs is a significant help while experimenting with different formulations and optimizations, as implementation bugs can be found already at an early stage.
For eThor we leveraged the official EVM test suite and our own propertybased test suite for assessing the correctness of of the abstract semantics and abstract properties. Out of 604 relevant EVM test cases, we terminated on 99%. All tests confirmed the tool's soundness and the possibility of specifying the test suite within eThor confirmed the versatility of our approach beyond reentrancy.
The correctness and precision of eThor for the single-entrancy property were assessed on a benchmark of 712 real-world contracts. Within a 10 minute timeout, eThor delivered results for 95% of the contracts, with all of them confirming soundness, and yielding a specificity of 80%, resulting in an F-meassure of 89%. These results do not only demonstrate eThor 's practicability on real-world contracts, but also clearly improve over the state-of-the-art analyzer ZEUS. When run on the same benchmark, ZEUS yields a specificity of only 11.4% (challenging its soundness claim) and a specificity of 99.8%, giving an F-measure of 20.4% 7
Future Challenges
To bring forward the robust design and implementation of sound static analyzers, we plan on extending HoRSt in multiple ways: We want to integrate HoRSt with proof assistants in order to streamline and partially automate soundness proofs. Further, we want to add support for additional compilation targets, and enrich the specification language and compilation to go beyond reachability analysis, and to support restricted classes of hyperproperties.
For the particular case of eThor , we want to improve the precision of the analysis, e.g., to include a symbolic treatment of hash values, and to enable the joint verification of multiple interacting contracts. Further, we strive to create a public benchmark of smart contracts exhibiting different security vulnerabilities, as well as mitigations. This would enable the community to systematically compare the performance, correctness, and precision of different tools.
Beyond that, we plan to transfer the presented techniques to other smart contract platforms, such a Libra, EOS, or Hyperledger Fabric, which exhibit domain-specific security properties and different semantics.
|
2020-10-29T09:08:47.564Z
|
2021-01-14T00:00:00.000
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{
"year": 2020,
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"oa_url": "http://arxiv.org/pdf/2101.05735",
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"pdf_src": "Arxiv",
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"extfieldsofstudy": [
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105743280
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pes2o/s2orc
|
v3-fos-license
|
The Thermodynamics and the Inverse Isotope Effect of superconducting PdH and PdD under pressure
We present in this paper the thermodynamics of superconducting PdH and PdD under pressure. We make use of a general method to calculate the thermodynamics under pressure within the Migdal-Eliashberg theory. We have considered the crystal lattice to be zincblende taking into account the experimental evidence for both PdH and PdD at temperatures below 55 K. We have studied, in particular, the changes induced by pressure in the critical temperature, $T_c$, in the specific heat jump at $T_c$, in the energy gap at $T=0K$, in the deviation function $D(t)$ and in the isotope effect coefficient, $\alpha$. We get a very good agreement with experiment where this data exist. This method represents a basis on which the thermodynamics of other hydrides under pressure can be calculated.
I. INTRODUCTION
In general, the volume of a solid is reduced when hydrostatic pressure is applied and as a result of the general stiffening of the lattice, the phonon spectrum shifts upwards to higher frequencies. This fact has an important influence on the properties of the solid. In particular, in superconductors, pressure induces changes in the whole thermodynamics. The critical temperature, T c , a very important parameter to decide on applications, can either increase or decrease. There is no criterion at present that would allow to predict the actual behaviour from the knowledge of the characteristics of the system at ambient pressure. For example, T c decreases under pressure for PdH, Pb, Sn and Hg 1-3 but increases in an important way in sulfur hydride, H 3 S. This system holds the record at present as a high-Tc superconductor (T c ≈ 200K) 4 .
Another important parameter is the isotope effect coefficient, it has been instrumental in understanding the mechanism responsible for Cooper pair formation in conventional superconductors. There is no general behavior for the isotope effect coefficient. In M gB 2 5 and the fullerides 6 , for example, the isotope coefficient is substantially reduced from the BCS value. In PdH, the compound of interest in this work, the isotope coefficient is large and negative (α ≈ −0.3) 7 and under pressure it diminishes steadily 3 . This is in contrast to H 3 S between 130 GPa and 140 GPa where the isotope coefficient goes down very quickly and then it goes further down but more slowly 8,9 .Further, 6 Li exhibits an unusually large isotope effect below 21 GPa and between 21 to 26 GPa, the superconducting isotope effect becomes inverse 10 . As a function of the doping concentration, the Oxygen and boron isotope coefficients in La 2−x Sr x CuO 4 11-13 and in M g 1−x Al x B 14 respectively also show a variation from the BCS value. In this paper, we will develop a method to calculate the changes under pressure in some important thermodynamic functions as the specific heat jump at T c , ∆C v (T c ), the energy gap at T = 0, ∆ 0 , the thermodynamic deviation function D(t), as well as the isotope effect coefficient, α, for PdH(D). This method uses the solution of the linearized Migdal-Eliashberg equations and the functional derivative of T c with the Eliashberg function.
In the past decade, numerous theoretical predictions have been made on the structure and superconducting behavior of stoichiometric and hydrogen-enriched hydrides with a variety of elements at high pressures. The surprisingly high observed T c in H 3 S raises the possibility that even higher transition temperatures may be attainable in hydrides. It is desirable to establish general characteristics that allow the understanding of the underlying mechanisms of high-T c superconductivity in hydrogen-rich materials 15 . To calculate in detail the behavior of both PdH and PdD under pressure constitutes a basis to identify these characteristics.
The rest of the paper is organized as follows. In the next section II, we present the theory and the basic equations that sustain our work. In section III we present the technical details. Section IV is devoted to our results and in a final section V, we draw our conclusions.
II. THEORY AND BASIC EQUATIONS
In this work, we use the solution of the linearized Migdal-Eliashberg equation (LMEE) 16,17 valid at T c . For an isotropic superconductor, the LMEE is Where∆ n is given by∆ Here ρ is the breaking parameter that becomes zero at T c . The frequencyω n is and iω n are the Matsubara frequencies defined on the imaginary axis, iω n = iπT (2n + 1) with n = 0, ±1, ±2 . . .. The e-ph coupling parameter λ nm is defined as λ nn is the known electron-phonon interaction parameter and µ * is the electron-electron repulsion parameter. The Eliashberg function is defined as where g qν,nm k+ q, k are the matrix elements of the electron-phonon interaction, ǫ k+ q,m and ǫ k,n are the energy of the quasi-particles in bands m and n with vectors k + q and k respectively. The functional derivative of the critical temperature with respect to the Eliashberg function is given as 18 .
To study the thermodynamics at temperatures below the critical temperature at each pressure we use the nonlinear Migdal-Eliashberg equation (NLMEE) From the solution of Eq.(7) the free energy difference ∆F (T ) between the superconducting and normal states can be computed. It is given by the critical magnetic field deviation function, D(t), given by where t = T /T c . The difference between the superconducting (S) and normal state (N) specific heat at constant volume, ∆C v , follows from the second derivative of the free energy
III. TECHNICAL DETAILS
Quite an amount of theoretical and experimental work has been done since the discovery of superconductivity in PdH and PdD. Most of the work done so far considers the rocksalt crystalline structure where the hydrogen atoms are located on the octahedral sites of the fcc lattice of Palladium 3,7,19-32 . However, there is another possibility. Neutron diffraction techniques have been employed to study the hydrogen-atom configuration in a single-phase sample of beta-PdH at several selected temperatures. The suggested low-temperature (T ≪ 55 K) structure of this compound is one which conforms to the space group R3m. This means that, depending on temperature (T ≪ 55 K), the hydrogen atoms move from their octahedral positions towards tetrahedral ones forming the zincblende structure [33][34][35][36] . For PdD something very similar occurs 37 . In a theoretical study, for pressures below 20 GPa at 0 K the stable structure was found to be zincblende 38 . So, following the facts just mentioned, in this paper we will consider PdH and PdD in the zincblende crystal structure that has not been yet considered.
A. Computational Details
The phonon spectra and the Eliashberg function where calculated within the density functional perturbation theory 39,40 as implemented in the Quantum -Espresso suite code 39 . We use the scalar relativistic pseudo potentials of Pardue and Zunger (LDA) 41 , a 150 Ry cutoff for the plane-wave basis and a 32 X 32 X 32 mesh for the BZ integration in the unit cell. For the force constants matrix we used a 16 X 16 X 16 mesh. The sum over k in Eq. (5) required a 72 X 72 X 72 grid.
