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103389218 | pes2o/s2orc | v3-fos-license | Pyrolysis gas as a carbon source for biogas production via anaerobic digestion
Carbon is an important resource for anaerobes to enhance biogas production. In this study, the possibility of using simulated pyrolysis gas (SPG) as a carbon source for biogas production was investigated. The e ff ects of stirring speed (SS), gas holding time (GHT), and H 2 addition on biomethanation of SPG were evaluated. The diversity and structure of microbial communities were also analyzed under an illumina MiSeq platform. Results indicated that at a GHT of 14 h and an SS at 400 rpm, SPG with up to 64.7% CH 4 could be bio-upgraded to biogas. Gas – liquid mass transfer is the limitation for SPG biomethanation. For the fi rst time, it has been noticed that the addition of H 2 can bioupgrade SPG to high quality biogas (with 91.1% CH 4 ). Methanobacterium was considered as a key factor in all reactors. This study provides an idea and alternative way to convert lignocellulosic biomass and solid organic waste into energy ( e.g. , pyrolysis was used as a pretreatment to produce pyrolysis gas from biomass, and then, pyrolysis gas was bioupgraded to higher quality biogas via anaerobic digestion).
Introduction
The global trend of energy use is moving towards sustainable development, and the waste-to-energy (WTE) concept is being highly promoted as a part of this effort. 1,2Nowadays, biomass is being utilized to generate renewable energy in many countries of the world. 3,4Biomass covers about 50 exajoule per year of the total primary energy demand of the world. 5Biomass resources have been paid increasing attention because of their renewability and biodegradability. 6In particular, the composition and energy utilization of biomass energy are very similar to those of fossil energy.Thus, it has the largest potential to substitute for conventional energy.Biomass materials have been used as structural materials or common materials from ancient times because their major components are natural polymers such as lignin, cellulose, starch, chitin, and protein, which guarantee enough strength and stability. 7According to China's unique energy structure and abundant biomass resources, the generation of high quality bio-gas by gasication of biomass becomes a research emphasis.
][10][11] One advantage of this process is that most of the organic components, especially the relatively dry and slowly biodegradable biomass (e.g., wood and woodchips) that are not suitable or effective for anaerobic digestion (AD) process, would be converted to pyrolysis gas.However, CO is a toxic gas for many organisms due to its high affinity for metal-containing enzymes.The presence of high concentrations of CO is a restriction in the utilization of pyrolysis gas in some applications. 7,11Moreover, low volumetric energy density (usually less than 13 MJ m À3 ) is a limitation for pyrolysis gas when used as a fuel gas. 8,12To convert the pyrolysis gas into high quality gas, a methanation process is necessary.Traditional catalytic methanation requires high pressure and temperature (230-700 C) and a metal catalyst, which involves high cost and energy consumption.A potentially more economical alternative is the utilization of a biological system to convert biomass pyrolysis gases to biomethane.Several trials have been reported to biologically convert CO, H 2 , and CO 2 to biomethane via biomethanation system. 13Wang et al. 14 reported that co-digestion of coke oven gas (COG) (92% H 2 and 8% CO) and sewage sludge could achieve in situ biogas upgradation and simultaneous COG biomethanation.Youngsukkasem et al. 15 found that using syngas and organic substances as feedstock for AD in a repeated batch mode, a good performance without any negative effect was observed.The conversion of H 2 and CO 2 to CH 4 is a common biological reaction occurring in the anaerobic digestion reactor.Only a small number of microorganisms (e.g., Methanobacterium thermoautotrophicum, Methanosarcina barkeri, Methanothermobacter thermautotrophicus, and Methanosaeta thermophila) can achieve CO biomethanation. 13,16,17Since the solubility of H 2 is 0.00016 g per 100 g of water above 1 atm and the solubility of CO is 0.0028 g per 100 g of water above 1 atm, anaerobic digestion of pyrolysis gas is typically limited by the gas-liquid mass transfer rate. 13From the view of thermodynamics, the reaction CO + H 2 / CH 4 is exothermic.However, in the biomethanation reaction, no signicant temperature changes were found due to the low conversion rate of biocatalysis as compared to traditional catalytic methanation.Actually, in the anaerobic digestion process, the system needs to be maintained at a constant temperature of 37 C to keep the microorganisms at a higher biocatalytic activity.The constant temperature is provided by an extra heat source.Thus, development of a suitable reactor and optimization of operating conditions are necessary.Moreover, a low cost and long-term AD operation is needed to industrialize the pyrolysis gas biomethanation technology.
Based on the abovementioned considerations, in this study, biomethanation was achieved in long-term AD tests where SPG was utilized as a carbon source for biogas production.AD can increase the LVC of SPG from 12.9 to 23.2 MJ m À3 , and Methanobacterium was dominant in the whole process.The conversion rates of CO and H 2 were 96.6% and 99.2%, respectively, which could effectively enhance gas production.The objectives of this study were as follows: (1) to investigate the effect of stirring intensity (SS), gas holding time (GHT), and H 2 addition on biomethanation of pyrolysis gas and (2) to evaluate the system stability using pyrolysis gas as a carbon source in longterm anaerobic digestion operation.
Experiment setup
Herein, three 1.69 L continuously stirred tank reactors (named R1-R3) with a working volume of 1.2 L were used.Non acclimated inoculum was obtained from the mixed effluent of anaerobic reactors treating various organic wastes (e.g., chicken manure, dairy manure, corn stover and food waste) in our laboratory.Before utilization, the residual solid matter in effluent was removed using a sieve.Then, liquid inoculum was pre-incubated at a constant temperature (37 C) incubator shaker (ZWYR-D2402, China) for degassing.The shaking speed was 120 rpm.Aer two weeks of pre-incubation, no gas was detected from inoculum.Then, the degassed inoculum was transferred to three reactors.The total solids (TS) and volatile solids (VS) of inoculum were 2.33 AE 0.02% FM and 1.03 AE 0.00% FM, respectively.To ensure an oxygen-free environment, the headspace of each reactor was purged with nitrogen gas for 2 min.The digestion temperature was 37 C. Simulated pyrolysis gas (H 2 : CO : CH 4 : CO 2 ¼ 35 : 30 : 15 : 20 on volume basis) was manually added to the reactors by a 100 mL syringe.Once added, pyrolysis gas was kept in the reactors for 24 h or 48 h depending on the experiment design (Table 1).To bioupgrade the generated gas from the reactor R2, pure hydrogen and generated gas from R2 were added to the reactor R3.To investigate the process stability of using simulated pyrolysis gas as the only substrate, no additional carbon source was added to this system.During steady-states, the liquid samples were obtained from R1-R3 for high-throughput 16S rRNA sequencing and analysis.Specic experiment design is shown in Table 1.
Cross-linking process
TS and VS were determined according to the standard methods. 18Gas samples were obtained every one or two days to detect the composition.A gas chromatograph (FULI 9790II, China) equipped with a thermal conductivity detector (TCD) was used with helium as the carrier gas.The temperatures of the injector, oven, and detector were 150, 130, and 160 C, respectively.The corrected CH 4 , CO, CO 2 , and H 2 content of the produced gas (i.e.CH 4corr , CO corr , CO 4corr , and H 2corr ) was calculated according to Li et al. 19 Biogas production of all reactors was measured daily by a 100 mL syringe.The pH was measured using a pH meter (SARTORIUS PB-10).The volatile fatty acids (VFA) and total inorganic carbon (TIC) were measured using a Mettler Toledo T70.Then, 7 mL sample was taken using a pipette (3.5 mL twice) into the titration beaker, and water was added to 40 mL scale line; then, the titration beaker was put on the titrator, the titration was started to adjust the pH of the sample to 5.0, 4.4, 4.3, and 4.0 with 0.025 mol L À1 H 2 SO 4 , and the volume of H 2 SO 4 used for each pH value was obtained.The VFA and VFA/TIC were then calculated according to Nie et al. 20 Lower caloric values (LCV, MJ m À3 ) of the simulated pyrolysis gas and generated gas were determined according to Li et al. 8 During steady-states, the liquid samples were obtained in phase 1 and 2 of the reactor R1 (named R1P1 and R1P2), phase 1 of the reactor R2 (named R2P1), and phase 1 of the reactor R3 (named R3P1) for sequencing using an Illumina MiSeq platform at Majorbio Company (Shanghai, China).Inoculum was also analyzed as a control.Microbial DNA was extracted from samples using the E.Z.N.A.®AxyPrepDNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer's protocols.The V4-V5 region of the bacteria 16S ribosomal RNA gene was amplied by PCR (95 C for 2 min, followed by 25 cycles at 95 C for 30 s, 55 C for 30 s, and 72 C for 30 s and a nal extension at 72 C for 5 min) using primers Arch519F and Arch915R.PCR reactions were performed in triplicate 20 mL mixture containing 4 mL of 5Â FastPfu Buffer, 2 mL of 2.5 mM dNTPs, 0.8 mL of each primer (5 mM), 0.4 mL of FastPfu Polymerase, and 10 ng of template DNA.
Operational taxonomic units (OTUs) were clustered with 97% similarity cut-off using UPARSE (version 7.1 http:// drive5.com/uparse/),and chimeric sequences were identied and removed using UCHIME.The taxonomy of each 16S rRNA gene sequence was analysed by RDP Classier (http:// rdp.cme.msu.edu/)against the silva (SSU115) 16S rRNA database using the condence threshold of 70%.Specic Illumina MiSeq sequencing procedures and processing of sequencing data can be found.
Effect of the gas holding time (GHT)
The time course of biogas production, biogas composition, pH, VFA concentrations, and VFA/TIC ratio are shown in Fig. 1-3.Table 1 summarizes the reactor performance during steadystates of each operation condition.Reactor R1 was operated at a low stirring speed (SS) of 120 rpm.Biogas production increased from 60 AE 5 mL d À1 to 114 AE 24 mL d À1 with the gas holding time (GHT) decreasing from 48 h to 24 h.The corresponding CH 4 content was decreased from 62.2 AE 1.3% to 49.9 AE 1.3%.The CO and H 2 conversion efficiency were 93.3 AE 1.2% and 98.5 AE 0.5% in phase 1 and 91.5 AE 2.3% and 85.1 AE 3.9% in phase 2, respectively.Change in the GHT can inuence the CH 4 content, and CO and H 2 conversion efficiency; this indicates that the contact time of gas and liquid is an important parameter for SPG biomethanation.For reactor R1, on increasing the injected gas ow from 325 mL d À1 to 505 mL d À1 (325 mL SPG + 180 mL H 2 ), the biogas production increased.However, high percentages of H 2 and CO were also determined in generated biogas (phase 3), indicating that the biomethanation efficiency in this operation was low.The corresponding H 2 and CO conversion efficiency were only 84.0 AE 3.6% and 77.5 AE 2.4%, respectively.The methane content in phase 3 of R1 was also low as compared to that in phase 1 of R1.
Effect of the stirring speed (SS)
For the reactor R2, H 2 and CO were not detected in the generated gas at SS of 400 rpm and GHT of 24 h; this indicated that they were efficiently utilized by the microorganisms in the reactor.Methane content was 64.7 AE 0.8% in R2 when the SS was adjusted to 400 rpm (Table 1 and Fig. 2), indicating that SS was a higher inuence factor on biomethanation of SPG as compared to GHT.Similar results were found by Luo and Angelidaki 21 indicating that increase in the stirring speed of the reactor from 150 to 300 rpm could increase H 2 consumption rate.As reported by Guiot et al., 13 gaseous feedstock is difficult to be captured by the microbes in the liquid phase.Further study should be focussed on the conversion efficiency of pyrolysis gas biomethanation and minimization of the gasliquid mass transfer limitation.From 60 to 72 days, reactor R2 was only maintained at 37 C without the input of SPG and output of generated gas due to the absence of researcher.On 73rd day, this reactor was recovered with feeding SPG, and it performed well in the next days.
Effect of hydrogen addition
In reactor R3, injected gas was composed by generated gas from reactor R2 (CH 4 content was 64.7 AE 0.8%) and additional H 2 .The addition of H 2 reached 116 AE 11 mL and 241 AE 33 mL per day in phase 1 and phase 2 of R3, respectively (Table 1).The corresponding CH 4 contents were 85.7 AE 1.0% and 91.1 AE 1.0%, respectively, which indicated that addition of H 2 could largely increase the CH 4 content (Fig. 2).Luo and Angelidaki 22 found that the addition of H 2 could achieve in situ biogas upgradation (methane content was from 78.4% to 90.2%) when using mixture of cattle manure and whey as feedstock.In this study, SPG was used as a carbon source.Moreover, through the addition of H 2 , SPG biomethanation was achieved.H 2 can be generated from electrolysis of water using extra electricity or unstable wind electricity, or water photolysis or methanol decomposition.The H 2 -assisted bioupgradation has several advantages, such as no CH 4 loss, low investment cost for additional equipment, and storage of excess electricity as methane, as compared to CO 2 -air strippers or autogenerative high pressure digestion (AHPD) method.
Calculation of mass balance
Due to the SPG containing 15% of CH 4 as a feed gas in the experiments, the mass balance discussion is necessity.Taking R1P1 for example, the SPG addition is 163 mL with 15% CH 4 (24.5 mL CH 4 added), and the biogas production is 60 mL with 62.2% CH 4 (37.3 mL CH 4 produced); thus, the pure CH 4 production is 12.8 mL.Similar calculation results for all the other phases are shown in Table 1.The results further inferred that CO, CO 2 , and H 2 were converted to methane by the anaerobic microbes.
Evaluation of the energy output
Although pyrolysis gas can be directly used as fuel gas, the volumetric energy density of pyrolysis gas is only about 50% of biogas.The conversion of pyrolysis gas to CH 4 is an important step to meet the increasing demand for natural gas.Aer SPG biomethanation, the energy output results are shown in Table 1.According to the lower caloric value (LCV) calculation equation, 8 the lower caloric value of injected gas (LCV in ) for R1 and R2 was determined to be 12.2-12.9MJ m À3 .Aer biomethanation process, the lower caloric value of generated gas (LCV out ) ranged from 20.4 to 23.2 MJ m À3 .For reactor R3, LCV out was 30.7-32.7 MJ m À3 aer the addition of H 2 .Herein, two-step approach (SPG biomethanation and H 2 addition) could be a minder and alternative way to bioupgrade pyrolysis gas into high quality biogas and convert toxic gas CO to CH 4 .
Process stability
During the long-term anaerobic digestion tests, using SPG as a carbon source, pH value was around 8.0 for R1 and R2 and 8.3-8.6 for R3.The pH value increased to higher than 8.0 in reactor R3 (Table 1 and Fig. 3) as H 2 addition was associated with the consumption of CO 2 in the reactors.Similar result was obtained by Luo and Angelidaki, 22,23 where manure alone was used as a substrate and pH value in reactor increased to higher than 8.0.In this study, no signicant inhibition was found although the pH value was not in the optimum range of 7.2-7.8.The VFA concentration showed an increasing trend with time and was determined to be 770-1058 mg L À1 for R1, 913 mg L À1 for R2, and 463-1727 mg L À1 for R3.The VFA/TIC ratios of R1-R3 were lower than 0.4, which were in a normal range. 19enerally, anaerobic reactors can perform well using SPG as a carbon source when the pH in the reactors remains above 8.0.
Microbial community analysis
Reaction equations of CO/synthesis gas biocatalysis are summarized in Table 2. CO and H 2 from synthesis gas can be converted to CH 4 in two ways: direct and indirect conversions.In the rst reaction, CH 4 was formed directly via conversion of CO, H 2 , and CO 2 by microorganisms. 22][26] Fig. 4 shows the rarefaction curves based on an operational taxonomic units (OTU) denition of 97% sequence similarity.New OTUs continue to appear even aer sampling 2000 sequences for archaea.However, the coverage values for archaea (>99%) indicated that most common OTUs were detected (Fig. 5).The Shannon diversity, CHAO1, and ACE richness index provides not only species richness but also the evenness of the species among all the species in the community.In the current work, inoculum had higher diversity and richness as compared to other samples.This may be because inoculum was obtained from the mixed effluent of anaerobic digesters treating various organic wastes, and samples from reactors R1-R3 were only fed with SPG.
At the phyla level, Euryarchaeota (>97%) was dominant in all these samples.The genus level identication of the archaea communities is illustrated in Fig. 6.Methanobacterium dominated in all samples.The amounts of other archaea communities are different in 5 groups; this is in accordance with the report of Luo and Angelidaki. 21Furthermore, we assigned the sequences to species level by choosing representative sequences from each dominant OTU.Results indicated that Methanobacterium formicicum was the dominant species in all reactors; this indicated that CH 4 was generated by an indirect way.Methanosarcina can mediate both hydrogenotrophic and acetoclastic methanogenesis. 14Therefore, it was also possible that Methanosarcina could utilize H 2 directly in R1.For reactor R3, the hydrogenotrophic genus Methanoculleus showed a higher relative abundance, which could be related with the utilization of H 2 . 28Based on the abovementioned nding, CO was converted to methane mainly by indirect pathway, and H 2 was converted to methane mainly by direct pathway.
Conclusions
Simulated pyrolysis gas (SPG) can be used as a carbon source for biogas production in long-term anaerobic digestion tests.Gasliquid mass transfer is the limitation for biomethanation of SPG.Gas-liquid mass transfer can be improved by mixing.The addition of H 2 to anaerobic reactor could bioupgrade SPG to high quality biogas (91.1% CH 4 ).Pyrolysis combined with anaerobic digestion is an attractive way to utilize biomass and produce higher quality biogas (mainly CH 4 ).Further research on the operation optimization to increase the efficiency of pyrolysis gas biomethanation should be performed.In addition, the effect of impurities on the bioupgradation conversion process should be concerned.
Fig. 1
Fig. 1 Profiles of the gas injection and generation rate (a, b, and c representing the reactor R1, R2, and R3, respectively).
Fig. 4
Fig. 4 Rarefaction curves based on the sequencing of archaea communities.The OTUs were defined by the 0.03 distance.
Fig. 5 Fig. 6
Fig. 5 Comparison of the richness and diversity of the 16S RNA gene libraries based on the 0.03 distance.
Table 1
Summary of the experiment design and performance in the reactors R1, R2, and R3 during steady states a Gas composition (H 2 : CO : CH 4 : CO 2 ¼ 4.5 : 1.5 : 0.75 : 1, on volume basis).b Day 11-13, the gas holding time was adjusted to 24 h.c Calculations were obtained based on 10 measurements during steady states.
Table 2
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119420504 | pes2o/s2orc | v3-fos-license | Laplace maps and constraints for a class of third order partial differential operators
We explore the existence of a class of generalised Laplace maps for third order partial differential operators of the form \[\partial_1\partial_2\partial_3+a_1\partial_2\partial_3+a_2\partial_1\partial_3+a_3\partial_1\partial_2+a_{12}\partial_3+a_{23}\partial_1+a_{13}\partial_2+a_{123}\] and related first order $3\time 3$ systems and show that they require the satisfaction of constraints on the invariants for such operators.
Introduction
The classical Laplace maps are transformations of an essentially algebraic nature between second order, linear, partial differential operators. They were developed by Darboux in [4] and can be thought about in a variety of ways with application to several areas. They are (generically invertible) maps between invariants associated with different operators.
The nomenclature surrounding this type of map is neither fixed nor always clear. So it is that the classical maps are sometimes described as Laplace transformations (rather than maps), as belonging to the more general class of Darboux transformations and as intertwining Laplace transformations (in order to distinguish this approach from other possible avenues of generlization). Such maps may also be considered to act either on the partial differential operators themselves or on the elements of their kernels, the homogeneous solution spaces.
In the theory of integrable sytems the Toda lattice [23] has been an influential paradigm. It is related to the theory of Laplace maps in that the maps generate three term recurrence relations on the (indexed) invariants which are exactly the equations of the Toda chain on Z over R 2 [26]. The vanishing of an invariant corresponds to the end of the chain and to a factorizable operator. From the kernel of this factorizable case all the invariants on the chain can be generated.
The Laplace maps can also be lifted to transformations of non-linear systems of hydrodynamic type because the Riemann invariants satisfy second order, linear partial differential equations [6,27].
In geometry the transformations describe maps between immersed surfaces and their accompanying conjugate nets (coordinates), a connection generalised to higher dimensions in [11,12]. This relation can be developed in discrete geometry also [5,16].
As geometrical objects the invariants themselves can be derived following the Cartan method of moving frames [20].
From a purely algebraic point of view, Laplace maps have been studied in work on factorization [3,10,21,22,24,25]. The general feeling appears to be that the classical instance of Laplace maps is not easily generalizable.
Further classes of generalization include Darboux transformations of type I [17,18,19] and of continued type [9]. A looser and more general notion of intertwining Laplace transformation than that presented in this paper can be found in [7].
This paper has two immediate predecessors. The first is [1] where Laplace maps for 3 × 3 systems are discussed. The current paper will slightly generalise that work. The second is [2] where a classification of certain invariants for a very large class of arbitrary order invariants is given.
Maps of this sort are usually characterised by an intertwining property of the form where one is interested in the kernel of A σ in relation to that of A: In this account we describe several classes of intertwining relation for third order partial differential operators in scalar and system form and show that intertwining maps of this type can exist only when certain constraints on the invariants of the operators are satisfied. This is perhaps, compared with the second order case, a disappointing result but it is compatible with the general difficulty of generalisation apparent in the literature. It is also the case that these constraints are not preserved as functions of the transformed invariants so that one may not successively apply the Laplace maps.
Further we compare the results for scalar and system forms. It is not generally true that scalar third order partial differential equations can be written in 3 × 3 system form nor vici versa. Again, certain conditions on invariants have to be satisfied and we look at the way the Laplace maps correspond under these conditions.
In section 2 we review the classical situation, presenting it in a form convenient to generalisation by introducing invariants as zeroth order differential operators constructed from the natural first and second order operators of the theory.
In the next section a technical trick is introduced which allows us to analyse polynomial identities between differential operators by reducing them to successively lower order identities and we introduce a deformation of the standard Laplace map.
Section 4 attempts to generalise the approach of section 2 to third order differential operators considering two scenarios: firstly first order and then second order intertwining operators. We find that constraints arise in each case, but they are fewer in number for the second order intertwiner. We then review the 3 × 3 system. Laplace maps for this case have been considered before but we clarify here the role of constraints needed for the Laplace map to function and we discuss the relation between scalar and system forms and their overlap when a Laplace map exists.
Throughout the paper our philosophy is to work with noncommutative polynomials generated by a finite set of (noncommuting) linear differential operators.
In particular our starting point is always a single linear operator of total degree n in derivations ∂ 1 , . . . , ∂ n but which is of degree one only in each distinct derivation. In [2] these were called hyperbolic but this suggests a reality condition that does not feature in the discussion.
Alhough in that paper systematic processes describe the construction of invariants, the calculations in the current paper are somewhat ad hoc and a deeper understanding of their structure would be required for discussion of higher orders to be feasible.
Finally we make some concluding remarks and suggestions for further exploration.
2 The Classical case
Scalar form
Consider the second order partial differential operator The coefficients belong to a differential field with derivations ∂ 1 and ∂ 2 and we assume no relations (algebraic or differential) between them. We write a i , j for the ∂ j derivative of a i etc. Note that the operator is symmetric in indices: We are interested in invariants of such operators under transformations of the form: L 12 → L g 12 = g −1 L 12 g where g is an arbitrary element of the differential field or an extension thereof. Invariants are constructed by defining and writing down the functions These are invariants by virtue of being differential functions (zeroth order operators) of the coefficients: Thus where I 12 = a 12 − a 1 a 2 − a 2,1 and I 21 = a 12 − a 1 a 2 − a 1,2 . Now suppose there is an element φ (in a field extension) such that L 12 φ = 0. Define φ σ = L 2 φ and φ Σ = L 1 φ. These satisfy the pairs and The σ− and Σ−Laplace transformed equations are those satisfied by φ σ and φ Σ obtained by eliminating φ from the above pairs.
. This implies transformations on the invariants: and correspondingly: These relations are all summarised in the simple intertwining relations For example, the first implies the following chain of argument.
By looking at leading order terms in differential operators, and finally, since L σ 12 = L σ 21 , We can however, also deform the intertwining relation by writing: where the primed operators are monic in ∂ 2 still but with coefficients distinct from the unprimed operators. We consider this case at the conclusion of the next section.
System form
The situation just described can always be represented in system form. We can write the pairs of equations for φ σ and φ Σ as and the intertwining relations become
Methodology
We adopt some ideas from the paper [2] where invariants of arbitrary order differential operators were constructed. Let I be a set of distinct labels. We associate with I a partial differential operator in derivations ∂ i for i ∈ I : Here the symbol ∂ J is the product of all derivations ∂ j for j ∈ J. All the coefficients a K for K ⊆ I are algebraically and differentially independent. We associate such an operator with any subset of I in a similar manner.
The coefficients are totally symmetric in their indices so that L I is a function on the set; that is, L I is totally symmetric in its indices.
For example if I = {1, 2, 3}, We consider the non-commutative polynomial ring over in these operators over some constants K : The invariants are the zeroth order differential elements of this ring. For example, in K {1,2} both L 12 − L 1 L 2 and L 21 − L 2 L 1 are zeroth order. Quite generally, for any index set I, because the differential operators transform as L J → g −1 L J g, for an arbitrary function g, any polynomial in the L J , F (L J |J ⊆ I), transforms similarly: F → g −1 F g. In the case where such an F happens to be a zeroth order operator (i.e. a function), it is therefore invariant.
In the paper [2] a large class of invariants is constructed.
We can describe such invariant elements by finding the kernel of a map Θ defined by θ being regarded as an arbitrary function. Θ acts as a derivation on the ring K I . We can then take a set of such maps {Θ i |i ∈ I} corresponding to differentiation of elements of K I with respect to indices by choosing θ = x i . We illustrate the methodology by a redescription of the classical case. Thus and so on. In particular we see that I 12 and I 21 are invariants because and similarly for I 21 .
We can use the Θ i maps to analyse the intertwining relation. By applying Θ 1 and Θ 2 successively we obtain, for the σ case for example, the following non trivial relations: The last tells us that L σ 1 = L 1 and we then rearrange the second to give I σ 21 = I 12 . The first equation can then be written Returning to the deformed case (3) we may analyse this is the same way. By derivation using Θ 1 and Θ 2 we obtain the tower: From these equations (in reverse order) follow: Note that this defomation is not equivalent to the undeformed case: we cannot gauge away the φ term in the primed operators by writing L 2 = g −1 L 2 g without introducing compensating terms in L 12 and so on.
From equation (5) we obtain, by putting Then φ is determined by the invariants I 12 and I 21 via the following equation: In the limit that φ, but not φ −1 φ, 2 , tends to zero, we recover the classical case: φ −1 φ, 2 = I −1 12 I 12 , 2 and φ = I 12 .
Otherwise this equation looks not easy to solve and is the object of further study.
Third order case
In the third order case, I = {1, 2, 3} there are six invariants on two labels, The Jacobi identity, yields a single identity On three labels we have the set Because of the total symmetry of L ijk we may make any of these our choice for one independent invariant. Indeed under transpositions of indices: One checks the invariance of I 123 by, for example, and likewise with Θ 2 and Θ 3 .
We call the expression of L ijk in terms of invariants and the L i , the invariant expansion of L ijk . We will discuss the system form in a moment. However, it should be emphasised that scalar and system forms are not interchangeable at order three unlike the order two case. We are generally able to write neither the scalar form as a 3 × 3 system nor such a system in scalar form.
First order intertwiner
In seeking to generalise the Laplace maps to the third order case we consider intertwining relations of the form L σi 123 L i = L σi i L 123 . For simplicity of notation we will consider i = 1 and write σ 1 = σ.
This definition of the first order intertwiner is a natural, formal generalization of the classical case. It would imply that if φ ∈ ker L 123 then L i φ ∈ ker L σi 123 . Apply the operators Θ 3 , Θ 2 , Θ 1 and Θ 2 1 to obtain the tower of equations Equations (7) and (8) are Laplace maps of the earlier kind (with the label 1 replacing 2) so that we immediately deduce: Equation (10) is consistent with the above and requires in addition, Equation (11) on the other hand implies a constraint on the invariants, a condiation that requires satisfaction, if the Laplace map is to exist. The condition is If we restrict attention to the differential ring in the coefficients of the differential operators, i.e. we do not employ any extension, then, up to a multiplicative scalar, we conclude that Unfortunately this relation is not preserved under the Laplace map: We seek to understand if further constraints arise from equations (9) and (6). Dealing with equation (9) first, we can write it, using the expansion in invariants, as an expression relating I σ 123 to I 321 : Finally we deal with equation (6). Once more using the invariant expansion of L 123 and L σ 123 : Incorporating what we have learnt already, Summarising: the existence of a Laplace map of the form L σ 123 L 1 = L σ 1 L 123 requires conditions on the form of the third order operator which can be expressed as the constraints on invariants arising from the three different expressions, (11) and (12) or, if the choice I 21 = I 31 is made These conditions amount to differential constraints on the coefficients a i , a ij and a ijk .
Deformed first order intertwiner
It is natural to ask if we can escape the constraints by considering the deformed relation: From (14) and (15) we will obtain Apparently we do not thereby escape constraints since the integrability conditions on φ require .
As before there is a limit in which φ but not φ,1 φ tend to zero and this corrresponds to the previous relations: I σ 12 = I 21 etc. Again, study of this situation is postponed to another time.
Second order intertwiner
A second possibility for implementing a type of Laplace transform is to consider a second order intertwiner, We proceed as before and indeed it turns out that this imposes fewer constraints on the invariants of L 123 . Θ 3 yields only trivial identities. The tower of identities obtained by applying Θ 1 and Θ 2 is: Again equations (23) and (24) are of the classical form for a Laplace transformation and they give us In particular, no constraint arises at this level. Equation (25) is dealt with using the invariant expansions The result is an expression for I σ 123 : We anticipate that equations (21) and (22) will give expressions for I σ 12 and I σ 21 and no constraints. Write (21) as Using the expansions the relation simplifies to Then Define and from equation (22), by transposition of 1 and 2, Note that, following from the properties of I ijk under transposition Using the relations L σ 2 I 32 = I 32 L 2 and L σ 1 I 31 = I 31 L 1 we get expressions for I σ 21 and I σ 12 : Finally we must analyse the relation (20) itself: At this point all the I σ invariants are defined in terms of the I invariants as are the L σ i so the most we can hope for here is a consistent set of relations but we might expect, instead, constraints to arise.
System form
In [1] Laplace type maps are developed for third order systems of the form In this section we provide a slight generalization of those results.
The set of transformations under which invariants are defined are taken to be conjugations of the matrix differential operator by diagonal matrices of three arbitrary functions A number of variant canonical forms are possible. For example, choosing g, 1 +h 11 g = 0 and g 1 = g, 1 , g 2 = gh 21 , g 3 = gh 21 h 32 we obtain (13)), 13 .
The deformed intertwining relation generalises that for the 2 × 2 case. For example, The Laplace maps respect the differential identities satisfied by the antisymmetric invariants. [12] The other algebraic constraint on the symmetric invariants (123) σ (132) σ = (12) σ (23) σ (31) σ provides a second (complicated) condition, not identically satisfied, on α and β, thus determining them. Note that in [1] the simplifying choice u 22 = u 33 has been made which leads to the relation α = β. It was not appreciated at that time that such a choice leads to constraints on the invariants. As is seen above allowing u 22 = u 33 leads to more complexity. This is currently being explored.
Scalarizability
Because the differential operator of the 3 × 3 system is defined over a noncommutative ring one cannot, in general, reduce to a single scalar equation as might over a field. But it is clear that the vanishing of any of the h ij is a sufficient condition to do so and we say that in such a case the system is scalarizable.
For example, if h 23 = 0 then we can write down a scalar equation for φ 2 of the form (L 3L1L2 −L 3 (12) − (13)L 2 + (132))φ 2 = 0, By comparing with the invariant expansioñ Finally we would like to see that the system Laplace map corresponds with a scalar one. It suffices to check that the invariants transform in the same way. The restriction (23) = 0 is consistent with (but not equivalent to) the choice α = β = 0 which follows from the assumption u 11 = h 11 and l 11 = h σ 11 , corresponding to the undeformed 3 × 3 system. This in turn implies as desired, sinceĨ 32 = 0.
Conclusion
This paper has examined in detail a natural generalization of the classical Laplace map on invariants of second order, partial differential operators to third order operators of both scalar and system forms and compared them in the overlap where a system is scalarizable. The basic obervation is that such intertwining maps of scalar operators can only exist where prior conditions exist on the invariants. It is shown by example that these can be compatible with the scalarizability of a system for which more general (unconstrained) Laplace maps exist. There is a further generalization of the notion of Laplace map which would allow us to avoid the constraints in the case of the second order intertwining relation. We call this a weak Laplace map: Consider the inhomogeneous partial differential equation L 123 φ = ψ and suppose that the intertwining relation holds only up to an unspecified function, K: L σ 12 L 123 − L σ 123 L 12 = K. Then the conditions (21) to (25) hold but not (20) and the relations between the I σ and the I are as in the last section but without the constraint (28). This allows us to define the weak Laplace map: where, φ → φ σ = L 12 φ, ψ → ψ σ = L σ 12 ψ − Kφ. There seems to be some scope here for further work. Apart from the study of weak maps and the deformed intertwining relations, other natural questions include: • What are the general constraints on the invariants of a 3 × 3 (or higher order) system that make it scalarizable?
• What conditions need to be satisfied by the invariants of a scalar operator in order for it to be written in system form?
• Does the second order intertwining map also correspond to a Laplace map of a 3 × 3 system?
Acknowledgements
I would like to thank the two anonymous referees for their thorough reading of the manuscript and their suggestions for clarification and improvement. This paper was written during the last weeks and days of my close friend and colleague, Jon Nimmo. It is appropriate to record here my appreciation of Jon's support, generous encouragement and humour over many years in Glasgow. | 2018-01-05T12:36:31.000Z | 2018-01-05T00:00:00.000 | {
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49233516 | pes2o/s2orc | v3-fos-license | Investigating the Relationship among the Level of Mobbing Experience , Job Satisfaction and Burnout Levels of Primary and Secondary School Teachersi
The aim of this study is to examine the level of mobbing experienced by primary and secondary school teachers and to determine how and to what extent this affects their job satisfaction and burnout levels. This research used a relational survey model. As a result of the study, it has been determined that there is a negative and medium-level relationship between the teachers’ level of exposure to mobbing towards their profession or social relationships and their job satisfaction levels; a positive and medium-level relationship between the teachers’ level of exposure to mobbing towards their profession or social relationships and their burnout levels; and a negative and low-level relationship between their internal and external job satisfaction levels and their burnout perceptions. According to the results of the path analysis, the teachers’ job satisfaction level decrease in line with an increase in their mobbing experiences related to their profession and social relationships, and thus, their burnout levels also increase. However, it has also been determined in the study that in line with an increase in the internal and external job satisfaction levels of teachers, their burnout perceptions decrease. Accordingly, it has been concluded that mobbing towards their profession and social relationships is a stronger predictor of burnout level in comparison to job satisfaction.