To solve the LMEE, we used a cut-off frequency, ω cutof f = 10ω ph where ω ph is the maximum phonon frequency, to cut the sum over the Matsubara frequencies.
B. The phonon spectra and the Eliashberg functions In figure Fig. 1 we show our calculated phonon spectrum and the Eliashberg function for the zincblende crystal structure. The phonon spectrum and the Eliashberg function for the rocksalt structure are also shown, the data were taken from reference 32 . For both crystal structures the phonon spectrum and therefore, the Eliashberg function, present two distinct frequency regions. The acoustic region, from the Pd vibrations, is in general similar for both structures and it ranges from 0 to approximately 250 cm −1 . In the two structures the optical frequency region from the Hydrogen and Deuterium vibrations, is separated by a frequency gap from the acoustic ones. The hydrogen optical region is higher in frequency than that of the deuterium one in both structures. Further, the optical region in the zincblende structure is shifted to higher frequencies as compared to the rocksalt structure.
IV. THE THERMODYNAMICS
The thermodynamics of the PdH and PdD systems under pressure has not been yet addressed theoretically. As we mention above, several theoretical works have been done for these systems at zero pressure considering the structure to be rocksalt. Here we will consider the PdH and PdD in the zincblende crystal structure. In order to check the validity of this hypothesis, we investigate the pressure dependence of the critical temperature and of the thermodynamic properties of PdH and PdD.
The method that we use to calculate T c as a function of pressure uses the functional derivative of T c with the Eliashberg function as a function of pressure and requires only to know the critical temperature at an initial pressure and the knowledge of the Eliashberg function at each pressure of interest 42 . Once we get T c and µ * we can solve the NLMEE to calculate the thermodynamics at each pressure.
A. The superconducting critical temperature
To calculate T c we start at an initial pressure where the critical temperature is known, T c (P i ). From it we obtain µ * (P i ) by fitting it to T c (P i ) solving the LMEE. Then we get the functional derivative of T c with the Eliashberg function, α 2 F (ω, P i ), δTc δα 2 F (ω) using the formalism of Bergmann 18 and of Leavens 43 . At another pressure, say P i+1 , we calculate T c (P i+1 ) from 42 where ∆T c (P i ) is given by and Since we already know T c (P i+1 ) and α 2 F (ω, P i+1 ), we can solve the LMEE to get µ * (P i+1 ). The procedure can be repeated to get T c at each pressure of interest. Notice that we get T c and µ * as a function of pressure in a consistent way with Eliashberg theory. We do not use any approximation.
In Fig. 2, we show the critical temperature at several pressures for PdH and PdD calculated with the method described above. We use for PdH, T c = 8.8 K at 0 kbar 3 as our starting data. As it can be seen from Fig. 2 in the whole pressure interval that we have calculated the agreement with the experimental values is excellent. In the case of PdD, we took as our starting temperature T c = 11.05 K at 0 kbar 3 . In this case, the agreement with experiment for T c as a function of pressure in the calculated interval follows closely the same trend as the experimental values and grosso modo agrees with it. Table I shows the electron-electron repulsion parameter µ * which depends indirectly on the mass of the ions 43 . It is reduced considerably in PdD as compared to PdH. This means that the coulomb electron-electron repulsion weakens in PdD.
B. The deviation function
To calculate the thermodynamics from the formulas given above, we use the α 2 F (ω, P ), µ * and T c calculated at each pressure in the previous section to solve the nonlinearized Eliashberg equations (Eq. ??). Figure 3 shows plots of the calculated deviation function, D(t), for 0, 20 and 40 Kbar pressures. This function is useful to magnify the difference between weak and strong coupling systems. In a strong coupling system, such as Pb 44 , the deviation function is everywhere positive. We find for both systems, PdH and PdD, to be intermediate coupling and show a tendency to weaker coupling with increasing pressure between 0 and 20 kbar but remains being intermediate coupling until 40 kbar, the highest pressure considered. In a weak coupling system the deviation function is negative definite everywhere.
C. The specific heat jump at Tc
It is conventional when analyzing the specific heat to add γT , an approximation to the normal state electronic heat, onto ∆C and normalize to γT , i.e., C es = ∆C − γT . Table II shows our results for the dimensionless quantity C es /γT . For this quantity the BCS weak-coupling limit at T c is 2.43. Our results with increasing pressure, are approaching, but do not reach the BCS limit (the weak coupling) as is expected from the behaviour of the calculated D(t) in In order to get the quasiparticle density of states and the gap energy we perform an analytic continuation to real axis [cita]. The quasiparticle density of states follows from Which is zero below the gap energy ∆ 0 at each temperature. Figure 5 shows the quasiparticle density of states at T c . For both, PdD and PdH, the energy at which the density reaches its maximum is displaced at lower energies under pressure, but the peak increases more in PdH than in PdD. Finally, in figure 4 we show ∆ 0 (T ), defined by the equation In this case the BCS ratio 2∆ 0 /k B T c at T = 0 is 3.52. Our results ranges from 3.728 to 3.706 for PdH at 0 and 40 Kbar respectively and from 3.754 to 3.747 for PdD at similar pressures.
V. THE ISOTOPE EFFECT COEFFICIENT
BCS theory gives for a compound with several atoms the following formula for the partial isotope effect coefficients α i ≡ −d ln T c /d ln M i where M i is the mass of the different atoms in the compound and T c the critical temperature. The total isotope effect coefficient is given by the sum of the partial ones, namely α tot = i α i .
A deviation from α = 0.5 could be a fingerprint of a non-conventional mechanism. For multiband superconductors this deviation is expected to vary between the BCS value and zero as long as the intraband couplings are affected. It can, however, exceed the BCS value when interband effects are dominant 45 . A multiband approach has been proposed to explain the anomalous isotope effect in pressurized H 2 S 46 . Unconventional isotope effects can also be due to lattice fluctuations. This fluctuations have been identified in cuprates as due to the role of the lattice in the pseudogap formation 11,12 or to the modification of the electron-phonon interaction in systems such as M g 1−x Al x B 14 and Y B 2 47 . It has also been shown that systems with anomalous isotope coefficients have anomalies at Lifshitz transitions 13,48,49 . The effects of the zero point motion has been proposed to explain the anomalous isotope effect in H 3 S 50 .
In palladium hydride the replacement of hydrogen by deuterium results in a higher superconducting temperature and in an anomalous isotope effect (α ≈ −0.3). For this system, most of the mechanism that have been proposed to explain it attribute the inverse isotope effect to vibrational effects of Hydrogen and Deuterium, such as anharmonicity 27,28,32 or to the zero-point motion 29 , both of which have an effect on the electron-phonon coupling without reaching to a satisfactory explanation of this phenomenon. For example, Errea et. al. 32 study the anharmonicity of the hydrogen vibrations within the rocksalt crystal structure and found the value of the isotope coefficient to be α = −0.38, however their calculated critical temperatures does not match the experimental ones (see Tab. III). In the work of Jena et. al. 29 they show that the electronic structure of PdH and of PdD is influenced by the zero-point vibration of hydrogen and deuterium. Even more, Yussouff et. al. 20 included anharmonicity on top of the zero-point effects and found the isotope coefficient to be α = −0.3926.
More recently, S. Villa-Cortés and R. Baquero [auto cita] have been proposed a different approach to explain the inverse isotope effect in palladium hydride. They considered the vibrational modes to be harmonnic and the crystal structure to be zincblende. In their approach they split the isotope coefficient into two contributions. One that comes from the change in the electron-phonon interaction, namely α el−ph , and other that comes from the change in the electron-electron interaction, namely α el−el . The last one gives us information on how the screening of the electron-electron interaction is affected by the isotope substitution. At 0 pressure they found the electron-phonon contribution to be α el−ph = 0.0556 and the electron-electron contribution to be α el−el = −0.369, this gives a total isotope coefficient α = −0.3134 in remarkable agreement to the experimental value (see Tab. III). For a detailed account of this approach see [autocita]. Here we extend our previous work to include the effects of pressure in the isotope effect.