Introduction
In an organisational environment, many different variables may have an effect on the job dissatisfaction and burnout of workers.One of these variables is mobbing.Mobbing is a concept that triggers stress, enhances its scope and renders it more serious [1].Mobbing is systematically exposing one or (rarely) several employees to emotionally deleterious behaviours in the workplace by one or more employees (rarely more than four) each day or for some months [2].The studies carried out by Işık [3] and Tayyar [4] showed that there is a significant relationship between mobbing practices and job stress level and job performance.Mobbing behaviour at work, which occurs as a result of the accumulation of psychological factors leading to tension and conflicts in the organisation, is one of the main problems that distorts organisational order, decreases performance, harms motivation, impairs commitment to work and has a negative effect on job satisfaction and working environment [5], [6], [7].Besides its negative effect on workers, mobbing negatively effects the organisations and causes loss of time and energy [8].
Burnout is a main factor affecting the job stress levels of employees; this and the accompanying loss of productivity are among the unfavourable conditions commonly in many social organisations.Burnout means an individual becoming insensitive to other people they meet as a result of their work, feeling exhausted and feeling less successful [9].Burnout has a negative effect on occupational performance.Even if the individual does not leave their job, it reflects the unfavourable experiences of their work.Rapid change of expectations concerning the organisation and the environment, and the individual's inability to keep up with this rapid change, enhances the feeling of burnout.Crowded places, limited opportunities, administrative pressure, low motivation of the administrators, unfavourable working conditions and personal and family matters are among the prominent reasons for burnout among workers [10].It is considered that emotional stress factors observed, especially in jobs necessitating face-to-face contact with people, are related to burn out, as different from other factors [11].
Another main factor affecting the job stress levels of the employees is burnout.According to Izgar, when the employees achieve their expectations in their working life, they feel more positive and this helps them develop a positive attitude towards their work [12].Job satisfaction corresponds to the attitude of an individual towards his work and covers his knowledge, beliefs, feelings, behaviours and considerations [13].Job satisfaction is a pleasurable or positive emotional state resulting from the appraisal of one's job or job experiences [14].Studies examining the relationship between the job satisfaction levels of workers and their psychological and physical health indicate that those with higher job satisfaction levels are healthier and have less stress in comparison with other employees [15].Moreover, high job satisfaction levels affect psychological and physical health and enhance the self-confidence of an employee [16].From a different perspective, job dissatisfaction leads to anxiety, depression, abstention, disciplinary problems and high employee turnover rates [17].
Research carried out in Turkey especially during 1980s on the job satisfaction [16], [18], [19], [20], [21], [22], [23], [24] and as of the mid-1990s on burnout [25], [26], [27], [28], [29], [30], [31] and on the job stress levels of teachers [3], [23], [32], [33], [34], [35] showed that the job satisfaction levels of teachers are low while their burnout levels are high that the teaching profession is more stressful in comparison with the other professions and that teachers face stress intensely in their working environments.Research carried out in other countries [1], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46] revealed that individuals who are exposed to mobbing have lower levels of job satisfaction, higher levels of burnout, bear indications of mental fatigue, have an increasing rate of psychological health problems, have higher levels of depression and anxiety, display more aggressive behaviours and experience serious health impairments.Korkmaz and Cemaloğlu pointed that in educational institutions with intense social relationships, mobbing weakens organisational trust and climate [47]; and Zapf argued that mobbing impairs organisational climate, leads to job stress and therefore increases accompanying problems [46].Moreover, Araujo et al. and Toker Gökçe determined that the mobbing at work, organisational climate and psychological and physical health of workers are related, while Jenkins et al. determined that mobbing at work and job performance of the workers, their motivation and organisational trust are related [37], [42], [48].Kocaoğlu reported that mobbing has a negative effect on the motivation of the people exposed to it [6].Sürgevil stated that there is a negative relationship between job satisfaction and burnout [49].Some researchers consider that job satisfaction decreases as a result of burnout.Although both concepts point to internal, negative and psychological experiences, burnout differs from job satisfaction by corresponding to a decrease in energy for work.Moreover, burnout is distinguished from job satisfaction as it is stated to mean psychological tension caused by occupational stress [50].
Matters such as the differentiation of social roles, conflicts experienced in relationships among workers, existence of competition in an organisational environment, self-actualisation efforts of the workers, high expectations negatively affecting the psychological health of workers, cause stress.In this regard, this study aims at examining the level of exposure to mobbing among the primary and secondary school teachers, and how and to what extent this affects their job satisfaction and burnout levels.By penetrating into the living spaces of organizations, making a close analysis of mobbing which has a negative effect on quality and success and examining the variables that may be related with mobbing may facilitate the understanding of the problem.It is important that teachers are aware of mobbing, an obstacle to a favourable working environment and of its harmful effects.This research aims to highlight this harmful effect and to ensure a favourable organisational climate for teachers, to increase the learning success of the students by these means, and therefore, to make a contribution to the quality of education.
Mobbing
According to Leymann, mobbing is psychological terror by one or several people towards another person or other people, by systematic hostile and unethical practices [1].Mobbing is a kind of systematic and long-term behaviour that directly targets an employee and may cause psychological and physiological damage [51] and is any kind of behaviour towards an employee such as maltreatment, threatening, violence and humiliation displayed by senior staff, subordinates or colleagues of an equal rank [52].As understood from the above-mentioned definitions, mobbing is a concept that exists since the beginning of the history of working life; however, there is no commonly accepted understanding about its definition.Moreover, some people who are exposed to mobbing at work consider this a routine conflict in the workplace and an everyday problem.This misleads the research and renders it difficult to make a diagnosis [53].
Work-related Mobbing
Branch defines work-related mobbing as hiding important information from the employee, continuously monitoring their work and gossiping about them [54].Mobbing may be seen in various ways, for example implying that the employee is a liar, teasing them, gossiping about them or hindering their communication with other people, subjecting them to a heavy workload, alienating them, subjecting them to verbal threats and supervising them often and unnecessarily [8].According to Einarsen and Raknes, work-related mobbing covers the prevention of teachers to obtain information that may affect their success, humiliation by employing them below their competences, assignment of tasks below their abilities, gossiping about them and ignoring, excluding and disregarding them [40].Leymann (1993) lists some of the work-related mobbing behaviours as [1], [53], [55], [56], [57]: (1) not assigning the important tasks, (2) limitation of tasks, (3) asking for meaningless tasks, (4) assigning tasks below competences, (5) continuously assigning new tasks, (6) assigning tasks that impair Investigating the Relationship among the Level of Mobbing Experience, Job Satisfaction and Burnout Levels of Primary and Secondary School Teachers self-esteem, (7) assigning tasks not complying with his nature, (8) forcing them to undertake a difficult task, (9) make them pay for losses.
Mobbing Directed at Social Relations
Receiving attention in a group, having a place there and feeling important to the group, are among the most basic needs of an individual.Social relationships are an important means of communication that makes existence meaningful [6], [58].Mobbing may sometimes be directed at hindering the social relationships that are among the basic needs of an employee [58].According to Einarsen and Raknes, mobbing directed at social relationships covers using offensive terms and behaviours about someone's personality, attitude and private life, unpleasant jokes, excessive teasing and ridiculing, shouting at them and displaying momentary anger, pointing at them and bullying behaviours such as attacking someone's personal sphere, pushing or intercepting them [40].Leymann (1993) summarises mobbing related to social relationships as follows [1], [53], [55], [56], [57]: (1) no-one talks with the employee and they are excluded from the group, (2) they are not allowed to speak with other employees and are deprived of the right to meet with other people, (3) they are left isolated and alone (4), other people are prohibited to talk with them, (5) they are ignored.These behaviours will not only affect those who are exposed to mobbing but also employees witnessing the process and cause them to lose their trust in the organisation.
Burnout
The concept of burnout was first defined in 1974 by Freudenberger, and in the last 20 years much research has been carried out in various fields of work.Freudenberger defined burnout as being unsuccessful, fraying, decreasing in energy and power and a state of exhaustion in the internal resources of a person, as a result of unsatisfied desires [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72].Today, the most widely recognised definition of burnout is the three-dimensional definition developed by Maslach and Jackson and it pioneers the studies in this field [9].According to Maslach and Jackson, burnout means 'emotional exhaustion, depersonalisation towards other people at work and reduced feeling of personal accomplishment.'Burnout that teachers or schools administrators face is addressed in three dimensions as emotional exhaustion, depersonalisation and personal accomplishment [9], [62].Depersonalisation points to the interpersonal dimension of burnout [60] and is observed as attitudes and behaviours devoid of feelings displayed by an individual towards the people they serve, ignoring them as an individual.The person displays an inhumane, teasing, humiliating, strict, stony and indifferent attitude [11], [73].Emotional exhaustion is defined as a state of burnout caused by excessively burdening the employee with work [74].It is stated that burnout starts with emotional exhaustion and the other dimensions are felt later on and that emotional exhaustion is the most important determinant of burnout [29].
Emotional exhaustion is the feeling of burnout due to excessive work load.The individual feels emotionally exhausted and thinks that they cannot fulfil their responsibilities towards the institution [69].Feeling that their emotional resources are exhausted, the individual begins to think that they are not as productive and responsible as before, and when they are not able to overcome this state, they may not want to go to work, may be late, may be absent or may resign [75].After reduced personal accomplishments, the individual with negative thoughts about other people begins to think negatively also about themselves [76], [77].The individual whose personal accomplishments have reduced feels that they cannot progress and even are regressing in their profession [78] and begins to feel guilty.This may cause the individual to value themselves negatively, to believe that they are not effective in the organisation, to lose self-respect and to sink into depression [11].
Job Satisfaction
Since the 1940s, job satisfaction has been among the most commonly discussed topics in the field of management.According to Balcı (1985), job satisfaction means the feelings aroused in an individual in relationship to whether their requirements, expectations and desires concerning the different dimensions of work are satisfied.Job satisfaction as a phenomena is an emotional reply caused by the interaction of values related to work and acquisitions from work [79], [80], [81], [82].Job satisfaction, one of the most effective means of increasing the productivity of a worker in the working environment, reflects the attitude of the employer towards the work; when this attitude is positive, the level of job satisfaction is also high and vice versa.However, in order to increase productivity in this sense, organisations should consider all psychological, economic and social expectations of the employee.Adaptation to work and the rules of the organisation will improve job satisfaction and productivity [83].Keeping the communication channels open between the administrators and workers and providing the opportunity to the workers to express themselves are important for the enhancement of job satisfaction.If the worker participates effectively in the decision-making process, they begin to embrace work more and feel more satisfied [84].
Job dissatisfaction is depressing for the worker and may cause them to have negative feelings.When job dissatisfaction leads to anxiety that is intense and permanent, the psychological health of the worker may be effected negatively and result in unfavourable behaviours within the organisation, such as weariness, leaving the job, absenteeism and quarrelsomeness [82].In his study, Balcı determined that school administrators feel job dissatisfaction, although not at high levels [16].There are many factors affecting the job satisfaction of the worker.Individual factors or personal factors are elements leading to different job satisfaction levels [85].Internal factors represent themselves and the need for them emerges automatically.These factors are related with meeting superior needs such as personal requirements and reputation [23].Organisational (external) factors are grouped into the nature of the work, management style, supervision type, feeling of security, communication, opportunities of income, improvement and promotion, competition, colleagues and organisational environment [85].External factors include management, technical matters, colleagues, as well as requirements concerning payments, promotion and advancement, and guarantees [23].
1.1.6.The Relationship among the Variables of Mobbing, Job Satisfaction and Burnout Existence of mobbing attack at work may be related with organisational culture.If policies to observe, prevent or punish mobbing attacks are not established in an organisation, the attackers have the opportunity to apply mobbing and accordingly the organisation will have to bear the consequences [86].Research regarding the effects of mobbing at work on job satisfaction [39], [87], [88] indicated a negative relationship between mobbing and job satisfaction and showed that the psychological health of those who are subjected to mobbing is affected by decreasing job satisfaction.In the study carried out by Çivilidağ among university academics, it was found out that there is a negative relationship between the mobbing experiences of academics at work and their job satisfaction and perceived social support levels [89].Moreover, in Doğan's study on the relationship between mobbing behaviours and job satisfaction, a negative and medium-level relationship was determined between these two variables [90].A study carried out by Okan among secondary school teachers revealed no significant relationship between the mobbing experience of teachers and their job satisfaction levels [91].However, Dinçer's study pointed that the level of exposure to violence is rather high among nurses, mobbing behaviours affect their job satisfaction levels and thus, they think of leaving the job [92].
Burnout is an important result of violence at work.Burnout, which is simply defines as the 'exhaustion of psychological and physical energy' [60], not only affects the working life of the individual but also their whole life [93].Alkan's study revealed a positive relationship between the mobbing behaviours and the burnout levels of teachers [94].Karakuş and Çankaya stated that stress and burnout levels of teachers also increase in line with an increase in the mobbing experience [95].Alkan reported that there is a significant and positive relationship between the mobbing and only the 'emotional exhaustion' sub-dimension of burnout while there is not a significant relationship between the 'personal accomplishment' and 'depersonalisation' sub-dimensions [94].Similarly, the study of Filizöz and Ay revealed a positive relationship between mobbing and the emotional exhaustion and depersonalisation sub-dimensions of burnout and indicated that there is not a significant relationship between mobbing and personal accomplishment [96].In Bucaklar's study carried out among teachers, it was found out that there is a positive relationship between mobbing and burnout [97].A positive relationship was determined between mobbing and burnout in the study by Varhama and Björkqvist in Finland among municipality workers and that by Einarsen, Matthiesen and Skogstad in the health sector in Norway [98].Koustelios and Tsigilis found that there is a negative and significant relationship between internal job satisfaction and burnout [99].The study carried out by Gençer among teachers and that carried out by Bayram, Gürsakal and Bilgel among academics found a negative and significant relationship between the job satisfaction levels of workers and their burnout levels [100], [101].
Feeling of satisfaction in the organisation by employees is mostly possible through an inherent communication among them.This satisfaction affects the commitment of the employee and thus, the level of commitment plays an important role for the maintenance of the organisation [102].Considering the effect of mobbing at schools, which increases the stress and burnout levels of teachers and decreases organisational commitment, job and life satisfaction, and the trust in the administration and the organisation [90], [95], [103], [104], [105] the question of which behaviours cause mobbing at which level becomes important.Therefore, determining the mobbing behaviours at schools and trying to lessen the accompanying negative effects on the employees and the organisation constitute the first step of the fight against mobbing.This research aims at examining the level of exposure to mobbing among primary and secondary school teachers, and how and to what extent this affects their job satisfaction and burnout levels.
Purpose of the Study
This research was carried out to examine the level of exposure to mobbing among primary and secondary school teachers, and how and to what extent this affects their job satisfaction and burnout levels.To this end, the following questions were posed: 1) What is the level of exposure to mobbing (in the dimensions of work-related mobbing and mobbing directed at social relationships) among primary and secondary school teachers?2) What is the level of job satisfaction (in the sub-dimensions of internal satisfaction and external satisfaction) among primary and secondary school teachers?3) What is the level of burnout (in the sub-dimensions of depersonalisation, emotional exhaustion and personal accomplishment) among primary and secondary school teachers?4) Is there a significant relationship between the level of exposure to mobbing among primary and secondary school teachers and their job satisfaction and burnout levels?5) How and to what extent does the exposure to mobbing among primary and secondary school teachers affect their job satisfaction and burnout levels?
Investigating the Relationship among the Level of Mobbing Experience, Job Satisfaction and Burnout Levels of Primary and Secondary School Teachers
Research Model
This research used the relational survey model, a research model for detect the existence and/or level of covariance between two or more variables [106].In this research model, there are three variables, two independent and one dependent.Work-related mobbing is a part of the independent variables of the research model.Another independent variable is the mobbing directed at social relationships.However, as job satisfaction has a direct and indirect effect on burnout, it was addressed as both independent and dependent variable.During the path analysis in the research, sub-dimensions of mobbing were not measured separately; and the processed data were addressed as two separate mobbing types, 'work-related mobbing' and 'mobbing directed at social relationships'.Other independent variables of the research are internal and external satisfaction, which are the sub-dimensions of the job satisfaction of teachers.Dependent variables of the research are the depersonalisation, emotional exhaustion and personal accomplishments that are the sub-dimensions of burnout.Accordingly, how the level of exposure to work-related mobbing and mobbing directed at social relationships among primary and secondary school teachers affect their perceived job satisfaction and burnout levels is examined in the proposed model.It is presumed in the study that the perception of teachers regarding their experience of work-related mobbing and mobbing directed at social relationships affect job satisfaction directly and burnout levels both directly and indirectly through job satisfaction.
Population and Sample
The research population of this study comprised 1946 teachers in total, working in 48 primary school (1058 primary school teachers) and 44 secondary (888 branch teachers) schools in the central district of Siirt and in its district centres during the 2014-2015 academic year.In order to obtain more reliable research data and as the number of teachers included in the research population is not high, sampling was not made.The data of 1116 teachers who did not fill in the forms or who filled the form in deficiently were excluded from the evaluation.The data of 830 teachers were found eligible for analysis.Thus, nearly 43% of the target population provided usable data for analysis.36.1% of the teachers who participated in the research were female and 63.9% were male.When the distribution by the seniority variable is examined, it is seen that the percentage of teachers in the seniority group 1-5 years (47.6%) and (33.6%) years is higher than other groups, while the percentage of teachers in the 16-20 years (7.9%) and 21 years or above is lower than other groups.The percentage of class teachers who participated in the research was 53.2% and that of branch teachers was 46.8%.