In Fig. 6 we show our results for the isotope coefficient under pressure. We procced in two ways. First we use the calculated T c under pressure for PdH and PdD (see Fig. 2) to calculate the isotope coefficient from α(P ) = −d ln T c (P )/d ln M . In general it tends to diminish under pressure. Second, we calculated both contributions for the isotope effect, the electron-electron and the electron-phonon interactions. In general α T otal follows closely the same trend of the calculated isotope effect under pressure. In general the magnitude of the inverse isotope coefficient can be explained by the change in the electron-electron interaction, α el−el , but the slight variation under pressure comes from the variation of the electron-phonon interaction under pressure.
VI. CONCLUDING REMARKS
In conclusion, we have used a novel method 42 to calculate the superconducting thermodynamics under pressure within the Migdal-Eliashberg theory. We have found that if we consider the zincblende crystal structure as the proper one for PdH and PdD at low temperatures (T ≪ 55 K) in agreement with experiment, the experimental values of the critical temperature and of the inverse isotope effect under pressure are well reproduced. This method represents a basis on which the thermodynamics of other hydrides under pressure can be calculated. We have found that the enhancement of the critical temperature, and then the inverse isotope effect, can be explained by taking the screening of the electron-electron interaction by the phonons into account. Further we have calculated the dependence under pressure of the specific heat at T c , the energy gap at T = 0, the deviation function D(t) and the density of the quasiparticle states. As a general remark we can say that the changes induced by pressure in the thermodynamics tends to weaken the system.
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2018-03-22T23:54:12.000Z
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2017-08-30T00:00:00.000
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Effect of Plant Density and Hand Weeding on Weed Control and Yield of the Vegetable Corn
Weed infestation has an adverse impact on the yield of vegetable corn. This study, therefore, aimed to investigate the effects of plant density and hand weeding on controlling weeds and yield of vegetable corn. The experiments were conducted in the field condition in a randomized complete block design with three replications. The planting densities were 79,365 plants ha (D1); 92,593 plants ha (D2); 111,111 plants ha (D3); and 138,889 plants ha (D4). The hand weeding treatments were no weeding (NW), hand weeding once at 3-4 leaf stage of vegetable corn (HW1), and hand weeding twice at 3-4 leaf and 8-9 leaf stages of vegetable corn (HW2). The results showed that the highest planting density combined with hand weeding was generally effective in controlling weeds. Furthermore, the increase in planting density combined with hand weeding significantly improved the physiological traits, which consequently increased the cob yield. The yield was optimum at D3 combined with hand weeding once. Thus, the results suggested that the optimum yield of vegetable corn could be obtained at a planting density of 111,111 plants ha combined with hand weeding once at 3-4 leaf stage, an increase of the cob yield by 2.01 tons ha.
Introduction
Corn (Zea may L.), which is planted in 1.03 million ha producing around 4.87 million tons in 2018, is the second most important food crop in Vietnam after rice, (FAO, 2020). Corn is a multipurpose crop (e.g., used as human food, animal and poultry feed, and in industrial products) (Bibi et al., 2010). Corn can be planted and harvested young to take advantage of its fresh, sweet, and tender ears for vegetable purposes, called vegetable corn. Vegetable corn is a very young cob with undeveloped seeds (Nguyen et al., 2009), which has flavour and is rich in folate, providing 13% potassium, 14% B6 riboflavin, 17% vitamin C, and 11% fibber every four ounces (Fakir & Islam, 2008). With the increasing interest in healthy foods worldwide, vegetable corn is a promising food due to its quality and safety.
There are several production constraints of corn, i.e. unavailability of seeds of improved and high-yielding varieties, high cost of agricultural practices and inputs, and susceptibility to various pathogens and insects, etc. Weed infestation is also considered an important constraint to corn production as weeds generally compete with the crop for light, nutrients, water, space, and allelopathy which reduce the yield and market value of the crop (El-sobky & El-naggar, 2016;Khanh et al., 2018). Worldwide, yield losses in maize due to weeds are estimated to be approximately 37% (Kumawat et al., 2019) and reach as high as 90% (Dalley et al., 2006). Although various weed control measures (e.g., hand weeding, mechanical weeding, and the use of herbicides) are effective against weed infestation in corn, each control measure has its limitations. For instance, herbicidal control needs repeated applications due to the reemergence of the weeds from the soil seed bank which may cause herbicide resistance in the long run with the same mode of action herbicides. Furthermore, excessive use of herbicides allows for the accumulation of toxicity in agricultural products, which has negative impacts on human health, soil, and water systems, and causes damages to biodiversity (Al-Samarai et al., 2018). Thus, there is a great demand for environmentally friendly approaches to weed management as alternatives to chemical weed control to maximize sustainability in agricultural production.
In this regard, hand weeding is still an effective conventional weed control method, if done properly. Dutta et al. (2016) showed that hand weeding significantly reduced weed dry weight and increased weed-control efficiency (88.3%), leading to increased nutrient uptake, thereby increasing the yield of vegetable corn by 69.9% over the weedy check. Kotru (2012) also showed that hand weeding was as effective as herbicide application, which increased vegetable corn yield attributes and total yield (by 33% and 29.2%, respectively) and reduced weed biomass and N-removal by weeds, relative to the weedy check. Furthermore, Kumar et al. (2017) also showed that hand weeding resulted in the lowest weed density and dry weight of all major weed species, followed by other herbicide applications, resulting in a significant increase in rabi maize yield. Overall, these studies generally showed that hand weeding is an effective nonchemical approach in weed management.
The frequency of hand weeding also greatly influences maize growth and yield but may not be economically feasible if yield largely depends on the timing of weed, especially at the critical stages of crop development (Abouziena et al., 2007). Vu & Ha (2015) reported that hand weedings at 3-4 leaf and 8-9 leaf stages of maize significantly lower weed density and weed biomass than single hand weeding at 3-4 leaf stage. Abouziena et al. (2007) indicated that there were no significant differences between hand weeding twice (at 3 and 6 weeks after sowing) or thrice (at 2, 4, and 6 weeks after sowing) in controlling weeds as well as yield. In addition, manual weeding is not always feasible due to the lack of labour in the proper time, higher labor cost, and unworkable condition of the labor (Kumar et al., 2017). Therefore, it is necessary to integrate different weed control techniques to achieve effective and sustainable weed control in vegetable corn production.
Planting corn at higher densities may increase its competitive ability upon the weeds. Higher planting densities have been used to suppress weeds and to increase the yield of the crop (Shapiro & Wortmann, 2006). In addition, optimum planting density plays an important role in efficient utilization of resources and the competitive balance between weeds and maize plants (El-Sobky & El-Naggar, 2016). Marín & Weiner (2014) and Youngerman et al (2018) found a strong negative relationship between corn density and weed biomass at different experimental sites when weed biomass would decrease as corn density increased. An increase in crop density could lead to the enhancement of the collective shade of weeds by the crops which suppressed weeds growth and prevented them from reaching the crop's initial size advantage (Marín & Weiner, 2014). Nguyen et al. (2009) recorded the statistically higher marketable yield of baby corn at the highest plant population (167,000 plants ha -1 ). Moreover, Ghosh et al. (2017) revealed that baby corn yield reached the highest value at the density of 100,000 plants ha -1 and slightly decreased at the density of 120,000 plants ha -1 . According to Eskandarnejad et al. (2013), with increasing the plant population from a specific level, competition for light and nutrients became intense and the growth of plants was declined and crop yield would be reduced as a result.
Considering the above mentioned facts and views, the experiment was conducted to study the effect of hand weeding and planting density on the suppression of weeds and the yield of vegetable corn.
Materials and Methods
The experiment was conducted in spring cropping season in 2020 at the experimental fields of the Faculty of Agronomy, Vietnam National University of Agriculture. The seed of vegetable corn used in the experiment was LVN23, a good domestic variety and found more popular in the Vietnamese market (Nguyen, 2016). The seeds were produced by the Vietnam National Maize Research Institute.
The experiment was laid out in a Randomized Complete Block Design (RCBD) comprising 12 treatments with three replications. The planting density included four levels: 79,365 plants ha -1 with 60cm x 21cm (D1); 92,593 plants ha -1 with 60cm x 18cm (D2); 111,111 plants ha -1 with 60cm x 15cm (D3); and 138,889 plants ha -1 with 60cm x 12cm (D4). The hand weeding treatments were: no weeding (NW), hand weeding once at 3-4 leaf stage of vegetable corn (HW1), and hand weeding twice at 3-4 leaf and 8-9 leaf stages of vegetable corn (HW2). The selected planting densities were based on the previous study of Nguyen (2016), while the hand weeding was based on the previous study of Vu & Ha (2015).
One seed was sown and raised in a black plastic bag, after seven-day-old seedlings were transplanted into the field. Hand weeding was done by the use of a hand hoe. The fertilizer doses of 120 kg N ha -1 , 100 kg P2O5 ha -1 , and 100 kg K2O ha -1 were applied. All of the P2O5 was applied as basal during transplanting, while the N and K2O were split equally into three applications during transplanting, at 3-4 leaf, and at 8-9 leaf stages of vegetable corn.
Weed species found during the experiment were identified at harvest from the non-weeding plot. Weed density (WD) was randomly determined within three quadrants (0.25m 2 ) placed from each plot at three growth stages, including 3-4 leaf stage, 8-9 leaf stage, and harvest stage. The weeds within each quadrant were oven-dried at 80 o C for 48h and weighed to determine dry mass at the harvest. Weed control efficiency (WCE) was calculated based on the weed dry matter recorded in each treatment at the harvest stage of the crop using the formula suggested by Mani et al. (1973) as follows:
WCE (%) = [(Weed dry weight in the unweeded (control) plot -Weed dry weight in the treatment plot)/Weed dry weight in the unweeded (control) plot] * 100.
The physiological traits such as leaf area index (LAI) as well as dry mass of the crop were measured at the 7-9 leaf stage, tasseling stage, and last harvest stage using five-plant samples. The shoots were cut from the base and ovendried at 80 o C for 48h. The tassels and cobs were separated and weighed separately from the rest of the shoots.
Leaf area index (LAI) was calculated as the leaf area divided by the ground area, where leaf area was calculated by the length x the maximum width x 0.75 x the total number of leaves (Nguyen Van Loc & Nguyen Van Minh, 2019). At the harvest stage, the yield components such as the number of cobs were counted by averaging the number of harvested cobs from 10 randomly chosen plants of each experiment plot. Cob weight and cob yield were measured by weighing and adding up the total weight of young cobs that had green husk (Nguyen et al., 2009).
Statistical analysis
The data were subjected to ANOVA for the planting density, hand weeding, interaction of planting density and hand weeding, and replication using IRRISTAT 5.0. The treatment mean differences were analyzed using least significant difference (LSD) at the 5% significance level.
Weed species
Weed species found in the experimental fields during the cropping period are presented for each weed group (i.e., grass, sedge, and broadleaf) (Suk et al., 2005). The weeds were Eleusine india, Cynodon dactylon, Echinochloa colona, and Leptochloa chinensis under grasses, Cyperus rotundus under sedges, and Eclipta alba, Rorippa indica, Portulaca oleracea, Physalis angulata, and Alternanthera under broadleaf ( Table 1). Spring season was characterized by incessant rain which resulted in higher weed density, especially Cynodon dactylon, Eleusine india, and Leptochloa chinensis (data not shown). Vu & Ha (2015) also reported that Cynodon dactylon, Eleusine india, and Leptochloa chinensis were the prevalent weeds in the maize field.
Weed density
The effects of plant density and hand weeding on the weed density at 3-4 leaf stage, 8-9 leaf stage, and harvest stage of vegetable corn are presented in Figure 1. The results showed that the response of weed density to the plant density as well as the hand weeding was not significantly different (P≤ 0.05) at the 3-4 leaf stage. However, at the 8-9 leaf stage and harvest stage of vegetable corn, the weed density was significantly influenced (P≤ 0.05) by plant density (Figure 1a). The weed density decreased significantly with increasing plant densities from D1 to D4. This can be explained as an initial size advantage of crops in competition with annual weeds which is favored by the increased degree of size asymmetric competition caused by an increase in the crop density (Weiner et al., 2001).
In addition, there was a significant difference in weed density between no weeding and hand weeding at the 8-9 leaf stage and the last harvest stage of vegetable corn. In comparison with non-weeding treatment, hand weeding treatments significantly decreased weed density (Figure 1b). Khan et al. (2012) reported that hand weeding was the most effective way to control the weed density in the maize field. Furthermore, our results showed that hand weeding twice at 3-4 leaf and 8-9 leaf stages of vegetable corn significantly decreased the weed density in comparison with hand weeding once at 3-4 leaf stage of vegetable corn. These results confirmed the finding of a previous study that hand weeding twice significantly decreased weed density as compared with hand weeding once (Vu & Ha, 2015). At the 3-4 leaf stage of vegetable corn, weed density was not significantly influenced (P≤ 0.05) by the interacting effects of plant density and hand weeding. However, there were significant differences in weed density among the combination treatments at the 8-9 leaf stage and the harvest stage of vegetable corn ( Table 2). The combination of the higher plant densities (111,111 and 138,889 plants ha -1 ) and hand weeding once or twice resulted in lower weed density compared to the combination of the lowest plant density (79,365 plants ha -1 ) and no weeding. The results suggested that higher plant density combined with hand weeding increased weed suppression. showed that weed dry weight differed significantly (P≤ 0.05) amongst the plant density treatments as well as hand weeding treatments. The higher plant densities had a lower weed dry weight compared to the lowest plant density. The results were consistent with the work of Ahmed et al. (2014) that weed dry weight decreased significantly with an increase in the seedling rate of crop. In terms of hand weeding treatment, there was a significant difference in weed dry weight between weedy check and hand weeding. Hand weeding once and twice significantly decreased weed dry weight in comparison with the weedy check. Similar findings were presented in the study of Vu & Ha (2015). In addition, the ANOVA analysis showed significant two-way interactions between plant density and hand weeding on weed dry weight ( Table 1). Weed dry weight was the lowest at the combination of higher plant densities and hand weeding twice.
Weed control efficiency (WCE)
The efficiency of treatments on the control of weeds in terms of dry weight in comparison with the control plot is shown in Table 2. The adoption of different plant densities and hand weeding controlled the weed efficiency as evident from the weed control efficiency, which ranged from 40.7 to 86.2%. Weed control efficiency (WCE) increased under hand weeding twice in comparison with hand weeding once, regardless of plant densities. In addition, WCE tended to increase with increasing plant density under both hand weeding once and hand weeding twice conditions. These results are in accordance with the findings of Madavi et al. (2017) who reported that higher plant density resulted in higher WCE compared to the lower density. Furthermore, the higher WCE was attributed to the lower weed dry weight. A similar finding was reported in the study of Deshpande et al. (2006). Moreover, Gnanasoundari (2013) also suggested that more reduction of weed dry weight by reducing the weed density in the treatments resulted in higher WCE.
Leaf area index (LAI) of vegetable corn
Leaf area (LA) is one of the most important growth traits as it influences the capture of solar radiation which is important for the rapid growth of plants (Valentinuz & Tollenaar, 2006). Thus, plant density of corn per unit area may affect the leaf size and its overall canopy closure which may aid in the competitive ability of corn against weeds. Vegetable corn grown at 92,593 plants ha -1 and 111,111 plants ha -1 had greater LA than 138,889 plants ha -1 (data not shown). The smaller LA at the higher plant density was possibly due to the intra-specific competition between the corn plants for growth resources (Murphy et al., 1996). However, in terms of leaf area index (LAI), analysis of the data showed that LAI increased significantly with increasing plant density, which was observed in all measured growth stages of vegetable corn (Figure 3b). It was previously shown that increasing plant density significantly increased LAI of maize (Nguyen et al., 2009;Timlin et al., 2014;Lykhovyd et al., 2019;and Han et al., 2020). The increased LAI achieved more light interception and photosynthetic assimilation per unit land area, thus increasing maize biomass yield (Xu, 2018).
In terms of hand weeding, there was no significant difference in LAI between weedy check and hand weeding plots at the 7-9 leaf stage of vegetable corn (Figure 3a). However, at the other measured stages, there was a significant difference in LAI between weedy check and hand weeding. The highest LAI was found in hand weeding twice and the lowest LAI was in weedy check condition, but LAI was not significantly different between hand weeding once and hand weeding twice as well as between hand weeding once and weedy check.
The experimental results showed significant differences in the interacting effects of plant density and hand weeding on LAI at all of the observed stages (Table 3). At the 7-9 leaf stage of vegetable corn, the highest LAI was obtained from the highest plant density, regardless of hand weeding conditions. However, at both the tasseling stage and harvest stage, the highest LAI value was at the highest plant density combined with hand weeding once and twice treatments (D4HW1 and D4HW2).