Negative Acts Questionnaire
In order to determine the frequency of exposure by the teachers to negative acts addressed under the scope of the mobbing dimensions as work-related mobbing and mobbing directed at social relations, the Negative Acts Questionnaire Scale (NAQS) developed by Einarsen and Raknes was utilised [40].NAQS is a five-point Likert scale answered and graded as daily (5), weekly (4), monthly (3), now and then (2), never (1).In the validity and reliability study, two factors were determined in the NAQS.Total variance explained by the first factor, work-related mobbing, was 39.40% and the eigenvalue of this factor was 9.03.Variance explained by the second factor, mobbing directed at social relationships was 12.41% and the eigenvalue of this factor was 4.63.Total variance explained by both dimensions was 51.81%.The first factor was related to mobbing directed at social relationships and composed of 15 items.Factor loads varied between .45 and .80.The second factor concerns work-related mobbing.In this dimension, six items were included.The factor loads of the items under work-related mobbing varied between .47 and .74.In order to determine the reliability of the NAQS, the Cronbach Alpha coefficient was utilised.The Cronbach Alpha coefficient of internal consistency was measured both for the whole scale and for each sub-dimension separately and specified as criteria of reliability.Internal reliability coefficient of these two sub-dimensions was .89 in the dimension of mobbing directed at social relationships and .76 in the dimension of work-related mobbing.In all dimensions of the scale, item-test correlations of 21 items varied between .49and .80.
Maslach Burnout Scale and Reliability Study
The Maslach Burnout Scale developed by Maslach and Jackson [9], which is used widely to determine the burnout levels of teachers, is composed of 22 items and addresses burnout in three sub-dimensions.In the Maslach Burnout Inventory (MBI), items under the sub-dimensions of emotional burnout and depersonalisation were scored alike while the items under personal accomplishment were subjected to adverse scoring and the total score was obtained [59].A high score obtained in the sub-dimensions of emotional exhaustion and depersonalisation and a low score obtained in the personal accomplishment scale point to burnout.Medium-level scores in all three scales correspond to medium levels of burnout.Validity and reliability coefficients of MBI in teachers were determined by Maslach and Jackson.The reliability coefficient of the scale was .88 for emotional exhaustion, .83for personal accomplishment and .72 for the depersonalisation sub-dimension.Girgin and Baysal [107] tried to adapt the scale for Turkey.Girgin determined the reliability coefficient as .87 for emotional exhaustion, .74 for personal accomplishment and .63 for depersonalisation.However, Baysal determined it as .74for emotional exhaustion, .77for personal accomplishment and .75 for depersonalisation [107].Under this research, in the validity and reliability study of the MBI, it was determined that the scale has a three-factor structure.Total variance explained by the first factor, emotional exhaustion, was 24.30% and the eigenvalue of this factor was 8.07.Variance explained by the second factor, the depersonalisation dimension of burnout, was 13.91% and the eigenvalue of this factor was 5.93.Variance explained by the third factor, the personal accomplishment dimension of burnout, was 8.02% and the eigenvalue of this factor was 4.24.Total variance explained by three dimensions was 46.23%.The first factor was related to emotional exhaustion and composed of nine items.Factor loads varied between .49and .83.The second factor concerns the depersonalisation dimension.In this dimension, five items were included.Factor loads in the depersonalisation dimension varied between .52 and .79.The third factor refers to personal accomplishment and includes eight items.Factor loads in the personal accomplishment dimension varied between .55 and .84.In the research, the Cronbach Alpha reliability coefficient of the MBI was determined as .89for emotional exhaustion, .68 for the depersonalisation sub-dimension and .77for the personal accomplishment sub-dimension.
Minnesota Job Satisfaction Scale Short Form
In order to determine the job satisfaction levels of teachers, the Turkish version of the Minnesota Job Satisfaction Scale developed by Weiss, Davis, England and Lofquist [108] was utilised.The scale was translated into Turkish by Baycan [109].The Minnesota Job Satisfaction Scale comprised 100 questions but in this research the short form of the scale, which covers 20 items, was utilised and each item was scored in a five-point Likert scale (1 = very dissatisfied, 2 = dissatisfied, 3 = undecided, 4 = satisfied, 5 = very satisfied).The Minnesota Job Satisfaction Scale has two sub-dimensions, internal and external.Internal job satisfaction was measured through 11 items while external job satisfaction through items.Numerical values of the five-point Likert scale under job satisfaction scale are as follows: Very dissatisfied, 1.00-1.79;Dissatisfied, 1.80-2.59;Somewhat satisfied, 2.60-3.39;Satisfied, 3.40-4.19;and Very satisfied, 4.20-5.00.In the validity and reliability study, two factors were noted for job satisfaction.Total variance explained by the first factor, internal job satisfaction dimension, was 34.40% and the eigenvalue of this factor was 14.023.Variance explained by the second factor, external dimension of job satisfaction, was 16.81% and the eigenvalue of this factor was 6.83.Total variance explained by both dimensions was 51.21%.The first factor was composed of 11 items and the factor loads varied between .54 and .85.The second factor was composed of nine items and the factor loads varied between .49and .78.In order to determine the reliability of job satisfaction scale, the Cronbach Alpha coefficient was measured.Internal reliability coefficients of these two sub-dimensions were .91 for internal job satisfaction and .78for external job satisfaction.The reliability coefficient of the whole scale was .87.In all dimensions of the scale, item-test correlations of 20 items in the sub-dimensions of the scale varied between .49and .85.
Data Collection and Analysis
Scales were applied to 1946 teachers in total, working in 48 primary schools in the central district of Siirt and in its district centres.After the application, forms that were not filled or filled deficiently were sorted out and the data in the remaining 830 forms were evaluated.AMOS 22 and SPSS 21 statistical package programmes were used for data analysis.Descriptive statistics of the variables were produced using SPSS 21 while AMOS 22 was used to test the research models.The research hypotheses were tested at .01 and .05significance levels.
Findings Regarding the Exposure of Teachers to Mobbing Behaviours and Their Job Satisfaction and Burnout Levels
Table 1 displays the descriptive statistics concerning the mobbing experience of primary and secondary school teachers who participated in the research, and their job satisfaction and burnout levels.
As seen in Table 1, the average of the scores given by the primary and secondary school teachers, which was x = 1.56,S=78 for the work-related mobbing and x = 1.37, S = 53 for the mobbing directed at social relationships reveal that the teachers experience mobbing 'now and then'.It may be stated that the internal job satisfaction levels of primary and secondary school teachers are good ( x = 3.54, S = .74)while their external job satisfaction levels are at a medium level ( x = 3.12, S = .65).
Considering the distribution by burnout levels of the primary and secondary school teachers, in Table 1 the highest score is observed in emotional exhaustion ( x = 3.07, S = .81),while the lowest is seen in the depersonalisation ( x = 2.08, S = .59)sub-dimension.It was determined that the personal accomplishments of the teachers are at a medium level.In other words, these may be construed as teachers have exhausted their internal resources, have difficulty in getting into contact with other people and considering themselves as incompetent of a good performance.
Findings Regarding the Correlations between the Sub-dimensions of Mobbing, Job Satisfaction and Burnout
Table 2 displays the correlational relationships between the mobbing, job satisfaction and burnout variables.
Investigating the Relationship among the Level of Mobbing Experience, Job Satisfaction and Burnout Levels of Primary and Secondary School Teachers As seen in Table 2, on the basis of the perceptions of the primary and secondary school teachers, there is a negative and medium-level significant relationship between the exposure to work-related mobbing and mobbing directed at social relationships and their internal (r = −.39,p < .01;r = −.35,p < .01)and external (r = −.31;p < .01;r = −.30;p < .01)job satisfaction levels.Similarly, a positive and medium-level relationship was reported between the exposure to work-related mobbing and mobbing directed at social relationships among the primary and secondary school teachers and the emotional exhaustion (r = .52,p < .01;r = .41,p < .01)and depersonalisation (r = .41,p < .01;r = .43,p < .01)sub-dimensions of burnout.Moreover, a negative and low-level relationship was noted between the exposure to work-related mobbing and mobbing directed at social relationships among teachers and the personal accomplishment (r = -.17,p < .01;r = -.15,p < .01)sub-dimension of burnout.The analysis carried out revealed a negative and low-level relationship between the internal and external job satisfaction levels of teachers and the emotional exhaustion (r = −.22,p < .01;r = −.24,p < .01)and depersonalisation (r = −.21,p < .01;r = −.15,p < .01)sub-dimensions of burnout.In addition, it was specified that there is a positive and low-level relationship between internal job satisfaction levels and the personal accomplishment (r = .24,p < .01)sub-dimension of burnout while there is not a significant relationship between external job satisfaction levels and the personal accomplishment (r = .03,p < .01)sub-dimension of burnout.
How and to What Extent does the Exposure to Mobbing Behaviours among Primary and Secondary School Teachers Affect Their Job Satisfaction and Burnout Levels?
Path analysis was carried out to determine how and to what extent does the exposure to mobbing behaviours towards primary and secondary school teachers affect their job satisfaction and burnout levels.Through path analysis, direct and indirect effects of predictor variables on predicted variables were determined.As a result of the analysis, some fit indices related to compliance of the model to data were examined.Most frequently used fit indices are x 2 , GFI, AGFI, CFI, NFI, IFI, RMSEA and AIC.Among these fit indices, x 2 is sensitive to sample size and therefore, it is necessary to utilise x 2 together with other indicators.In terms of fit indices, certain criteria are applied, such as x 2 /sd value should be lower than 5, GFI should be higher than .90,IFI and CFI should be higher than .95, and RMSEA should be lower than .06[110], [111].Moreover, if GFI, NFI, CFI and AGFI values, as fit indices are higher than .90and RMSEA is lower than .05,this indicates that the model complies well with the data [112].In this research, fit indices measured in relationship to the compliance of the model (NFI = .96,CFI = .95,IFI = .98,RMSEA = .067,x 2 /sd = 48/10 = 4.8 < 5.00) show that the model complied well.Standardised path coefficients regarding how and to what extent does the exposure to mobbing behaviour among the primary and secondary school teachers affect their job satisfaction and burnout levels are given in Figure 1.
Figure 1 shows that when the standardised path coefficients are examined, work-related mobbing has slightly better indicators, in comparison with the mobbing directed at social relationships and that the internal job satisfaction dimension (−.94) is the best indicator of job satisfaction.With regard to burnout, the emotional exhaustion dimension (1.00) is a stronger indicator than the depersonalisation dimension (.87).Work-related mobbing (β = .51)is more effective on burnout, in comparison with job satisfaction (β = −.23).This finding indicates that work-related mobbing is a stronger predictor for burnout than job satisfaction.A negative and medium-level relationship is observed between work-related mobbing and job satisfaction.Moreover, mobbing directed at social relationships (β = .31)is more effective on burnout, as against job satisfaction (β = −.23).This finding reveals that mobbing directed at social relationships is a stronger predictor for burnout than job satisfaction.However, work-related mobbing and mobbing directed at social relationships have a direct effect on burnout while work-related mobbing has an indirect significant effect through job satisfaction.In line with the increase in the exposure to work-related mobbing and mobbing directed at social relationships among the teachers, their job satisfaction levels decrease and thus their burnout levels increase.Nevertheless, the research showed that the effect of work-related mobbing (β = −.38) and mobbing directed at social relationships (β = −.32) on job satisfaction is negative and significant.Based on this finding, it may be deduced that the job satisfaction levels of the teachers may decrease upon their exposure to work-related mobbing and mobbing directed at social relationships.In other words, in line with the increase in the exposure to work-related mobbing and mobbing directed at social relationships among teachers, their job satisfaction levels decrease and burnout levels increase.When it is considered that the direct effect of job satisfaction on burnout is negative and significant (β = −.23), it may be stated that the burnout perceptions of the teachers may be decrease in parallel to the increase in internal and external job satisfaction levels.Moreover, 28% of the total variance of the job satisfaction variable is explained on the basis of work-related mobbing and mobbing directed at social relationships.In addition, 16% of the total variance of the burnout variable is explained on the basis of work-related mobbing and mobbing directed at social relationships and a direct effect of the job satisfaction latent variable, as well as an indirect effect of work-related mobbing and mobbing directed at social relationships through the job satisfaction variable.
Discussion
As a result of the research, it has been determined that Investigating the Relationship among the Level of Mobbing Experience, Job Satisfaction and Burnout Levels of Primary and Secondary School Teachers according to the perceptions of primary and secondary school teachers; teachers are exposed to unfavourable acts connected with the work-related mobbing dimension 'now and then'.This finding is in parallel with the findings of the research carried out by Cemaloğlu [113], Gündüz and Yılmaz [114], Einarsen and Rakness [40], Karyağdı [115], Kul [116], Kılınç [117], Okçu [118] and Onbaş [119].
When the studies concerning the frequency of the teachers' exposure to mobbing are examined under the related literature, it can be seen that different findings were obtained in these studies.Cemaloğlu [120], Hoel et al.
[121], Hubert and Veldhoven [122], Dick and Wagner [41] and Toker [123] determined that the level of exposure to mobbing among the teachers is at medium level.
Considering the replies of the teachers regarding the acts connected with the work-related mobbing dimension, it can be seen that the teachers are exposed to mobbing but their level of exposure is low.As a result of the research, it has been determined that the perceptions of the primary and secondary school teachers regarding the mobbing directed at social relationships dimension is at the 'now and then' level.This result confirms the findings of the research carried out by Cemaloğlu [113], Cemaloğlu and Ertürk [8], Ertürk [124], Ergener [125], Gündüz and Yılmaz [114], Kılınç [117] and Onbaş [119], Sağlam [126] Toker [123] and Okçu [118].Exposure to mobbing directed at the social relationships dimension among the teachers indicates that they are exposed to mobbing from top to bottom and horizontal mobbing, and that these two types of mobbing are more common in educational institutions.Intimidation of teachers by their colleagues in schools may be caused by the differences in the education understanding.Exposure to mobbing by the administrators or their colleagues has a negative effect on the climate in educational institutions.However, it is difficult to claim that teachers are exposed to mobbing systematically and intensely.
It has been determined that the internal job satisfaction levels of the teachers are at a good level while their external job satisfaction levels are at a medium level.It has also been specified that the worker with a high level of job satisfaction is healthier in comparison with the other workers and that they feel less stressed.Result of the research is remarkable in this regard.Many researches, especially those carried out after the 1980s, have revealed that the job satisfaction levels of the teachers are not at the required level [16], [18], [19], [20], [21], [22], [23], [24].
When the results concerning the burnout levels of the primary and secondary school teachers are examined, it has been found out that emotional exhaustion sub-dimension has the highest value while the depersonalisation has the lowest.The finding of this research is also supported by those obtained by Budakoğlu [127], Kepekçioğlu [128], Yıldız [129], Özgüner [50], Cerit [130] and Acar-Arasan [131].Moreover, research on burnout, carried out by Koçak [64], Cemaloğlu and Erdemoğlu Şahin [26], Kayabaşı [132], Coşkun [133], Ergül, Saygın and Tösten [134], Torun [135], Budak and Sürgevil [60], Avşaroğlu et al. [72], Tümkaya [136] reveal that teachers are exposed to mobbing.It is stated that emotional exhaustion found out to be high among primary and secondary school teachers may be caused by the problems in the triangle of school, student and parents; mostly by the inadequacies and disappointments related with the teaching profession.Moreover, it is also noted that excessive work load and stress may cause teachers to feel exhausted emotionally.
As a result of the research, it has been determined that there is a negative and medium-level relationship between the exposure to work-related mobbing and mobbing directed at social relationships among the teachers and their job satisfaction (internal and external) levels.Much research on the effect of mobbing at work on job satisfaction has been carried out both domestically and abroad [39], [87], [88], [89], [90], [91], [92] and has revealed that there is a negative relationship between mobbing and job satisfaction and that mobbing affects the psychological health of those exposed to it and decreases their job satisfaction.Şahin [137] stated that mobbing mostly affects the job satisfaction of the employee and their tendency to act unproductively.
A positive and medium-level relationship was reported between the exposure to work-related mobbing and mobbing directed at social relationships among the teachers and their burnout levels at the emotional exhaustion and depersonalisation sub-dimensions.Moreover, the relationship between the exposure to work-related mobbing and mobbing directed at social relationships among the teachers and their burnout levels at personal accomplishment sub-dimension is found to be negative and at a low level.In much research carried out both domestically and abroad [39], [94], [96], [97], [98] it has been determined that there is a positive relationship between the mobbing and burnout.
As a result of the analysis, a negative and low-level relationship was noted between the internal and external job satisfaction levels of the teachers and their burnout perceptions at emotional exhaustion and depersonalisation sub-dimensions.Moreover, it has been reported that there is a positive and low-level relationship between the internal job satisfaction levels of the teachers and their burnout levels at the personal accomplishment sub-dimension and that there is not a significant relationship between the external job satisfaction levels of the teachers and their burnout levels at the personal accomplishment sub-dimension.The research carried out by Koustelios and Tsigilis [99], Gençer [100], Bayram, Gürsakal and Bilgel [101] revealed a negative and significant relationship between the job satisfaction levels of the workers and their burnout levels, and thus it supports the results of this research.
Conclusions
According to the path analysis carried out under this research, in line with the increase in the exposure to work-related mobbing and mobbing directed at social relationships among the teachers, job satisfaction levels of the teachers decrease and, accordingly, their burnout levels increase.In other words, the job satisfaction levels of the teachers increase and correspondingly their burnout levels may decrease when the level of exposure to work-related mobbing and mobbing directed at social relationships among the teachers decreases.Moreover, it was also detected in the research that in line with the increase in external and internal job satisfaction levels of the teachers, their burnout perceptions may also diminish.In addition to this, it can be concluded that work-related mobbing and mobbing directed at social relationships are stronger predictors of burnout than job satisfaction.