Dry matter weight of vegetable corn
The results of Figure 3 and Table 3 showed that the dry matter weight of vegetable corn increased over time under all treatments. The maximum dry matter weight was observed at the harvest stage (Figure 3 and Table 3).
At the 7-9 leaf stage, there was no significant difference in dry matter weight of vegetable corn between no weeding and hand weeding as well as among different plant densities (Figure 4). However, at the tasseling stage and harvest stage, hand weeding once and twice remarkably increased dry matter weight of vegetable corn in comparison with no weeding. The highest dry matter weight of vegetable corn was observed in the hand weeding twice plots at both the abovementioned growth stages, while the lowest dry matter weight of vegetable corn was found in the no weeding plots. Similarly, in terms of plant density, the dry matter weight of vegetable corn increased significantly with increasing plant density at both of these measured stages. Khan et al. (2017) and Han et al. (2020) also reported that increasing plant density significantly increased shoot dry biomass of corn, which may be due to the reduced competition from the weeds at the higher densities. The interacting effects of hand weeding and plant density on dry matter weight of vegetable corn are shown in Table 3. There were significant differences in the dry matter weight among the treatments at all of the measured stages. Higher plant density significantly increased the dry matter weight compared to lower plant density, regardless of the hand weeding. The highest plant density combined with hand weeding once and twice had the highest dry matter weight at the tasseling stage and harvest stage.
Yield components and yield of cobs
The results showed that there was no significant difference in the cob weight among plant densities, wheares there was a significant difference in the number of cobs ( Table 4a). The maximum number of cobs was obtained at the densities of 111,111 and 138,889 plants ha -1 , while the minimum number of cobs was observed at the density of 79365 plants ha -1 . Therefore, increasing plant density increased the number of cobs, which then led to an increase in cob yield. Higher plant densities (92,593; 111,111 and 138,889 plants ha -1 ) resulted in significantly higher cob yield compared to the lower plant density (79,365 plants ha -1 ), but there was an insignificant difference in cob yield between the density of 111,111 and 138,889 plants ha -1 . Similar results were observed in the studies of Nguyen et al. (2009) and Khan et al. (2017). This might be explained by the fact that corn yield can be boosted with increased planting density, however the excessive density can also cause yield loss due to intra-specific competition (Shapiro & Wortmann, 2006). In addition, Matta et al. (1990) reported that moderately high densities may be useful to minimize intraspecific competition between the crop plants and wisely suppress weeds to achieve higher grain production. Similar results in terms of the effects of hand weeding were observed for the number of cobs, cob weight, and cob yield. Hand weeding once and twice remarkably increased cob yield in comparison with no weeding; however, there was no significant difference in cob yield between hand weeding once and hand weeding twice.
Statistical analysis showed significant effects of plant density and hand weeding on the number of cobs and cob yield. There were also significant two-way interactions between plant density and hand weeding on the examined traits ( Table 4b). The maximum number of cobs was observed in the plots with the density of 138,889 plants ha -1 combined with one and two hand weeding treatments and was followed by the density of 111,111 plants ha -1 combined with one and two hand weeding treatments, whereas the minimum number of cobs was noted in the lowest plant density combined with no weeding treatment. There was no significant difference in the cob weight among treatments, but there was a significant difference in the number of cobs, which led to the significant effect of all examined treatments on cob yield (P≤ 0.05). The cob yield was the lowest at the density of 79,365 plants ha -1 combined with no weeding treatment (1.35 tons ha -1 ) and the highest at the density of 111,111 plants ha -1 combined with hand weeding twice (2.23 tons ha -1 ) treatments. However, there was no significant difference in cob yield among the density of 111,111 and 138,889 plants ha -1 combined with hand weeding once and twice (D3HW1, D4HW1, D3HW2, and D4HW2, respectively). The results suggested that, to decrease the labour in manual weeding, the vegetable corn should be planted at the density of 111,111 plant ha -1 combined with hand weeding once, which led to an increase in the cob yield due to the suppression of the growth of weeds.
Note: Columns with the same letter within each treatment are not significantly different at P>0.05. D1,D2,D3,and D4: planting density at 79,365;92,593;111,111;and 138,respectively. HW,HW1,and HW2: no weeding, hand weeding once, and hand weeding twice, respectively. Table 4a presents the effects of plant density and hand weeding on the green biomass of vegetable corn. The results showed that green biomass differed significantly (P≤ 0.05) among the plant density treatments as well as hand weeding treatments. The higher plant densities had higher green biomass compared to the lowest plant density. Similar results were observed in the studies of Nguyen et al. (2009) who indicated that increasing plant density significantly increased green fodder yield for all of the hybrid vegetable corns examined. In terms of hand weeding treatment, there was a significant difference in green biomass between weedy check and hand weeding. Hand weeding once and twice significantly increased green biomass compared to the weedy check. However, there was no significant difference in green biomass between hand weeding once and twice. The result of ANOVA analysis showed Table 4b). The maximum green biomass was observed in the plots with the density of 138,889 plants ha -1 combined with one and two hand weeding treatments (32.22 and 32.36 tons ha -1 , respectively), whereas the minimum green biomass was noted in the lowest plant density combined with no weeding treatment (18.78 tons ha -1 ).
Conclusions
The results showed that increasing the plant density combined with hand weeding once and twice suppressed the growth of weed, which then led to an increase in the yield of vegetable corn. The lower weed density and weed dry weight s were found at the higher plant density combined hand weeding twice treatment. Similarly, higher plant density combined with hand weeding resulted in higher cob yield compared to the lowest plant density combined with no weeding treatment. However, there was no significant difference in cob yield among the densities of 111,111 and 138,889 plants ha -1 combined with hand weeding once and twice. Therefore, the results suggested that under the lack of manual weeding labour condition, vegetable corn should be planted at the high density of 111,111 plants ha -1 combined with hand weeding once at 3-4 leaves, which suppressed the growth of weed and obtained the high cob yield (2.01 tons ha -1 ).
|
2021-08-04T00:04:07.603Z
|
2020-12-31T00:00:00.000
|
{
"year": 2020,
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"oa_url": "https://vjas.vnua.edu.vn/index.php?journal=vjas&op=download&page=article&path[]=205&path[]=78",
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|
49707795
|
pes2o/s2orc
|
v3-fos-license
|
First Molecular Diagnosis of a Patient with Unverricht-Lundborg Disease in Korea
Unverricht-Lundborg disease (ULD) is a form of progressive myoclonus epilepsy characterized by stimulation-induced myoclonus and seizures. This disease is an autosomal recessive disorder, and the gene CSTB, which encodes cystatin B, a cysteine protease inhibitor, is the only gene known to be associated with ULD. Although the prevalence of ULD is higher in the Baltic region of Europe and the Mediterranean, sporadic cases have occasionally been diagnosed worldwide. The patient described in the current report showed only abnormally enlarged restriction fragments of 62 dodecamer repeats, confirming ULD, that were transmitted from both her father and mother who carried the abnormally enlarged restriction fragment as heterozygotes with normal-sized fragments. We report the first case of a genetically confirmed patient with ULD in Korea.
INTRODUCTION
Unverricht-Lundborg disease (ULD) (EPM1, OMIM 254800) is the "purest" type and the most common form of progressive myoclonic epilepsy (PME). 1,2 It is characterized by symptoms of myoclonic jerks and generalized tonic-clonic seizures (GTCSs), which are caused by photic or touching stimulus and usually occur at the age 6−16 years. 3 This disease is an autosomal recessive disorder, and the CSTB gene encoding cystatin B, a cysteine protease inhibitor, is the only gene known to be associated with ULD. 4 The 12-nucleotide dodecamer repeat (5'-CCC-CGC-CCC-GCG-3') expansion in the promotor region of CSTB accounts for approximately 90% of cases worldwide and 99% of the EPM1 cases in Finland. 5 Loss of lysosomal linkage and an endogenous neuroprotective role of CSTB are known as the main pathological mechanisms of the disease. 6 Here, we report a patient with ULD and her families diagnosed by Southern blot analysis. It is the first case of a genetically confirmed ULD in Korea. This study was approved by the Institutional Review Board of Severance Hospital.
CASE REPORT
A 20-year-old woman presented with both uncontrolled hand tremor and seizure attacks. The perinatal and infancy periods of the patient were not eventful, and early developmental milestones were normal. She developed myoclonic movements of the whole body at the age of 10 years. The myoclonic movements became aggravated gradually with more severe involvement of both upper limbs. They occurred frequently every day, more severe on settling into sleep and awakening, and their severity was reduced after turning off the light. They were provoked upon sudden movements, such as standing up. Also, tremulous movements of both hands (small amplitude jerks) occurred persistently. They increased in severity as the hand moved closer to its target; therefore, detailed hand movements, such as when operating a cell phone, were diffi-cult. Also, she could not run because of provoked myoclonic movements since the age of 13 years. Dysarthric speech and gait disturbance were recognized around that time and became gradually aggravated. She had developed mild intellectual disability since the age of 10 years. The results of neuropsychological tests (Korean-Wechsler Intelligence Scale for Children-III) taken at the age of 15 years showed mild mental retardation [full-scale intelligence quotient (IQ)=65, verbal IQ=61, and performance IQ=75]. Also, she showed emotional liability. GTCSs following a series of myoclonic movements occurred for the first time, a half year after the occurrence of myoclonus. GTCSs were mainly provoked by bright lights or emotional stress and occurred once a week. At that time, she began to receive treatment with antiepileptic drugs (AEDs). Her seizures, including myoclonic movements and GTCSs, were aggravated with oxcarbazepine, carbamazepine, and lamotrigine treatment, and significantly improved, particularly in GTCS, with valproic acid treatment. Frequency of GTCS was reduced to three or four times a year.
At the age of 20 years, she was admitted to our hospital due to aggravation of GTCS control (two times per month). She had been treated with levetiracetam (2000 mg/day), valproic acid (500 mg/day), clonazepam (0.75 mg/day), baclofen (20 mg/day), and lorazepam (1.5 mg/day). On neurologic examination, the almost continuous, tremulous movements of both hands were observed. Relatively large amplitude myoclonic movements occurred intermittently, particularly when moving suddenly, and were also provoked with arm stretching and hand extension. Also, she showed a mild degree of dysarthria, dysmetric limb ataxia, and truncal ataxia on tandem gait. The patient's 15-year-old sister had normal cognition and no neurologic symptoms. Her father had experienced several seizures after head trauma in his 30s. After taking AEDs for three years, seizures no longer developed. Also, this family had no ancestors from other countries. Other family history was unremarkable ( Fig. 1).
Continuous video-electroencephalography (EEG) was performed. The EEG showed intermittent generalized sharp waves with posterior emphasis, although the myoclonic movements, including the almost continuous, small amplitude jerks, were not time-locked to EEG discharges. Background rhythm on EEG was normal with well-regulated 10-Hz posterior dominant rhythm. Photic stimuli could not provoke electrical and clinical seizure. Brain magnetic resonance imaging and magnetic resonance spectroscopy revealed no abnormalities. Somatosensory-evoked potential and visual-evoked potential tests showed normal latencies, shapes, and amplitudes.
CSTB gene-associated dodecamer repeat expansion was analyzed by Southern blot analysis after obtaining informed consent. Genomic DNA was extracted from peripheral blood leukocytes using the Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Southern blot analysis was performed at Athena . The patient showed only abnormally enlarged restriction fragments of 62 dodecamer repeats, confirming ULD, that were transmitted from her father and mother who carried the abnormally enlarged restriction fragments as heterozygotes with normal-sized fragments (Fig. 2). Her sister, also a heterozygote, carried the expanded fragment as a carrier.
We changed AEDs for seizure control. Upon discontinuing clonazepam, baclofen, and lorazepam, the patient was treated with valproic acid (1250 mg/day), levetiracetam (2000 mg/day), clobazam (10 mg/day), and zonisamide (300 mg/day). GTCS did not recur in the following 9 months. Further, her small amplitude jerks of both hands much decreased.
DISCUSSION
The present patient's clinical manifestations were compatible with PMEs related to several etiologies. ULD has rather milder symptoms than other PMEs. Our patient experienced gradually worsening, but mild neurological symptoms and preservation of normal EEG background activity. Therefore, these clinical features and the disease course of our patient were regarded as the typical clinical manifestations of ULD. If there is a severe progression in cognitive or visual symptoms, other forms of PME, such as myoclonic epilepsy with ragged red fibers, neuronal ceroid lipofuscinosis, Lafora's disease, and sialidosis, should be suspected. 7 Recent study suggests that longer CSTB expansion mutations have a modulating effect on age at disease onset, myoclonus severity, and cortical neurophysiology in ULD. 5 Abnormal CSTB gene-associated dodecamer repeat expansion was confirmed by Southern blot, leading to the diagnosis of ULD. Normal alleles of the CSTB gene contain two to three dodecamer repeats and full-penetrance alleles of more than 30 repeats. 8 The size of fully expanded alleles and GC-rich sequences in the dodecamer repeat region make conventional PCR extremely difficult and require laborious Southern blot analysis. More convenient diagnostic testing (the dodecamer repeat analysis method, such as repeat PCR assay) needs to be developed in the future.
Although the prevalence of ULD is high in the Mediterranean and Finland, sporadic cases have been diagnosed worldwide, including Japan. 5,9 However, the recognition of ULD is still lacking in areas of low prevalence due to varying availability of molecular diagnostic techniques and since it is difficult to diagnose ULD with clinical symptoms alone. 10 To our knowledge, our patient is the first case to be confirmed genetically in Korea. In patients with intractable myoclonus or myoclonic epilepsy, especially with onset after the age of 6 years, analysis of the CSTB gene using Southern blot analysis will help to define the syndrome. Also, adequate choices of AEDs might be important in treatment of symptoms in ULD. Carbamazepine, oxcarbazepine, and lamotrigine may paradoxically worsen myoclonus as seen in our patient. An inverse pharmacodynamics effect is suggested as the main mechanism of aggravation of myoclonic seizures. 11
|
2018-07-17T00:17:54.958Z
|
2018-07-04T00:00:00.000
|
{
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"oa_url": "https://doi.org/10.3349/ymj.2018.59.6.798",
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|
210118594
|
pes2o/s2orc
|
v3-fos-license
|
Convolution neural network–based Alzheimer's disease classification using hybrid enhanced independent component analysis based segmented gray matter of T2 weighted magnetic resonance imaging with clinical valuation
In recent times, accurate and early diagnosis of Alzheimer's disease (AD) plays a vital role in patient care and further treatment. Predicting AD from mild cognitive impairment (MCI) and cognitive normal (CN) has become popular. Neuroimaging and computer-aided diagnosis techniques are used for classification of AD by physicians in the early stage. Most of the previous machine learning techniques work on handpicked features. In the recent days, deep learning has been applied for many medical image applications. Existing deep learning systems work on raw magnetic resonance imaging (MRI) images and cortical surface as an input to the convolution neural network (CNN) to perform classification of AD. AD affects the brain volume and changes the gray matter texture. In our work, we used 1820 T2-weighted brain magnetic resonance volumes including 635 AD MRIs, 548 MCI MRIs, and 637 CN MRIs, sliced into 18,017 voxels. We proposed an approach to extract the gray matter from brain voxels and perform the classification using the CNN. A Gaussian filter is used to enhance the voxels, and skull stripping algorithm is used to remove the irrelevant tissues from enhanced voxels. Then, those voxels are segmented by hybrid enhanced independent component analysis. Segmented gray matter is used as an input to the CNN. We performed clinical valuation using our proposed approach and achieved 90.47% accuracy, 86.66% of recall, and 92.59% precision.
Introduction
Alzheimer's disease (AD) is a progressive dementia, which causes a loss of connection between nerve cells in elders. Owing to AD, the brain shrinks, hippocampal size decreases, and the brain ventricles enlarge. As AD progresses, it debases memory, thinking ability, and the person's expressions to the problem in day-to-day activities. Understanding AD, mild cognitive impairment (MCI), and cognitive normal (CN) manifestation is one of the most challenging tasks faced by neurologists from the past few years. Physicians are using different clinical methodologies to perform classification of AD. Clinically, cerebrospinal fluid (CSF) concentration deals with AD. The level of norepinephrine increases in the CSF as the disease progresses. The CSF is collected using a ventricular puncture; the physician makes a hole in the skull and collects the CSF directly from one of the brain ventricles [1]. It is a laborious procedure, and it may have a risk of bleeding in the brain. With the development of medical imaging techniques, neuroimaging plays a major role in the diagnosis of structural and functional changes in the brain and encompasses computer tomography, magnetic resonance imaging (MRI), positron emission tomography, functional MRI, and single-photon emission CT. MRI is used to analyze structural changes caused by AD, CN, and MCI manifestation because of its ease of accessibility. The most common MRI sequences are T1-weighted and T2weighted scans. T2-weighted scans are used in this work. Neuroimaging techniques help visualize the anatomical changes in the brain. In Fig. 1, change in the hippocampal size and enlargement of ventricles are observed in the MRI image AD brain having cortical atrophy compared with the MRI image of CN brain and MCI brain.