In conclusion, mobbing negatively affects the human resources, quality and success by silently penetrating into schools, and has also a negative effect on the job satisfaction levels of the teachers and thus, increases their burnout levels.It may be ensured that teachers train in a healthy organisational climate by taking measures in this regard and applying sound policies accordingly.By this means, the learning success of the students may be enhanced and therefore, by making a contribution, the quality of education may also be improved.Based on the results of the study, the following suggestions may be made: 1) Research to establish the reasons for low job satisfaction levels and high burnout levels among the teachers may be carried out by utilising different methods (qualitative research).2) More comprehensive research on the reasons of mobbing at schools may be carried out by utilising different methods (qualitative research).3) Similar research may be carried out in the secondary schools and higher education institutions in different provinces and regions.
Figure 1 .
Figure 1.Results of standardised path analysis
Table 1 .
Descriptive statistics concerning the mobbing experience, and the job satisfaction and burnout levels of the teachers, on the basis of the perceptions of primary and secondary school teachers
Table 2 .
Correlational relationships between the mobbing, job satisfaction and burnout variables | 2018-06-16T12:57:28.469Z | 2017-01-01T00:00:00.000 | {
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79038481 | pes2o/s2orc | v3-fos-license | Pattern of breast cancer risk factors among pre and post‐menopausal women at a Primary Care Clinic in Nigeria
Context: The incidence of breast cancer is increasing worldwide. In black women, breast cancer is associated with aggressive features and poor survival. Objective: Identification of risk factors such as early age of menarche, obesity and family history of breast cancer may permit preventive strategies. Study Design: A cross‐sectional comparative study design was used and questionnaires were administered to 400 adult women at a tertiary health centre in Nigeria. The data was analyzed with the Statistical Package for the Social Sciences version 17; the level of significance set at alpha = 0.05. Results: There was significant association between pre‐menopausal and post‐menopausal women with positive family history of breast cancer with P = 0.010. Majority of the respondents with a positive family history of breast cancer were menopausal (P = 0.010). There was a statistically significant association between menopausal status and ever consuming alcohol‐based herbal concoctions (P = 0.010) and in those whose partners smoked cigarettes (P = 0.001). Majority of respondents were not currently on any form of contraceptives. Parity, breastfeeding and use of hormonal contraceptives were all statistically significant (P < 0.001, P < 0.001 and P = 0.004, respectively). Almost all the women in our study, 97%, had never had a mammogram. There was a significant association between pre‐menopausal and post‐menopausal women with positive family history of breast cancer (P = 0.010). Conclusion: With increasing incidence of breast cancer worldwide and late presentation in developing countries with high morbidity and mortality, effective screening for risk factors will go a long way in reducing the incidence of breast cancer.
Introduction
Breast cancer is a non-communicable disease of huge public health importance and is the most common cancer in women in Nigeria and worldwide. [1,2] Globally, the incidence of breast cancer is increasing and even more so in societies that previously had a low incidence of the disease. [3] The highest number of deaths from breast cancer are from the United Kingdom, where approximately 42000 new cases are diagnosed annually, with a mortality rate of 15000 per Pattern of breast cancer risk factors among pre and post-menopausal women at a Primary Care Clinic in Nigeria annum. [4] In the United States of America, the incidence rate of breast cancer is 200,000 per annum, with a mortality of about 40000 per year. [4] The prevalence of breast cancer in Nigeria was 116 per 100,000 and 27840 new cases were expected to develop in 1999. [2] In a more recent retrospective study by Agbo et al., reported in 2014 from the northern part of Nigeria, the prevalence rate of breast cancer was found to be 10.4 cases per 100,000. [5] The incidence rate of breast cancer in Nigeria has steadily increased from 15.3 per 100,000 in 1976 to 33.6 per 100,000 in 1992. [6] Jedy-Agba et al., in 2012, analyzed data from the Ibadan Population Based Cancer Registry (IBCR) and the Abuja Population Based Cancer Registry (ABCR) and reported an age standardized incidence rate (ASR) of breast cancer of 52.0 per 100,000 women in Ibadan, Nigeria and 64.6 per 100,000 women in Abuja, Nigeria. [7] Akarolo-Anthony et al., in 2010, reported that in Nigeria, there was a rise in the number of women at risk of breast cancer from approximately 24.5 million in 1990 to approximately 40 million in 2010 and will likely reach over 50 million by 2020. [8] Reasons adduced for the high incidence of cancer now in Nigeria could be increased reporting and improved diagnosis. [4] There is also a trend toward westernization in developing countries, with the change in demographic profile and lifestyle, and the changing socioeconomic profile of the country.
In assessing the risk of developing breast cancer, a multidisciplinary team approach should be employed. Medical and surgical history, history of exposure to possible carcinogens and a detailed family history of cancer is obtained while physical examination and radiologic imaging studies are also done. [9] The factors to be elaborated upon for pre-menopausal women include increasing age, the density of the breast, history of breast or ovarian cancer in any family member and previous procedures on the breast. [10] For post-menopausal women, areas of emphasis are age, breast density, race, ethnicity (especially Ashkenazi Jewish), family history of breast cancer, a breast procedure in the past, body mass index, menopause occurring naturally, hormone therapy, and a previous false-positive mammogram. [10] Notable risk factors for breast cancer include age at menarche, first child birth, and menopause; parity; number of breast biopsies done in the past; histological findings of atypical hyperplasia or lobular carcinoma in situ in breast tissue; use of oral contraceptive pills and nulliparity. [9] There are also environmental influences important in the aetiology of breast cancer, as well as genetic factors. [3,11] Approximately 5-10% of breast cancers are familial and mutations in breast cancer susceptibility genes -BRCA 1 and BRCA 2 -are responsible for 80% of the familial cases while sporadic cases are rare. [1] Other risk factors include previous thoracic radiation therapy and the use of hormones with history of present or past use of oestrogen and progesterone. Body mass index, breast density at mammography, alcohol consumption, diet and physical activity are also implicated. [9] In a case-control study of the epidemiological risk factors for breast cancer done in the Oncology clinic of University College Hospital (UCH), Ibadan, the mean patient age of 43 years was obtained and it was also found that the incidence of breast cancer in Nigeria was increasing. [3] The incidence of breast cancer increases with age and doubles every 10 years till menopause. [12] In the Western world, the age specific incidence of breast cancer appears to increase with age. Approximately 50% of breast cancers occur in the age group 50-65 years and 30% occur over the age of 70 years. [4] Breast cancers in Nigerian women is, however, noted to present a decade earlier than in developed countries. [4,13] The age at development of breast cancer is reducing with worse prognosis in black women, and in these group of women, breast cancer has aggressive features and poor survival. [6] The global age-standardized mortality rate for female breast cancer is shown in Figure 1, with some of the highest mortality rates found in Africa. Determining a woman's breast cancer risk will allow early detection and prompt treatment. Low technology, low-cost management options and examining the value of genetic risk factors of breast cancer in determining a population at risk are required. [3] The incidence of breast cancer is increasing rapidly in Nigeria, therefore, there is a need for determining the pattern of breast cancer risk factors among pre-and post-menopausal women and encouraging appropriate follow-up of clients with high risk of breast cancer.
Materials and Methods
This was a cross-sectional comparative study and was conducted among 400 adult female patients above the age Ibadan is the largest city in west Africa, and has a population of 2.9 million people. [14] Women who were too ill to go through the rigours of the study and pregnant women were excluded.
The sample size was estimated by using the formula for simple proportions with an expected average prevalence of combined breast cancer risk of 33%. [15] The following assumptions were also made: Type 1 error was 0.05, the area under the normal curve was equivalent to 1.96 at 95% confidence interval and the precision was 5%.The consecutive sampling method was adopted in recruiting the participants. Validated questionnaires with sections on assessment of sociodemographic characteristics, gynaecological history, breast cancer screening, family history and lifestyle factors were administered by the interviewer after informed consent was obtained from the participants. The University of Chicago Cancer risk clinic new patient risk assessment questionnaire was adopted. Data were collected over a period of 7 months. Body mass index was used to define and classify obesity. [16] Approval was obtained from the Head of Department of Family Medicine, UCH, Ibadan and the ethics committee of the joint University of Ibadan (UI)/UCH institutional review board. The UI/UCH EC registration number obtained was NHREC/05/01/2008a. All patients recruited were given health education and referred appropriately to general surgery and/or radiotherapy specialists within the health facility for further evaluation and management, if indicated.
The data collected from the participants were coded serially, cleaned and entered into a computer using the software programme; Statistical Package for Social Sciences for data analysis (SPSS ® version 17) was used for data analysis. Discrete variables were summarized using proportions and Chi-square test were used to explore statistical relationships.
Results
The frequency distribution of sociodemographic characteristics of the 400 respondents is shown in Table 1. Over two-thirds of the women were pre-menopausal with the highest proportion of women being between 25 and 34 years old. The greatest proportion of women who were post-menopausal was those above 55 years of age. All the single respondents were pre-menopausal. Majority of the married respondents were pre-menopausal (69.5%), whereas many of the widowed respondents were post-menopausal (79.5%).
Among the pre-menopausal group of respondents, the highest level of education of the respondents was tertiary education (81.6%) followed by secondary education (79.6%). However, among the post-menopausal group of respondents, non-formal education (76.3%) was the most common followed by primary education (60.4%). Most of the pre-menopausal respondents were students (94%), whereas majority of the post-menopausal respondents were traders (38.3%). Many (71.4%) of the premenopausal women were Christians whereas the highest proportion among the post-menopausal women were Muslims (33.3%). The Igbo tribe was the most predominant among the post-menopausal respondents (33.3%), whereas it was the least among the pre-menopausal group of respondents (66.7%).
The distribution of lifestyle and risks factors of the respondents is depicted in Table 2. Out of the 400 respondents in this study, 280 (70%) were pre-menopausal whereas 120 (30%) were post-menopausal. Only a small proportion 15 (3.8%) of the respondents had a positive family history of breast cancer with the majority 9 (60.0%) being post-menopausal. There was significant association between pre-menopausal and post-menopausal women with positive family history of breast cancer with P = 0.010.
Only 2 (66.7%) of the pre-menopausal women and 1 (33.3%) post-menopausal women had ever undergone surgical procedures such as hysterectomy and oophorectomy. There was no significant association between pre and post-menopausal women and ever having any surgical procedures (P = 0.899). Majority of the respondents, 9 (69.2%) and 11 (68.8%), respectively, were currently consuming alcohol at least once a week and had ever consumed alcohol once a week for six months or longer were pre-menopausal.
Twenty-two (5.5%) of the respondents had taken alcohol-based herbal concoction, out of which 12 (54.5%) of the respondents were post-menopausal. There was a statistically significant association between ever consuming alcohol-based herbal concoctions (P = 0.010). Only one (0.3%) of the respondents had ever chewed tobacco and was pre-menopausal. Fourteen (3.5%) of the respondents had their spouses smoking cigarettes. There was a statistically significant association between pre-menopausal and post-menopausal women whose partners smoked cigarettes (P = 0.001).
The highest proportion 158 (40.1%) of the respondents were of normal weight. This was followed by overweight 112 (28.4%) and obese respondents were 93 (23.6%) with class 1 being predominant 60 (64.5%). There was no statistically significant association between pre-menopausal and post-menopausal women who were obese using the body mass index category (P = 0.118).
The gynaecological history of the respondents is shown in predominated in this set. Intrauterine contraceptive device (IUCD), injectables and oral contraceptives were the three most frequently used forms of hormonal contraceptive by pre-menopausal women, i.e. 24 (47.1%), 21 (77.8%), 10 (76.9%), respectively; whereas, IUCD was the single most common form of hormonal contraceptive used by postmenopausal women. Majority of women in our study were not on any form of contraceptive. Parity, breastfeeding and use of hormonal contraceptives were statistically significant (P < 0.001, P < 0.001 and P = 0.004, respectively), however, the duration of breastfeeding was not statistically significant.
Most women in our study 97% never had a mammogram and two-thirds 273 were pre-menopausal (70.4%). Two-hundred and thirty-three women (58.3%) had examined their own breasts for lumps whereas 167 (41.8%) did not examine their breasts. In both groups, the pre-menopausal fraction constituted approximately two-third of the women (70.4% and 69.5%, respectively).
The proportion of women who examined their breasts for lumps and those who did not examine their breast were 58.3% and 41.8%, respectively. The proportion of women in both groups were alike 70.4% and 69.5% in the pre-menopausal category and 29.6% and 30.5% in the post-menopausal category, respectively.
The majority of women in our study 88.5% (354) had never detected a breast lump. Most of the women in this group were also pre-menopausal, 69.5% (246), with only 30.5% (106) in the post-menopausal group. Approximately 12% (46) of the 400 women evaluated had detected a lump. In this category, the pre-menopausal women also predominated 73.9% (34).
Discussion
The highest proportion of women (26.8%) in this study were those between age 25-34 years for pre-menopausal respondents and greater than 55 years for the post-menopausal respondents. This is comparable to a study in Nigeria in which the women were found to be in a similar age group bracket. [17] In another Nigerian study, the highest prevalence of breast cancer was in those whose age group was 26 to 45 years, [18] whereas majority of cases in another study were in the age group 40 to 49 years. [19] Most of the married women in the study were pre-menopausal, whereas many of the widowed women were post-menopausal. This was comparable to a study in southwestern Nigeria. [2] The highest level of education of the pre-menopausal respondents in this study was tertiary education, whereas in the post-menopausal women it was non-formal education. This was in line with the study by Meshram et al. in 2009. [19] Studies have linked the risk of developing breast cancer with positive family history in first degree relatives (FDR). [20] This finding was in agreement with our study, which revealed family history as a strong risk factor to developing breast cancer (P = 0.01). Cancer in FDR was considered more important in post-menopausal women. [21] Ovarian cancer risk is lowered more than 90% in women with BRCA1 or BRCA2 mutations who decided to have risk-reducing salpingo-oophorectomy. In this same population, prophylactic removal of the ovaries was found to be associated with approximately 50% reduction in the risk of breast cancer. [22] Our study, however, did not show any relationship between breast cancer and salpingo-oophorectomy or hysterectomy.
The risk of breast cancer is said to increase by approximately 10% for each 10 g of daily alcohol consumption. [23] Prior studies of BRCA1/BRCA2 mutation carriers, however, did not find any increased risk with alcohol intake. [24,25] Our study found no significant interaction between alcohol, duration of consumption and the period of onset of breast cancer in either pre-menopausal or post-menopausal women. This was also buttressed by the Tecumseh Community Health Study (TCHS). [26] There was, however, a strong association between consumption of alcohol-based native medication and risk of developing breast cancer noted in our study, with (P = 0.01).
Weight gain and being overweight are well-known risk factors for breast cancer. In general, overweight women are most commonly observed to be at a greater risk of post-menopausal breast cancer and at lower risk of pre-menopausal breast cancer. Sedentary lifestyle may also be a risk factor. [27] In our study, there was no statistically significant association between obesity using body mass index and being pre and post-menopausal. One study [28] found a reduced risk of breast cancer among BRCA1/BRCA2 mutation carriers who smoked, however, an expanded follow-up study failed to find an association. [29] Our study however revealed strong association between women whose spouses smoked cigarettes (P = 0.001).
In the woman's contraceptive and Reproductive Experiences Study, women who reported having at least one full-term pregnancy before the age of 25 years were at 36% reduced risk of breast cancer compared to nulligravida. Parous women in the study with a late age at first birth had an increased risk of both ERPR-positive and ERPR-negative tumors with each additional birth although neither result was statistically significant. [30] Low parity and late age at first birth were confirmed as significant and independent determinants of breast-cancer risk. [31] Nulliparity was also associated with a 30% increase in risk compared with parous women, and for every two births, the risk was reduced by approximately 16%. [31] A woman's risk of developing breast cancer has been shown in various studies to be related to her exposure to hormones produced by her ovaries. Reproductive factors that increase the duration and/or levels of exposure to ovarian hormones, which stimulate cell growth, have been associated with an increase in breast cancer risk. These factors include early onset of menses, late onset of menopause, later age at first pregnancy and nulliparity. Pregnancy and breastfeeding are considered as protective against breast cancer as both reduce a woman's lifetime number of menstrual cycles, and therefore her cumulative exposure to endogenous hormones. [32] In addition, pregnancy and breastfeeding have direct effects on breast cells, causing them to differentiate, or mature, so that they can produce milk. Some researchers hypothesize that these differentiated cells are more resistant to becoming transformed into cancer cells than cells that have not undergone differentiation. [33,34] Breastfeeding for an extended period (at least 1 year) is associated with a decreased risk of both hormone receptor-positive and hormone receptor-negative breast cancer. [35,36] International Agency for Research on Cancer (IARC) has classified the current or recent use of combined oestrogen-progestogen oral contraceptives (OCs) as a cause of breast cancer. [37] OCs contain synthetic sex hormones, which may explain the link between OC use and breast cancer risk. An estimated 1% of female breast cancers in the UK are linked to OCs because breast cancer risk is generally low in the OC-using population (typically younger women). [38] A meta-analysis showed that current users of OCs have approximately 24% higher breast cancer risk compared to never users and that breast cancers in OC users tend to be less advanced compared with those in OC never-users. [39] The relative risk of breast cancer declines after OC cessation such that 10 years after cessation no excess risk remains. [39,40] Breast cancer risk does not appear to increase with longer duration of OC use. Young age at first OC use is associated with a larger increase in breast cancer risk. [39] The risks associated with OC use appears to be similar across OC formulations. [39,40] Some types of benign breast disease are linked with increased breast cancer risk. Among women with benign breast disease, cancer is more common in the breast with the benign disease than in the opposite breast. [41] Breast cancer risk is generally not increased in women in non-proliferative disease (NP). Proliferative disease without atypia (PDWA) has a breast cancer risk of 44% higher in women compared with women with NP. [42] However, breast cancer risk does appear to be increased in women with NP or PDWA and a FDR with breast cancer. [42] Atypical hyperplasia (AH) has close to a threefold higher risk for breast cancer in women compared with women with NP. Lobular AH is associated with higher breast cancer risk than is ductal AH. Having a FDR with breast cancer does not appear to further increase breast cancer risk in women with AH. [42] Breast cancer is a significant problem among women worldwide with associated high morbidity and mortality. The incidence of breast cancer is increasing worldwide with late presentation most especially in developing countries. Effective screening will go a long way in reducing the incidence of breast cancer. Effective screening through detailed family and gynaecological history with regular breast examination and radiological screening (sonomammogram and conventional mammogram) will allow for early diagnosis and prompt treatment of breast cancer, hence reducing morbidity and mortality associated with breast cancer. Mammograms should be encouraged as it has been reported in a Nigerian study that the level of awareness of mammograms is very low in Nigeria. [43] There was strong association between breast cancer and family history of breast and ovarian cancer among women attending the primary care clinic. This underscores the need for provision of screening services at the clinic and effective health education to promote preventive practices and inculcate a screening culture among women. | 2019-03-16T13:08:04.489Z | 2016-05-01T00:00:00.000 | {
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208745517 | pes2o/s2orc | v3-fos-license | Application for novel electrochemical screening of antioxidant potential and phytochemicals in Cornus mas extracts
ABSTRACT Cornelian cherry (Cornus mas L.), a fruit found in Central Europe, is used as a traditional ingredient in fruit preserves. In this study, we analyzed seven cultivars of Cornelian cherry growing in Poland and the differences between them in sugar content, total polyphenol content, and total chlorophyll content. Moreover, we measured the antioxidant potential of Cornelian cherry extracts was also measured. Polyphenols were found to be highest in Słowianin, Wydubieckij and Jolico cultivars. The presented research outcomes were extremely different from the results obtained for the other cultivars of Cornelian cherry. The electrochemical analysis of extracts proved that compounds contained in Cornelian cherry had the reducing against peroxyl radicals, which makes these extracts an important class of nutritional antioxidants. Our study showed that electrochemical assessment can replace the standard colorimetric tests in the evaluation of the antioxidant potential of analyzed compounds.