It is evident that the texture of the brain changes as the disease progresses from CN to MCI to AD. Shape transformation in the brain is used as a morphological signature of the brain structure. Morphological changes in the brain texture, structure, and volume are used to classify the healthy brain from a diseased brain [2,3]. AD is caused by degeneration of brain cells and changes in the brain volume. The early effect of AD is observed based on changes in the hippocampus, the size of which is used to classify the AD stage [4]. Change in the white matter (WM) is estimated to analyze the area of the brain affected due to AD [5]. The gray matter (GM) is used to analyze AD [6]. AD is classified using the volume of interest [7]. Image volume has more number of voxels and high dimensionality. The huge information is reduced via wavelet transformation where the classification is carried out in a voxel-by-voxel manner instead of classifying the entire data [8]; selecting an appropriate voxel and relevant area will result in good specificity and sensitivity [9]; voxel-based features are used to classify AD stages [10].
Related work
Since the last few years, computer-aided diagnosis (CAD) is used to assist and give a second opinion to the physicians. Many researchers are developing different CAD systems to diagnose AD. Most physicians use physical tests and the Mini-Mental State Examination [2,11] to verify the stage of AD. Clinically AD classification is performed by collecting different parameters and by developing biomarkers to test the AD stage. A 5-stage route map was developed for CSF-based diagnosis of preclinical AD using Ab ratios rather than Ab42 [12]. Recent CAD systems use machine learning as a computational technique to analyze patterns of medical data. Different machine learning approaches such as regression, classification, and clustering are used in the CAD system. Machine learning approach gives better classification accuracy based on the features that are extracted from the images; to detect the structural and textural changes in the brain MRI, single modalities and multimodalities are used as features [13]. Brain volume, shape, voxel intensity, CSF measurement, and genetic information are used as features to perform the classification of AD, using random forest [14]; those features are correlated using PCA, the dimensionality of the features is reduced, and they are classified using sup-port vector machine (SVM) and particle swarm optimization [14,15]. As AD progresses, it affects brain tissues such as the WM, GM, and hippocampus. The WM and GM are segmented from brain MRI using learning vector quantization, an unsupervised approach, and classification was performed using SVM. Texture changes in the WM and GM are used to differentiate AD from MCI and CN; texture changes are measured using first-order statistical parameters that are extracted from the histogram, and then the second-order statistical features are extracted from GLCM and Gabor filters [16]-these features are used to differentiate AD from CN using KNN [16] and SVM [17] classifiers. Hybrid features generated by combining texture and volume information, such as texture features along with GM volume, are used to perform the classification of AD using SVM-random Fourier expression (SVM-RFE) [18]. Hybrid features extracted from segmented brain image and clinical data are used for multiclass classification of AD from MCI and CN [19]. As the features are more, the classification accuracy increases, but it makes accurate training of a classifier more complicated; greedy score is used to select the important features, and kernel-based discriminative method is used to perform feature selection of complex features [20]. Hippocampal volume is used to differentiate AD and MCI [21]. Hippocampal volume is verified patchwise [22]; patch-based image features are selected by professional and medical experts with knowledge in medical segmentation. Texture features are extracted patchwise using Gabor filter, and classification is performed using a weak classifier [23]. In all the aforementioned approaches, features are extracted manually, and it requires expert knowledge in selecting the features.
In recent years, deep learning framework has achieved greater success in many fields. An artificial neural network has more influence on the development of deep learning architecture. There are many machine learning approaches adopted to perform classification of medical images using CAD. The advantage of neural networks is that the CAD system used in recent days [24]. In deep convolutional neural networks, hierarchical layers are connected and have the advantage over artificial neural networks. Deep learning achieves good performance in medical image analysis [25]. Deep radiomic features are extracted from the threedimensional MRI image using entropy convolution neural network (CNN) to perform AD classification [26]. Multimodal three-dimensional CNN is used to extract the features and perform AD classification [27]. Features from stacked autoencoders and low-level features in combination help to build the classification model [28]. Extracting texture from the center slices of the MRI image and using those as input for performing AD classification using bootstrap algorithm as the region of interest is used to collect the features from MRI [29]. Transfer learning using the VGG-16 pretrain model is used to perform the classification of AD-NC-MCI [30]. Hippocampal volume patches are used to perform the classification using the hybrid classifier CNN and recurrent neural network [22].
In this article, we propose a CNN classifier for automatic classification of AD from MCI and CN using GM.
We evaluated the architecture performance using T2weighted MR images collected from a standardized data set, Alzheimer's Disease Neuroimaging Initiative (ADNI). The contribution of the article is summarized as follows: Fig to remove the unwanted tissues from the voxels. b) We specifically used GM for atrophy detection. In this article, GM tissues are segmented using hybrid enhanced independent component analysis (ICA). c) CNN architecture is trained using segmented GM voxels. d) The trained CNN is evaluated using independent MRI voxels collected from a local MRI center and correlated with clinical information, which achieves remarkable accuracy.
The aim of this work is to develop a computer-based diagnosis system that provides additional support for the medical staff to support their diagnosis evidence.
Materials
In our work, a total of 1820 MRI images are obtained from the ADNI database (adni.loni.usc.edu). We used 1.5-Tesla, T2-weighted MRI volumes, which are of 420 ! 462 ! 32 voxels. We collected AD, MCI, and CN MRIs of individuals of different age groups, both male and female. MRIs and their demographic representation are shown in Table 1. Overall, we collected 635 AD MRIs, 548 MCI MRIs, and 637 CN MRIs.
Methodology
In our work, MRIs are initially sliced into voxels. These voxels are preprocessed to correct for geometric distortion and reduce noise using Gaussian filter. Nonbrain tissues are removed from the voxels using the skull stripping algorithm. Structural and texturual changes in the brain are used to differentiate healthy and diseased tissues, and enhanced ICA is used to perform segmentation of the brain into the WM, GM, and CSF. Brain tissue atrophy is used to detect AD stage. AD is a progressive disease, in which the brain experiences changes in GM and WM texture and volume, as well as expansion of ventricles. In our work, we classify the AD based on GM atrophy. We used CNN as a classifier which is used in different computer vision techniques, since the past couple of years. Our classification approach has 3 major sections: (1) preprocessing; (2) train, test, and validation of the classifier; and (3) perform clinical valuation-as shown in Fig. 2.
Skull stripping algorithm
Skull stripping is the most important preprocessing technique. For accurate classification of images, unwanted and nonbrain tissues are initially removed from the voxels and the brain tissues are left. The proposed skull stripping algorithm has a sequence of steps. Before applying skull stripping, the image is enhanced using a Gaussian filter, and detailed information and noise are reduced.
The enhanced voxels are convolved by a 3 ! 3 filter as given in equation (1).
The filtered image is segmented into brain and nonbrain tissues using thresholding technique; selection of threshold value is crucial to generate the initial binary image. Morphological operation is performed on the binary image to remove the unwanted regions. Active contour is applied on the binary image to separate foreground from background and generate a final binary mask. On multiplying the final binary mask with the original voxel, the skull is stripped and unwanted tissues such as skull, scalp, dura, eyes, and so forth are removed from the voxel. The skull stripping algorithm steps are as follows.
Segmented algorithm
Blind separation of brain tissues in the MRI is carried out by using an unsupervised segmentation approach. In our work, we have used hybrid enhanced ICA. K-means and expected maximization (EM) are combined to form a hybrid strategy to cluster the brain tissues in the MRI. This combination achieves the capability of providing clusters for welldistributed image pixels and compactness through EM.
In our proposed hybrid enhanced ICA, the concept of mixture model is introduced and it is characterized into mutually exclusive classes. In the modified GMM approach, spatial information is added to GMM using Markov random field (MRF) and takes spatial dependency into account. In EM, the expected step is computed using log likelihood with mean and variance calculated using modified K-means and latent variable calculated through Gibbs density function. The aforementioned parameters are used as input parameters to hybrid enhanced ICA to perform the segmentation of the brain MRI voxels. The algorithm is further explained using the following steps:
Classifier
The CNN is used for classification. In our article, we used 224 ! 224-sized gray segmented images as input to the CNN. The performance of the CNN depends on the network architecture and weights that are set. The architecture of the CNN depends on the specific task, and the requirements of the data for the network need to be known. The size of the MRI slice, filter size, number of kernels, padding, and strides determine the particular convolution layer size. Our classifier has 5 convolution layers with 32, 64, 128, 256, and 512 filters with different sizes (4,4), (5,5), (3,3), (3,3), and (3,3), respectively, at different stages of stride 1, padding, followed by max pooling layers used to extract features and 6 fully connected layers used to perform classification. The network is trained using back-to-back propagation with 200 epochs; we used Adam optimization. Equations (2), (3), (4), (5), (6), (7), and (8) show the layerwise parameter calculation and activation functions used at convolution layer and fully connected layers.
g(x, y) is the input image into k identically independent GMMs with parameters q k 5 {m k , E k } Step1: Represent g(x,y) in vector{g i : i 5 1, 2, 3, 4. N} Step2: Modified K-means to find the prior information of the Gaussian mixture model such as mean and covariance {Mixing Coeficient p k : i 5 1, 2 ,3 ,4 . N}, g i is the gray level.
a. Partition of N pixels into K equal sets b. Center of each set as a centroid c1, c2, c3, .c k c. Find the distance between Euclidean distance between g(x, y) and the cluster centers d. Find the centroid that is close to the particular g(x, y) e. Recalculate the centroids of each clusters f. Repeat the steps from c to e g. If the distance between g(x,y) and new cluster center is less than or equal to the previous distance, then g(x,y) will be in the same cluster otherwise it moves to another cluster based on the distance. h. The process continues until the clusters are convergence i. Collect mean and covariance of the clusters q k 5 {m k , E k } Step2: q k 5 {m k , E k } for i 5 1: pixels for k 5 1: number of k Probability density of the mixture model is considered as pðx i jp; qÞ5 P c k51 p k i p ðx i jq k Þ end end Step 3: log likelihood of the density function is calculated to find the probability of pixels that belong to the particular Gaussian ln pð5pðXjqÞ5 P N i51 ln pðx i jqÞ , q 5 {z, m, s}, z, latent variable and calculated using expectation and maximization Step 4: E step for I: Q ðiÞ ðZ ðiÞ Þ : 5Pðz i jx i ; qÞ Step 5: M Step for all z q:5argmax q P i P Z i Q i ðz i Þ log pðx i ; z i ; qÞ Q i ðz i Þ , Q i the posterior distribution of (z i ) Step 6: Prior distribution of p is given by MRF model through Gibbs density function pðpÞ5expv 2b P N i51 V Ni ðpÞ a , a is a normalizing constant, V Ni (p) is the clique potential function Step 7: Process stops when q new 2 q old error Convolution width 5 Input MRI Slice Size width 2Filter width 1ð2XPaddingÞ Strides Width 11 (2) soft max function 5 e xi P n j51 e xj for i 5 1; 2; 3; .n (8)
Model development and training
In our work, we used MATLAB R2015b to perform slicing, skull stripping, and segmentation of the image. To train deep neural network data, parallel processing is needed, so we used an open-source software package Python, version 3.0, Google Colab to perform the training and validation of the classifier (GPU: 1xTesla K80, having 2496 CUDA cores, compute 3.7, 12 GB [11.439 GB useable] GDDR5 VRAM). We used Keras library over TensorFlow modules to design our proposed model.
Creating training and test set
Our total data set has 18,017 GM segmented images. We shuffled and split the data set in the ratio 80:20 as training and test data sets. We used this data set for multiclass classification and binary classification; the data set is summarized in Table 2.
Results
In this work, a total of 18,016 MRI axial slices are used; these are generated from 1820 T1-weighted MRIs collected from the standard AD data set, the ADNI. All the voxels are preprocessed by enhancing them using rotationally invariant Gaussian filters. Irrelevant tissues such as scalp, skull, ears, dura, and eyes are removed from the MRI voxels using skull stripping algorithm. We focused on the GM for atrophy detection. GM tissues are segmented using hybrid enhanced ICA. Atrophy in the GM is used to differentiate AD from MCI and CN. Our proposed CNN model has 5 convolution layers and 5 fully connected layers; each convolution layer is followed by dropout, padding, and max pooling layers with ReLu as an activation function. Preprocessed images are augmented to increase the sample size and train the CNN model. We used Keras with TensorFlow to build the proposed CNN model using Python. Our proposed approach performs binary classification and multiclass classification to fit the model in a batch size of 128 in 200 epochs using Google Colab. It takes around 7 hours to train the model. The total architecture is summarized in Table.3. We train the model by Adam optimization with a learning rate of 0.001, beta1 of 0.9, and beta2 of 0.999. Our classifier is trained with 200 epochs. We used it to perform binary classification and multiclass classification. During binary classification, we first trained the classifier with AD-CN segmented images and the model resulted in 99.75% training accuracy. Then, we trained the classifier with segmented AD-MCI voxels, which achieved 98.72% training accuracy. Later, it was trained with segmented CN-MCI images which resulted in 99.87% training accuracy. Multiclass classification was performed by the trained classifier, using segmented AD-MCI-CN images which achieved 99.50% training accuracy. Training, testing accuracy, and loss graphs are shown in Fig. 3, Fig. 4, Fig. 5, and Fig. 6. It is required that our proposed framework is trained and the prediction is made with utmost accuracy.
We compared the performance of the proposed system with that of different models discussed in literature review as shown in Table 4. It is observed that our classifier achieves remarkable performance both in binary classification and multiclass classification. Fig. 7 shows parameters of both binary and multiclass classifications.
We further performed the clinical evaluation using our proposed approach on 21 independent MRI slices collected from Poorvi MRI Center, Chirala and compared the predicted results generated by our system with results diagnosed by the physician. The confusion matrix of clinical analysis is shown in Fig. 8.
We calculated some important performance measurement parameters as follows using Equations (9), (10), (11), and (12): We achieve 90.47% accuracy, 86.66% recall, and 92.59% precision in comparison of our system with physician decision. Bar diagram of the clinical evaluation is given in Fig. 9.
Conclusion
Effective diagnosis of AD helps the patient to get a featured treatment. Many researchers are focusing on this challenging task; they had developed many CAD systems to perform the diagnosis of AD. In our workflow, we developed a deep learning approach to perform the classification based on GM segment, using hybrid enhanced ICA.
Our proposed framework has more strengths than the previous techniques. We use heterogeneous MRI volumes of different age groups and gender. In our experiment, we used T2-weighted MRI to perform the classification. The GM has neuron cell bodies and non-neuron brain cells called glial cells. The GM undergoes development and growth throughout childhood and adolescence; it is used to carry glucose to the brain, and changes in this affect the memory, speech, and motor controls. In our work, we mainly focused on the use of the GM to classify AD. In our work, we observed that the framework is not affected with noise and data augmentation. Our deep learning model got trained and was validated and tested on the MRI collected from the database, and we performed binary classification such as AD-MCI, AD-CN, and MCI-CN and multiclass classification such as AD-MCI and CN. We further compared the classifier performance with the physician's decision and achieved good results. No other framework performed the comparison of the system with the physician's decision. Our system is recommend not to replace but to support the physician decision.
RESEARCH IN CONTEXT
1. Systematic review: In our work we had used AD data collected from online repository to train the model and test the model using images collected from local MRI center. We used 21 MRI slices of different age groups of 60 to 92 years both male and female, and compared the test result of model with a physician decision based on MSME score, to evaluate the system accuracy.
2. Future directions: System is further improved by adopting multiple image data such as T1, T2 and meta data along with the proposed system to improve evaluation of AD at clinical level.
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2020-01-02T21:48:51.309Z
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2019-01-01T00:00:00.000
|
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"oa_license": "CCBYNCND",
"oa_url": "https://doi.org/10.1016/j.trci.2019.10.001",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "2d8772f217e18657c448c7cd5f6d39787215c777",
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"extfieldsofstudy": [
"Medicine",
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}
|
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