Introduction
The Cornus genus includes~40 species that are found in the natural state in the temperate climate zones. Cornelian cherry (Cornus mas L.) is a cultivated plant species that has attractive and concurrently useful fruits. Note that cornelian cherry can be mostly found as a shrub growing up to 9 m high as well as, although rarely, as a small tree (Czerwińska & Melzig, 2018). Cornelian cherry (Cornus mas L.) is recognized as a source of polyphenols, tannins, anthocyanins, and iridoids, all of which are present in both its fruits and leaves (Szczepaniak, Kobus-Cisowska, Kusek, & Przeor, 2019;Kucharska, Szumny, Sokól-Letowska, Piórecki, & Klymenko, 2015). Cornelian cherry is a plant grown in the Eastern and Southern regions of Europe and the Middle East, where it has been used in domestic cuisines for centuries (Czyżowska et al., 2017;Rop, Mlcek, Kramarova, & Jurikova, 2010). Cornelian cherry's edible parts are reddish, oval fruits, sweet, and sour in taste (Kucharska, Sokół-Łętowska, & Piórecki, 2011). Cornus mas is regarded as a source of natural radical scavenging anti-inflammatory and anti-toxic agents; however, this potential is deviated by natural causes, i.e. cultivar issue and genetic variation (Cornescu & Cosmulescu, 2017;Pantelidis, Vasilakakis, Manganaris, & Diamantidis, 2007). The primary substances identified and labeled in Cornelian cherry fruits (which are responsible for their biological activity) are vitamin C, anthocyanins, flavonoids, and iridoids (De Biaggi et al., 2018;Kucharska et al., 2015). In the literature, the issue of deviation between different Polish cultivars of Cornelian cherry has been examined using standard radical tests (Adamenko, Kawa-Rygielska, Kucharska, & Piórecki, 2018;Kostecka, Szot, Czernecki, & Szot, 2017;Kucharska et al., 2011). There have been no studies about the antioxidant profile analyses of Cornus mas using electrochemical tools and techniques.
Electrochemical assays can provide much more consistent and accurate results compared to colorimetric tests (de Macêdo et al., 2017;Keyrouz et al., 2011). Our aim was to fill this gap and show differences in polyphenols, sugars, and chlorophyll content between the seven cultivars grown in Poland and how these differences affect the antiradical properties of the examined cultivars. The fruit extracts were analyzed in two different solvents, i.e. water and 40% ethanol, to verify the differences in the migration of bioactive compounds during the extraction phase. We conducted standard colorimetric tests, i.e. total phenolic content (TPC), 2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging together with two separate electrochemical analyses, to verify the similarity between the comparisons performed by our team.
Material
The ripe fruits of Cornelian cherry were harvested in September 2018 in the orchard farm Szynsad in Dąbrówka Nowa, Błędów, Mazowieckie, Poland (51°47′01″N 20°43′04″E). We collected Cornelian cherry in production orchard where the fruit harvest took place from the third year of cultivation. Soil in the orchard was characterized by an average abundance of macronutrients. The approximate value of pH for soil, marked in 1M KCl, was 6.1. The content of humus was 1.1%. The average amount of precipitation in the growing season was 320 mm per square meter, with an average daily temperature of 14.9°C. The fruits of seven cultivars cultivated in Poland: Bolestraszycki, Wydubiecki, Szafer, Jolico, Florianka, Słowianin, and P5. The fruits were stored in refrigerated conditions (cooling temperature = 4°C ) until the extracts were prepared.
Extraction
Cornelian cherry extracts were composed of fresh fruits in which the skin was cut, to allow proper maceration of the plant material. Then fruits were macerated with distilled water or 40% (v/v) ethanol at ratio fresh fruit: extractant 1:5 (w/v) for 30 min at 40°C. Then, the extracts were seeped through paper filters (Alfachem, Poznań, Poland). The prepared extracts were stored in dark tubes until examination at −21°C. Two extractants were chosen in relation to their applicability in food, i.e. aqueous extractsfor the production of functional foods, and ethanol-water extractsto use as tinctures.
Total phenolic content
TPC was evaluated according to Cheung et al.'s (2003) method with our some modifications. The tested extract (5 ml) and Folin-Ciocalteau reagent (5 ml; Chempur, Piekary Śląskie, Poland) were added to 50 ml volumetric flasks. Then, the two solutions were mixed for 5 s, followed by the addition of the supersaturated solution of Na 2 CO 3 and NaHCO 3 to the homogenized mixture. The sample was incubated in a darkroom for 90 min, and then the liquid was collected by decanting. The analysis was performed using the Specord 40 spectrophotometer (Analytic Jena, Germany), and the absorbance was measured at λ = 765 nm. Each sample was measured six-fold and for each tested extract were conducted three independent measurements. Gallic acid (Sigma-Aldrich, Germany) was used to prepare the calibration curve at five different concentrations: 22.21 mg/ml; 26.65 mg/ml; 31.09 mg/ml; 35.54 mg/ml and 44.42 mg/ml. The standards were measured six-fold. The linearity of the calibration curve (r 2 ) was 0.9523, and the curve was validated using internal cross-validation method. The total average recovery observed was 102.9%. The results were referenced to the dry mass of the extract and expressed in milligrams of gallic acid equivalents (GAE)/100 g dry mass (d.m.).
Antioxidant potential
We determined the inhibition capacity of ABTS radical according to Bartosz's (2003) methodology with some modifications. After the addition of 980 µl of ABTS solution, its absorbance was measured at λ = 414 nm. Then, 20 µl of the extract was added, and the absorbance of the solution was measured after 40 s. The standard ABTS radical inhibition curve was determined based on Trolox concentration. The linearity (r 2 ) of the calibration curve was 0.9829, and inhibiting capacity of the ABTS radical was defined by the sample absorbance at λ = 414 nm measured at t = 0 s (A') and the sample absorbance at λ = 414 nm measured at t = 40 s (A), as following Equation 1.
The inhibition capacity of DPPH radical was determined according to Kobus-Cisowska et al.'s methodology (2019) with some modifications. We added. 100 µl of the tested extract to 2.9 ml of 0.1 mM DPPH solution in a tube, and then the sample was mixed on a shaker for 3 s at 1500 rpm, which was deposited in a darkroom for 30 min. The calibration curve was performed based on Trolox solutions, and the linearity of the curve (r 2 ) was 0.9075. The inhibition capacity of the DPPH radical is expressed under Equation 2.
where A′ is the absorbance of the blank sample at λ = 517 nm (measured at t = 0 min), and A is the absorbance of the sample at λ = 517 nm (measured at t = 30 min). The results for tests with ABTS and DPPH were presented as milligrams of Trolox equivalent (TE)/100 g d.m. The radical inhibition activity of DPPH and ABTS was performed using the Metertek SP-450 spectrometer (Metertek, Taiwan).
Total chlorophyll content
Total chlorophyll content (TCC) was determined according to Arnon (1948) and the absorbance spectra were obtained at λ = 652 nm using the Specord 40 spectrophotometer (Analytic Jena, Germany). TCC was calculated according to Equation 3 and expressed in mg/g d.m.
where A 652 is the absorbance at a wavelength of 652 nm; V is the total volume of the extract [ml] (the total volume needed to determine the dry matter was 1 ml); and W is the mass of the sample equivalent to dry mass of the extract [g]. All measurement was conducted in threefold repetition.
Semi-qualitative assay of sugars
Sugars were determined by high-performance liquid chromatography (HPLC) on Hitachi-L-2000 Series LaChrom Elite chromatograph (set: automatic sample feeder L-2200, double pump L-2130, refractometric detector RID L-2490 and UV detector L-2400). We used the Rezex ROA Organic Acid H+ (8%)-300 × 7.80 mm column (Phenomenex) for the measurements. We used 0.0225 N H 2 SO 4 as an eluent at an isocratic flow rate of 0.6 ml/ min. The measurements were carried out at 30°C. The samples (10 μl) were applied to the column, and quantitative and qualitative identification was conducted using the external standard method, and integration with the measurements of the area under the curve and peak height was performed with the support of EZChrom Elite software (Agilent, USA).
Electrochemical assay
The antioxidant capacity of examined extracts was determined based on the value of the oxidation potentials of the electroactive compounds present in them. It was measured by dissolving the extracts in phosphate buffer (1 M, pH = = 7.0). The determination was carried out according to Filipiak et al.'s method (2001). Voltammetric measurements were performed using PGSTAT12 device with the GPES 4.9 control software (EcoChemie, The Netherlands). The measuring system comprised a reference electrode Ag/AgCl (3 M KCl) (Mineral, Poland), platinum as an auxiliary electrode (Mineral, Poland); and carbon paste as a working electrode (CPE), which was developed according to a previously described procedure (Ligaj, Tichoniuk, & Filipiak, 2008). Carbon paste was made by mixing graphite powder with mineral oil (Sigma) in the ratio of 70:30 (w/w). The surface of the CPE electrode was renewed after each measurement by rubbing the outer layer of the paste on tissue paper, and a new paste was applied and polished on a matte microscope slide. The measuring vessel had a diameter of 8 mm and a capacity of 3.5 ml. We added 500 µl of the examined aqueous extract to 500 µl of 0.05 M phosphate buffer with pH 7.0. The measurement cycle included the following stages: activation of the CPE electrode surface in phosphate buffer (60 s with the potential of +1.7 V); immersion of the electrode in the extract (120 s); and voltammetric measurement in the range from −0.6 V to +1.4 V. We performed two independent cyclic voltammetry (CV) and square wave voltammetry (SWV) measurements in three repetitions for each aqueous extract. SWV voltamperograms were smoothed using Savitzky-Golay's method (Press et al., 1992). From all SWV voltamperograms, we subtracted the baselines determined with moving average procedure (n = 3). We then calculated the oxidation and reduction potentials, corresponding to peak heights (currents), peak areas, and total peak areas for each of the extracts, and a new E dm coefficient was determined for CV and SWV measurements: Furthermore, electrochemical index (EI) for anode peaks measured by CV technique was determined according to Macedo et al.'s (2017) method with the abovementioned modifications. The average potential was assumed for the formula for nonseparated peaks with common intensity and area.
Statistical analysis
We performed a statistical analysis of all the results using Microsoft Excel 2013 software (USA). For TPC, ABTS, DPPH, and electrochemical tests, principal component analysis (PCA) was conducted to verify the relation between these factors and to present the deviation between the tested cultivars. The electrochemical results were treated as an additional factor to the model based on standard analytical techniques. To calculate PCA model qualitative factors, i.e. defined sample, extractant and cultivar were applied. Moreover, the p values for Levene's test of independent variables were calculated using Statistica 13 software (StatSoft, Poland).
Scavenging of TPC, ABTS, and DPPH radicals
Based on the current literature on the state of Cornelian cherry properties, the cultivar impact on the antioxidative potential of their aqueous and alcoholic extracts was analyzed. Selected Cornelian cherry cultivars were characterized in terms of TPC (mg GAE/100 g d.m.). The results are presented in Table 1. Moreover, we conducted the analyses of the scavenging ability of ABTS and DPPH radicals. The content of polyphenols in Cornelian cherry ranged from 289.69 to 2598.39, mg GAE/100 g d.m. and the highest value measured for was obtained for the alcoholic extract of cv. Jolico (Table 1). The other cultivars had significantly lower TPC values compared to cv. Jolico. The mean TPC of all analyzed samples was 839.48 ± 610.14 mg GAE/100 g dm. The poorest in polyphenolic compounds was cv. P5. Moreover, the alcoholic extracts had generally higher TPC values than the aqueous ones. The extracts can be divided into two groups: samples that were not significantly varied (BA, BW, FW, PA, SA, SW, XW, VW, and CW) and other those that varied distinctly.
The highest ABTS radical scavenging potential was presented for the aqueous extracts of Słowianin and Szafer cultivars (Table 1), and the inhibitory potential was higher for a majority of samples. The mean inhibition rate for all tested samples was 36.759% ± 26.793%. However, presenting ABTS scavenging results in relation to dry mass indicates that the predominant antioxidant properties had cv. Jolico and Słowianin. Moreover, there was no significant difference between the aqueous and alcoholic extracts. Cultivar P5 had the poorest ABTS scavenging properties presented as both TE concentration and percentage of the inhibition ratio. The results for cv. Jolico was significantly different from the average for all tested cultivars (Figure 1(a)). All the results obtained for TPC and antiradical activity against ABTS seem to be within the limits of double standard deviation (2σ), except CA sample. Levene's test results for independent samples indicate a significant relationship between TPC and antiradical activity against the ABTS radical (p = .000980 for α = 0.05). The highest antioxidant activity regarding DPPH radical was noted for the alcoholic extracts of cultivars Jolico and Wydubiecki (Table 1). The mean scavenging rate was 72.086 ± 15.009% and gave 2.932 mmol Trolox equivalents/100 g d.m. Alcoholic extract results from cv. Jolico and P5 significantly differ from the other tested cultivars (Figure 1(a)). Levene's test results showed no correlation between the antiradical activity against ABTS and DPPH radical (p = .068474 for α = 0.05). The relationship between total polyphenol content (TPC) and antiradical activity measured for DPPH radical seems to be significant (p = .001004 for α = 0.05).
Total chlorophyll content
TCC of the extracts tested ranged from 0.073 to 6.281 mg/g of dry matter (Figure 2). The highest chlorophyll content was found in the alcoholic extract of Wydubiecki cultivar, while the lowest content was found in the alcohol extract of Florianka cultivar. Note that the chlorophyll content was similar for most of the extracts that were studied, except the PA, SA and XA samples which exceeded the average level. Similar to TPC, the relationship between TCC and the inhibition ability of ABTS and DPPH radicals was verified. We found using Levene's test for independent variables strong statistical relationship between the TPC and antioxidative capacity against DPPH (p = .037801, α = 0.05) while the relation between TCC and antioxidative capacity against ABTS was not significant (p = .617441 α = 0.05).
12-30% of all sugars detected in this fruit; however, the share of glucose was the highest and varied from 43% to 50% of the total sugars (Figure 3). Table 2 shows the results of CV and SWV electrochemical analyses. The aqueous extracts of Cornelian cherry revealed four clear signal maxima appearing in the forward step (oxidation signals), which were identified on CV voltamperograms. These overlapping signals (current peaks) were located in the potential region from 0.2 to 0.65 V and were characterized by the current values from~ca. 0.14 to~ca. 0.67 µA, depending on the cultivar tested. Note that the other measured voltammetric signals had very low intensity, and the location of current peaks originating from the same electroactive analytes may slightly differ because of differences in the pH of extracts, obtained from the quantitatively differentiated set of compounds present in specific cultivars (Kilmartin, 2001). The cathodic current (associated with the reduction), we generally detected only one broad signal of low intensity, which was located in the region from −0.1 to 0.5 V. The highest total area of cyclic voltammetry peaks for Cornelian cherry samples relative to dry matter was found in the Słowianin cultivar; however, the lowest number of compounds in the extract volume was reported for the Florianka cultivar. The average value of this coefficient for all Cornelian cherry samples was 238.551 ± 112.123 V*µA/100 g d.m. SWV technique was used to confirm that the Jolico cultivar contains the highest quantity of electroactive components with antioxidant potential, while the P5 cultivar has the lowest concentration of the target components ( (Table 3); however, there is no clear relationship for TPC. The data in Table 3 indicate that the correlation between electrochemical signals measured by SWV technique is clearly dependent on chlorophyll content in the extracts and activity toward ABTS and DPPH radicals, although it is not a strictly linear relationship. PCA analysis shows that both CV and SWV results fit the analytical model based on standard photometric tests (Figure 1(b)).
Discussion
The average content of polyphenols is consistent with the data presented in previous studies ( Kucharska et al. (2011) and Rop et al. (2010) who indicated the TPC content for Szafer and Wydubieckij cultivars at 464.12 mg/100 g fresh mass and 811.00 mg/100 g fresh mass, respectively. Figure 1 shows that the high content of polyphenols in Cornus mas L. influences its high antioxidant properties to varying degrees, depending on the fruit cultivar, which is confirmed by the data presented in Figure 1(c). The antioxidant properties of polyphenols resulted from the presence of -OH groups, which reduces free radicals, as indicated in this study. According to Kobus-Cisowska et al. (2019a, 2019b, phenolic compounds in which the hydroxyl group is bound to an aromatic ring with acidic properties show special antioxidant properties, i.e. they easily capture the radical. The resulting free phenolic radical is stable enough (relocation of the radical because of a resonance) and is not able to disconnect the hydrogen atom from another one. However, according to Piekarska, Szczypka, Kucharska, and Gorczykowski (2018), anthocyanins and iridoids contained in Cornelian cherry fruit have a dominant influence on antioxidant properties. Iridoids are a large group of secondary metabolites and belong to the group of cyclopentane monoterpenes with a basic skeleton composed of a ring of cyclopentane and pyran. As reported by Czerwińska and Melzig (2018), iridoids, depending on their structure, show different pharmacological properties, including antibiotic, anti-inflammatory, or hypotensive properties. Furthermore, the studies have shown additionally the existence of statistically significant relationships between TPC and antioxidant capacity for ABTS and between TPC and antioxidant capacity for DPPH. The capacity is because of the strong polar extraction solvents used in the study and Cornelian cherry contains chemical compounds capable of extinguishing both cationic radicals and inert radicals. Vilano et al. (2007) and Nenadis, Wang, Tsimidou, and Zhang (2004) reported that catechins, phenolic acids, and kaempferol were characterized by the ability to scavenge DPPH radicals; however, the phenolic acids and quercetin have proven the extinction capacity of the ABTS radicals. Despite the big similarity in the substrates involved in the inhibition of these two radicals, their extinction turned out to be independent of each other, which may indicate the fact that other photochemically active compounds were present in Cornelian cherry extracts. As an element of novelty, electrochemical methods were used to evaluate the antioxidant activity of Cornelian cherry extracts. A statistically proven relationship between ABTS radical extinction and TCC can be justified by the presence of iron ions in the tested extracts. Iron ions can form complexes with chlorophyll ring, and thus better catalyze the oxidation of polyphenols. However, chlorophyll shows antioxidant properties that are three times weaker than Trolox (Wang & Wink, 2016). This is because of the easy exchange of Mg 2+ ions by chlorophyll molecules for ions of other bivalent metals (Kobus-Cisowska, Flaczyk, Rudzińska, & Kmiecik, 2014), which may explain the high antioxidant activity determined in the study of free radical tests (Table 1). Furthermore, chlorophyll α and β have clearly different reduction and oxidative potentials, which may result in better scavenging of these two radicals (Goedheer, Horreus De Haas, & Schuller, 1958).
All the factors tested in PCA (Figure 1(b)), excluding DPPH, are in the same part of the chart, that together with the high correlation (r 2 values) confirms the dependence of the data tested for these parameters. The location of the DPPH factor on the outside of the graph can be explained by low correlation rate measured between DPPH and the other factors. In the study of Cetkovska et al. (2015), a considerable correlation between TPC and DPPH scavenging capacity (r 2 nearly 0.8) was observed. Our research showed that TPC and DPPH are dependent, but weakly correlated, what leads to the final statement that DPPH scavenging is not solely caused by molecules active against Folin-Ciocalteau's reagent. Furthermore, Cetkovská et al. (2015) measured DPPH scavenging with the use of different analytical technique, i.e. electron paramagnetic spectroscopy, which makes our results not fully comparable.
Chlorophylls and carbohydrates as components of extracts may affect their activity, i.e. they may act synergistically or antagonistically with other components of the extracts. The close statistical distance between ABTS, DPPH scavenging and Table 2. CV and SWV peaks heights and areas for tested Cornus mas aqueous extracts and standards.
Tabla 2. Alturas pico y áreas de CV y SWV para extractos acuosos y estándar de Cornus mas. chlorophyll confirms the Levene's test results from point 3.3. Figure 1(c). Chlorophyll present in Cornelian cherry fruits may negatively affect the radical scavenging assays, by reoxidizing the radicals. This mechanism can be observed for the majority of samples subjected to DPPH radical scavenging test (Table 1). Samples richer in chlorophyll tended to be weaker DPPH scavenging agents, except sample XA. The basis for the electrochemical determination is the current that is formed during the redox reaction performed nearby the surface of working electrode. The electrochemical analysis of Cornelian cherry extracts showed the dominance of signals related to the oxidation of compounds because of the flow of anode current in the CV voltamperograms. The reduction signals were characterized by peaks with low intensity (low current values) and large width (wide potential range), which indicates that the compounds contained in the extracts or their derivatives obtained by oxidation (associated with the flow of anode current) were not reduced in the reverse scan. This could be because of many factors that have widely discussed Kilmartin's study (2001) in which detailed protocols for electrochemical analysis of natural antioxidants are presented. A similar phenomenon is observed, inter alia, on CV voltammograms for ascorbic acid in which the oxidation product of ascorbic acid (dehydroascorbic acid) is known to be relatively stable and may be reduced; however, the complete lack of its cathodic peak is observed. The location of current peaks in the region of relatively low potential ranging from 0.2 to 0.6 V (observed on SWV voltammograms) indicates that electroactive compounds contained in Cornelian cherry extracts show a high antioxidant activity. Their reduction potentials are lower than the reduction potentials of biologically damaging peroxyl radicals, and their addition in foods makes them an important class of nutritional antioxidants (Jovanovic, Steenken, Hara, & Simic, 1996). The intensity of the current peaks observed for aqueous Cornelian cherry extracts is ten-fold lower than those of de Macedo et al. (2017) for fruit extracts.for because of the possibly different dry mass content of extracts, which is reflected in the concentration of biologically active ingredients in the sample.
The glucose and fructose presence in tested extracts can affect their electrochemical profiles. In Ji et al.'s study (2017), glucose tested using screen printed electrodes possessed two characteristic peaks: the former measured at 0.2 V anodic current and the latter for the cathodic current at 0.3 V. Similar results provided Amani-Beni and Nezamzadeh-Ejhieh (2017) using CPE electrode modified with nanoscale CuO. The glucose peak was recorded in anodic current range of 0.1-0.2 V depending on the scan rate applied. In the cited study, the authors recorded an additional peak at cathodic sweep approx. 0.2 V. Fructose electrochemical signals are similar to signals of glucose, which causes their mutual distinction hardly without modifying the electrode detection layer (Amani Beni, & Nezamzadeh-Ejhieh, 2017;Nicolas, Pittson, & Hart, 2018). In our study, the over mentioned anodic peak was detected for samples VW, CW and surprisingly for quercetin (Table 2), while the cathodic signal was present in all tested samples (also quercetin and gallic acid), except BW and XW.
The examination of electrochemical signals for gallic acid and quercetin standards using the CV technique showed that these compounds could be found in P5 and Wydubiecki cultivars, as well as in Szafer, Słowianin, and Bolestraszycki cultivars, respectively ( Table 2). The location of peaks measured by SWV confirms the presence of quercetin in Szafer and Słowianin cultivars, indicating the presence of this compound in Florianka cultivar; however, the location of potentials in Bolestraszycki and Jolico cultivars indicates the presence of gallic acid. Table 3 lists the Pearson correlation factors and shows that the SWV technique represents the standard photometric tests such as TPC, TCC, and ABTS radical scavenging (r 2 values 84.95%, 85.49%, and 73.61%, respectively) compared to CV (31.20%, 52.96%, and 64.21%, respectively). In the case of the Folin-Ciocalteau test's results compared with Levene's p values indicate that the obtained relations were partially random, while the CV and SWV results set with other factors showed significant dependence.
Conclusions
SWV relates well with the following three parameters, i.e. TPC, TCC and ABTS radical scavenging. Relation with these factors was found to be less significant for the CV technique. The electrochemical detection using square wave voltammetry and our novel E dm parameter can effectively replace the abovementioned three methods. Cornelian cherry fruits and extracts obtained from them are potential sources of phenolic compounds and possess high antioxidant activity, which may act positively against oxidative stress and could be a raw material for producing supplements and functional foods.
Declaration of interest statement
We declare no conflict of interest. Table 3. Probability values for Levene's test of statistical independence (p) and Pearson correlation factor (r 2 ) between total polyphenol count, radical scavenging activities, total chlorophyll count and relative surface of peaks measured using SWV or CV techniques. | 2019-10-10T09:23:38.253Z | 2019-01-01T00:00:00.000 | {
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268201263 | pes2o/s2orc | v3-fos-license | An EWAS of dementia biomarkers and their associations with age, African ancestry, and PTSD
Background Large-scale cohort and epidemiological studies suggest that PTSD confers risk for dementia in later life but the biological mechanisms underlying this association remain unknown. This study examined this question by assessing the influences of PTSD, APOE ε4 genotypes, DNA methylation, and other variables on the age- and dementia-associated biomarkers Aβ40, Aβ42, GFAP, NfL, and pTau-181 measured in plasma. Our primary hypothesis was that PTSD would be associated with elevated levels of these markers. Methods Analyses were based on data from a PTSD-enriched cohort of 849 individuals. We began by performing factor analyses of the biomarkers, the results of which identified a two-factor solution. Drawing from the ATN research framework, we termed the first factor, defined by Aβ40 and Aβ42, “Factor A” and the second factor, defined by GFAP, NfL and pTau-181, “Factor TN.” Next, we performed epigenome-wide association analyses (EWAS) of the two-factor scores. Finally, using structural equation modeling (SEM), we evaluated (a) the influence of PTSD, age, APOE ε4 genotype and other covariates on levels of the ATN factors, and (b) tested the mediating influence of the EWAS-significant DNAm loci on these associations. Results The Factor A EWAS identified one significant locus, cg13053408, in FANCD2OS. The Factor TN analysis identified 3 EWAS-significant associations: cg26033520 near ASCC1, cg23156469 in FAM20B, and cg15356923 in FAM19A4. The SEM showed age to be related to both factors, more so with Factor TN (β = 0.581, p < 0.001) than Factor A (β = 0.330, p < 0.001). Genotype-determined African ancestry was associated with lower Factor A (β = 0.196, p < 0.001). Contrary to our primary hypothesis, we found a modest negative bivariate correlation between PTSD and the TN factor scores (r = − 0.133, p < 0.001) attributable primarily to reduced levels of GFAP (r = − 0.128, p < 0.001). Conclusions This study identified novel epigenetic associations with ATN biomarkers and demonstrated robust age and ancestral associations that will be essential to consider in future efforts to develop the clinical applications of these tests. The association between PTSD and reduced GFAP, which has been reported previously, warrants further investigation. Supplementary Information The online version contains supplementary material available at 10.1186/s13148-024-01649-3.
Background
Large-scale cohort and epidemiological studies suggest that stressful life events [1], psychological distress [2], and PTSD [3][4][5][6] confer increased risk for Alzheimer's disease (AD) and related dementias (ADRD) in later life.However, the causal mechanisms underlying this association, including the influence of genetic and epigenetic factors on the relationship between PTSD and ADRD, remain an open question.This study was designed to address this by examining age-and AD-associated biomarkers in an ageand ancestrally-diverse cohort with a high prevalence of trauma exposure and PTSD.
In the past decade, considerable progress has been made in the identification of ADRD biomarkers.The neuropathological features of AD include the presence of amyloid β plaques and neurofibrillary tangles containing hyperphosphorylated tau.Amyloid positron emission tomography (PET) has been shown to offer a valid in vivo tool for assessing the presence of amyloid β deposits, and levels of the amyloid-beta peptide Aβ42 (or the Aβ42/ Aβ40 ratio) in cerebrospinal fluid (CSF) also provide a valid indicator of the pathologic state of cerebral Aβ [7].However, the high cost and invasiveness of PET and CSF assessment have motivated the search for inexpensive, minimally invasive, and objective peripheral biomarkers of ADRD that can be used not only to aid in diagnosis, but also for prognostic evaluation, tracking treatment response, and monitoring of disease progression.
The development of ultra-sensitive immunoassay technologies, such as the single-molecule digital assay (Simoa) by the Quanterix Corporation, now permits the reliable assessment of central nervous system (CNS)derived proteins in blood samples at extremely low concentrations.The Simoa markers most relevant to ADRD, many not previously detectable in blood due to their low concentrations (or even known to exist in the periphery), include Aβ40 and Aβ42, glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), and phosphorylated tau at threonine 181 (pTau 181 ).Aβ peptides are the main components of senile plaques and cleaved from the amyloid precursor protein into 40 and 42 amino acid residual peptides termed Aβ40 and Aβ42, respectively.Lower plasma Aβ42 and a lower Aβ42/Aβ40 ratio is associated with greater brain amyloid pathology and thought to be attributable to the aggregation of Aβ42 into amyloid plaques [8].GFAP is a protein expressed in astrocytes and released during astrocytic activation.Elevated GFAP levels have been observed in association with traumatic brain injury and several neuroinflammatory and neurodegenerative CNS diseases [9].NfL is a cylindrical protein that provides structural stability to, and enables the growth of, myelinated axons.It has been shown to provide a reliable index of axonal injury across several neurological disorders [10].Finally, pTau 181 is a form of phosphorylated tau that aggregates into deposits that form the neurofibrillary tangles characteristic of Alzheimer's Disease and other tauopathies [11].Each of these biomarkers has also been shown to increase as a function of age [12][13][14].
On the basis of prior research suggesting that PTSD is associated with risk for dementia, accelerated DNAm age, and other indices of advanced cellular aging [15][16][17], we hypothesized that aging patients with PTSD would show elevated levels of some or all of the brain age-and dementia-associated plasma biomarkers Aβ40, Aβ42, GFAP, NfL, and pTau 181 .Further, in light of recent evidence that the risk that PTSD confers for ADRD increases additively as a function the apolipoprotein E gene ε4 risk allele (APOE ε4) [3], we also evaluated the main and interactive effects of this important genetic risk factor.Specifically, we hypothesized that the strength of the association between PTSD and the Simoa biomarkers would be greater among APOE ε4 carriers compared to non-carriers.
The National Institute on Aging and Alzheimer's Association have advanced a classification scheme for brain aging and Alzheimer's disease biomarker research based on three continuous dimensions, termed beta-amyloid deposition (A), pathologic tau (T), and neurodegeneration (N) [7].Known as the "ATN framework, " this system lends itself to latent variable modeling, which estimates the shared variance between indicators of a common dimensional construct, or factor (e.g., A, T, or N), and separates it from error variance in the indicators.This can be expected to increase power for association analyses relative to examining individual markers where true score variance and error variance are conflated.Theoretically, in the context of modelling associations among multiple correlated biomarkers, the resulting factors represent the broader biological construct underlying their covariation.Practically, this approach offers a method of data reduction that reproduces the observed relationships among multiple indicators using a smaller number of latent variables representing their commonalities.For the epigenome-wide association analyses reported here, this approach allowed us to distill the 5 Simoa biomarkers down to a smaller of number underlying factors, thereby reducing the study-wide multiple-testing burden.
This study was based on data from 849 ancestrally diverse individuals with a high prevalence of trauma exposure and PTSD spanning a wide age range with genome-wide DNA and DNA methylation (DNAm) data available for analysis.We began by performing factor analyses of the plasma Aβ40, Aβ42, GFAP, NfL and pTau-181 Simoa biomarkers the results of which yielded a twofactor solution with the Aβs loading on one factor and the three other markers loading on a second factor.We then performed epigenome-wide association analyses of each participant's scores on the two factors with the aim of identifying novel epigenetic loci associated with levels of the ADRD biomarkers.Finally, using structural equation modeling, we evaluated (a) the influence of PTSD, age, APOE ε4 genotype and other relevant covariates on levels of the ATN factors, and (b) tested the mediating influence of the EWAS-significant DNAm loci on these associations.
Participants and procedures
Analyses were based on existing clinical, genetic, and biomarker data collected under research protocols led by investigators at the Behavioral Sciences Division of the VA National Center for PTSD [18].Data for this report were from 849 individuals, including 580 US military veterans and a subset of 269 of their intimate partners, collectively ranging from 19 to 75 years of age.Clinical and demographic characteristics of the sample are listed in Table 1.Each of the original studies, and the research presented in this report, was reviewed and approved by the appropriate institutional review boards.In each study, participants had blood drawn for future genetic and biomarker assays.Blood was collected in EDTA tubes, centrifuged to separate plasma, serum, and buffy coat, then aliquoted and stored at − 80 °C until thawed for analysis.Participants also underwent psychiatric assessments using the Clinician-Administered PTSD Scale for DSM-IV or DSM-5 (CAPS [19,20]) and Structured Clinical Interview for DSM-IV or DSM-5 (SCID [21,22]), depending on the version of DSM in use at the time of study enrollment.For the CAPS, in addition to determining current diagnosis, frequency and intensity ratings of each symptom were summed to create a dimensional score reflecting current PTSD severity.We harmonized CAPS symptom severity across the two DSM versions by calculating each participant's symptom severity as a percentage of the maximum possible severity score for the relevant version of the measure (yielding scores ranging from 0-1).All diagnostic interviews were video recorded.Inter-rater reliability was assessed for approximately 25% of participants; kappa for each diagnosis reported here was greater than 0.78.History of psychological trauma was assessed using the Traumatic Life Events Questionnaire (TLEQ) [23], an inventory of 23 different types of traumatic experiences that were coded as positive if the participant endorsed (a) exposure to the event, and (b) experiencing "intense fear, helplessness, or horror" when it happened.Additional details regarding the samples, clinical assessments, and inter-rater reliability are available in previous reports [24,25].Neurocognitive function was not formally assessed; however, participants were terminated from the procedure if, in the clinician's judgment, cognitive impairment interfered with the participant's ability to complete the procedures.Finally, current psychiatric medication use was assessed using a self-report checklist and then classified into four categories: (a) SSRI/SNRIs, (b) other antidepressants, (c) typical/atypical antipsychotic, (d) sedatives, hypnotics, anxiolytics.
Genotype and DNA methylation data
Genetic data were generated using methods described in prior publications [18].Briefly, DNA was isolated on a Qiagen AutoPure instrument with Qiagen reagents and samples normalized using PicoGreen assays (Invitrogen, Grand Island, NY, USA).Each DNA sample was run on an Illumina OMNI 2.5 microarray and scanned using an Illumina HiScan System (San Diego, CA, USA) according to the manufacturer's protocol.Imputation was based on the Thousand Genomes Phase 3 reference panel [26].Ancestry was determined using a pipeline [27,28] that identified ancestral principal components using 100,000 randomly selected common single nucleotide polymorphisms (SNPs).APOE ε4 carrier status was called using the isoform-defining SNPs (rs7412 and rs429358) which were well-imputed (r 2 = 0.96 and 0.99, respectively).We used "best guess" imputed genotypes with a 90% confidence threshold for these SNPs to derive the APOE ε4 genotypes.DNA methylation (DNAm) studies involve measurement of a methyl group on the DNA strand at a cytosine-phosphate-guanine (CpG) site.DNAm data were generated using methods described in prior publications [29].In brief, DNAm was measured using the Illumina Infinium Methylation EPIC BeadChips.Zymo EZ-96 DNA Methylation Kits (D5004) were used to bisulfiteconvert batched samples.DNA conversion was accomplished via PCR using DAPK1 primers (Zymo) followed by gel electrophoresis of PCR products.Bisulfite-modified DNA was then whole-genome amplified, hybridized to the BeadChips, single-base extended, and stained using the Automated Protocol for the Illumina Infinium HD Methylation Assay.Assignment of individuals to chip and chip positions were balanced based on PTSD diagnosis and sex.We applied a quality control (QC) pipeline developed by the Psychiatric Genomic Consortium-PTSD Workgroup [30] prior to analysis (and recently updated as described at https:// github.com/ PGC-PTSD-EWAS/ EPIC_ QC).Proportional white blood cell (WBC) estimates (CD8-T and CD4-T cells, natural killer cells, b-cells, monocytes) were calculated from the methylation data for use as covariates.
Simoa markers
Simoa assays were performed at the Quanterix Accelerator Lab (Quanterix Corporation, Billerica, MA) using plasma samples.Samples were thawed and diluted per manufacturer's specifications, centrifuged to remove particulates and debris, then pipetted into 96 well plates, diluted 4x, and run in duplicate.All markers were tested using the HD-1 Analyzer.Aβ40, Aβ42, GFAP, and NfL were assayed using the N4PE advantage kit (Quanterix Item #103,670).pTau181 was assessed using the pTau181 advantage v2 kit (Quanterix Item #103,714).We initially intended to include plasma total Tau, but preliminary analyses showed weak bivariate associations between this analyte and the other Simoa markers (rs < 0.2).Furthermore, unlike the other five markers which are primarily brain-derived, the Tau protein is also expressed in peripheral tissues [31], and a recent study estimated that only 20% of plasma total Tau originates in the brain [32].Calibration was conducted with reference samples and QC procedures included evaluation of average enzyme per bead and coefficient of variation (CV).Samples that did not pass QC procedures were re-run, when possible, with the goal of minimizing freeze/thaw cycles.Samples were excluded (0-4.5%,varying by marker) if (a) the CV was > 25%, or (b) a duplicate was not available.Remaining concentrations were then multiplied by the dilution factor (× 4) prior to analysis.Results below the functional lower limit of quantification (fLLOQ) were set to the fLLOQ, and results above the functional upper limit of quantification (fULOQ) were set to the fULOQ.In total, data from 713 participants passed the DNAm and Simoa QC procedures, and of those, 704 also had genotype data.
Data analyses Exploratory and confirmatory factor analyses
Exploratory (EFA) and confirmatory factor analyses (CFA) of raw values for the five Simoa markers (Aβ40, Aβ42, GFAP, NfL, pTau181) were performed using Mplus v8.5 [33].EFA is a method for identifying the structure and dimensionality underlying the covariation of set of variables when that structure is not known a priori.In EFA, models with different numbers of latent variables (factors) are compared to determine which model best accounts for the covarion among the variables.It is similar to principal components analysis except that EFA can distinguish between true score variability and error and separates these two sources of variance.CFA, in contrast, enables examination of the degree to which data fit a predefined, or a priori hypothesized, structure for the number of factors underlying the covariation of variables and loading of individual variables on each factor.In both approaches, the fit of the model to the data is evaluated using several commonly used fit indices including the root mean square error of approximation (RMSEA), standardized root mean square residual (SRMR), and the comparative fit and Tucker-Lewis fit indices (CFI and TLI, respectively).The Bayesian information criterion (BIC) can be used for evaluating the fit of competing models.
We began by conducting an EFA of the five markers in a random half of the sample and evaluated 1 and 2 factor solutions using geomin rotation.(With 5 markers, we could only evaluate 1-and 2-factor solutions due to the fact that a model with more factors would have been statistically under-identified.)The robust maximum likelihood estimator (MLR) was used in all analyses.The results of the best fitting (two-factor) EFA were then used to inform the structure of a CFA that we tested in the other random half of the sample.After identifying a good fitting two-factor CFA, we executed the same model in the full sample (N = 849), again evaluated fit, then saved those factor scores for use in the EWAS.The factor scores reflect each individual's score on the latent variables (e.g., a higher score on the latent variable would account for higher values on the individual Simoa markers that load on that factor).
Epigenome-wide association analyses (EWAS)
We performed EWASs of scores from the two factors using linear models in the Bioconductor limma (Linear Models for Microarray Data) package [34], with the base 2 logit-transformed methylated proportion (known as an M values) as the response and the factor score as the predictor.Each EWAS included the following covariates: the top three ancestry principal components, age, sex, estimates of WBC proportions, a categorically coded batch variable representing the methylation project that each sample was assayed under, and a DNAm-based smoking score.The latter was based on effect-size estimates for the top-39 probes from a smoking EWAS [35] that we have previously shown to be an important covariate to include in DNAm association analyses [29].We computed false discovery rate (FDR) corrected p values [36], also known as Q values, to control for multiple testing (denoted "padj").Finally, we examined the genes corresponding to the top 500 sites from each EWAS for enrichment of specific gene ontology (GO) term categories using the gometh function from the R miss-Methyl package [37].This function is an extension of the GOseq method [38] which explicitly models the relationship between the number of CpG sites assessed within a gene and the probability of that gene appearing within the target list.
Structural equation model
Structural equation modeling (SEM) is a multivariate analytic method for simultaneously estimating the strength of associations between latent variables (e.g., in this case, the Simoa factors) and other observed variables in a causal structure containing direct (regressive paths) and/or indirect (mediated) paths in a single analysis.It is like path analysis, but involves paths between latent variables, as opposed to between observed variables.As with the factor analyses, this was performed in Mplus.This model examined (a) the strength of associations between the independent variables (i.e., the demographic, psychiatric and APOE ε4 genotypes) and the dependent variables (i.e., the Simoa factors), (b) the influence of the independent variables on the M values from the CpG sites identified by the EWAS, and (c) the effects of covariates relevant to each variable in the model.In addition, given the role of DNAm in mediating effects of environmental factors on many forms of gene and protein expression, we also modeled the EWAS-significant CpG sites as mediators of the associations between the independent variables and the Simoa factors.Additional file 1: Fig. S1 illustrates full structural equation model including the latent variables, regressive paths, factor loadings, factor correlations, and covariates.Given the large number of parameters to be estimated and evidence that Type 1 error can become inflated in SEM analyses [39], we utilized a conservative p value threshold of 0.001 for all direct regressive paths in the model.Finally, for significant CpG associations, we also evaluated the significance of the indirect (mediated) effect of the IV on the DV via the CpG loci using the "model indirect" command in Mplus which computed the products of (a) the effect of each IV on each CpG, and (b) the effect of each CpG on the Simoa factor, along with the p values for each indirect path.
Based on this, we then tested a CFA in the second random half of the sample (n = 421) in which Aβ40 and Aβ42 were set to load on one factor (that we term "Factor A, " i.e., "A" from the "ATN" framework) and pTau181, GFAP, and NFL on a second factor (that we term "Factor TN").This model fit the data well (Table 3) with all indicators loading on their respective factors at the p < 0.001 level.This model also yielded the lowest (i.e., best) BIC value compared to the EFA models.We then ran the 2-factor CFA in the full sample (n = 849) and found that this model also fit well in the full cohort with each indicator again loading significantly on its respective factor at the p < 0.001 level.The two factors were correlated at r = 0.62 (p < 0.001).Finally, we saved the factor scores from this analysis for use in the EWASs.Scatterplots of the associations between each factor, their indicators, and between the indicators themselves, are shown in Additional file 1: Figs.S2, S3.
Epigenome-wide association analyses
Lambdas for the EWASs indicated modest inflation (Factor TN = 1.201;Factor A = 1.249).After applying an FDR correction across the two analyses (total # of comparisons = 1,605,282), 3 loci were identified as significantly associated with Factor TN (cg26033520, cg23156469, cg15356923) and one was significantly associated with Factor A (cg13053408).See Table 4 for the EWAS-significant loci, Figs. 1 and 2 for the EWAS Manhattan plots, and Additional file 1: Figs.S4, S5 for the EWAS QQ plots.GO term enrichment analysis of the top 500 most significant probes from each EWAS yielded no FDR significant GO terms.The top-50 probes from each EWAS are listed in Additional file 1: Tables S1, S2.A comparison of the list of p < 0.001 significant probes from the two EWASs showed that 12.9 percent of these loci overlapped.Full summary statistics from each EWAS are available in the Additional files 2 and 3.
Structural equation model
The bivariate correlations between the two Simoa factors and each of the clinical, genetic, and demographic variables are listed in Table 1.Table 5 lists the bivariate correlations between the primary independent variables and each individual Simoa marker.Figure 3 shows the significant paths and parameter estimates (ps < 0.001) from the SEM.(Full results are available in Additional file 1: Table S3; scatterplots of the associations between the PCs are depicted in Additional file 1: Fig. S6).Fit statistics showed adequate model fit (χ 2 = 160.09,df = 68, p < 0.001; ).The EWAS-significant locus is highlighted in green In total, the model explained 54% of the variance in the Factor TN and 23% of the variance in Factor A. As shown in Fig. 3, age showed the strongest associations with both factors and was more strongly associated with Factor TN than Factor A. To test this difference empirically, we ran a follow-up model in which these age paths were held to equivalent and found that doing so significantly degraded model fit (Δ χ 2 = 87.32,Δ df = 1, p = 9.23e −21 ).Age was also significantly associated with cg15356923 and the indirect effect of age on Factor TN via this DNAm locus was significant (β = 0.041, p = 0.005).Sex was positively associated with cg26033520 (greater for females) and the indirect effect of sex on Factor TN via this locus was significant (β = − 0.058, p < 0.001).Lifetime trauma count was positively associated with cg130534081 and the indirect effect of trauma on Factor A via this locus was also significant (β = 0.022, p = 0.008).We also observed a significant positive residual correlation between DNAm levels at cg130534081 and cg23156469, implying additional covariation among these loci beyond that which could be attributed to the shared predictors of these loci.APOE ε4, PTSD, and their interaction were not significantly associated with any of the CpGs or either factor (ps > 0.001).
Table 5 Bivariate correlations between the primary independent variables and each Simoa marker The SEM analysis also revealed a significant positive association between Factor A and PC1 (β = 0.196, p < 0.001) controlling for age and the other variables in the SEM, indicating that individuals with a greater proportion of African ancestry had lower levels of Aβs relative to those of European ancestry.This difference was also significant in the categorical ancestry classification based on the genotype data (African vs.European ancestry clusters; p = 0.004) and for self-identified Black participants compared to self-identified White individuals (p = 0.012).All of these differences were significant when controlling for age.Finally, as a sensitivity analysis, we ran the full SEM with the addition of the four classes of psychiatric medications and found no significant associations between current medication use and either factor (all ps > 0.001).
Discussion
Research on blood-based biomarkers of ADRD is rapidly advancing, yet relatively little is known about how these markers covary, or what influence various genetic, epigenetic, demographic, and psychiatric factors have on their levels.In this study, we used factor analyses to identify two dimensions that explained the covariation among Aβ40, Aβ42, GFAP, NfL and pTau-181.Drawing from the National Institute on Aging and Alzheimer's Association "ATN" research framework, we termed the first factor, defined by Aβ40 and Aβ42, "Factor A".GFAP, NfL and pTau-181 loaded on a second factor that we accordingly termed "Factor TN. " Next, we performed an EWAS of each factor, the results of which identified 4 DNAm loci that survived multiple-testing correction across the two analyses.The most strongly associated CpG from the Factor TN EWAS, cg26033520, was located in the vicinity of the Activating Signal Cointegrator Complex Subunit 1 gene (ASCC1).ASCC1 transcribes a protein that plays a role in gene transactivation via its effects on several transcription factors.This locus was one of two EWAS-significant CpGs identified in a recent study of blood DNA methylation in Parkinson's disease patients, with the same direction of association [40].In a subsequent metaanalysis of epigenome-wide associations across Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS), this CpG also showed significant shared effects across all three disorders [41].ASCC1 exerts an inhibitory effect on nuclear factor kappa-B (NF-kB) [42], a well-established inflammatory transcription factor implicated in tau pathology, glutamate excitotoxicity, and reactive microglia and astrocytes [43].The top-hit from the Nabais et al. [41] EWAS, cg03546163 in FKBP5, was also among the top-50 most highly associated probes in our Factor TN results.
The other two EWAS-significant loci from the Factor TN analysis were cg23156469 and cg15356923, located in FAM20B and FAM19A4 (aka TAFA4), respectively.FAM20B transcribes a kinase that phosphorylates xylose residues and triggers the synthesis of peptidoglycan, the main component of the cell wall in most bacteria.Though the literature on this is gene is very limited, a SNP in FAM20B (rs4652345) was the 16th most strongly associated locus in a GWAS of individuals with polygenic risk extremes for Alzheimer's disease in the UK Biobank [44].FAM19A4 (aka TAFA4), on the other hand, transcribes a well-known neurokine that has been implicated in the regulation of immune responses and pain signaling within the nervous system [45].
The Factor A analysis identified one EWAS-significant locus, cg13053408, located in FANCD2OS (i.e., "FANCD2 opposite strand" aka C3orf24).As an opposite strand gene (i.e., located on the antisense or noncoding strand of DNA), this region of DNA serves as the template for making FANCD2 messenger RNA.FANC2D has been identified as a mediator of the effect of the amyloid precursor protein fragment APP intracellular C-terminal domain on FOXO3 [46] which has a well-established role in aging and many age-related disease processes [47,48] including Amyloid-β induced astrocytosis and astrocyte death [49].FANCD2OS overexpression has also been shown to modulate expression of the steroidogenic acute regulatory protein (STAR1 or STARD1) [50].STAR1 is an intracellular cholesterol carrier that has been observed to be elevated in the cortex of Alzheimer's disease and Down syndrome patients and correlated with Aβ42 deposition in regions of the hippocampus [51].Also relevant to our findings, a pair of SNPs in FANCD2 and FANC-2DOS (rs1552244 & rs9849434) were among the top-25 hits from an early Alzheimer's disease GWAS meta-analysis [52] (albeit not replicated in more recent studies with larger Ns).
We then submitted the EWAS-significant DNAm values to an SEM analysis that evaluated these CpG sites as possible mediators of associations between age, sex, trauma history, PTSD, APOE ε4 genotypes and the two Simoa factors.Age was positively associated with both factors and significantly more so with Factor TN than Factor A. These findings align with a rapidly growing body of research showing that these markers are present in plasma of adults across the lifespan, trending gradually higher through early and middle adulthood, and then increasing exponentially in old age [12][13][14].Mediation analyses identified three small but statistically significant indirect effects of age and sex on Factor TN, and number of lifetime traumas on Factor A, that were mediated by the CpGs.Though interesting and worthy of future examination, given the small size of the effects and novelty of the findings we are hesitant to over-interpret those results.
Genotype-determined ancestry PCs were included as covariates of the two Simoa factors and each CpG locus in the EWAS and SEM.The SEM revealed a significant positive association between Factor A and PC1 indicating that individuals with a greater proportion of genotype-determined African ancestry tended to have lower levels of Aβ40 and Aβ42.Similarly, self-identified Black participants showed significantly lower levels of Factor A than self-identified White individuals.Though not an a priori focus of the study, these findings are consistent with results of recent studies that examined racial differences in levels of plasma Aβs and brain amyloid levels.For example, Hajjar et al. [53] examined associations between self-identified race and genetic ancestry in 300 Black and 429 White individuals, the majority of whom had mild cognitive impairment and were over 60 years of age.Results showed that Black participants had significantly lower unadjusted levels of Aβ40 compared to White individuals and, as in our study, the percentage of African ancestry in the sample was negatively correlated with Aβ40 levels.Similarly, Deters et al. [54] examined racial differences in positron emission tomography (PET) measured amyloid in cognitively normal older adults and found that Black individuals had lower amyloid levels compared to White participants and this difference was enhanced as a function of the participant's proportion of African ancestry.Finally, in an earlier study of nearly onethousand adults in their seventies approximately 50% of whom were Black, Metti et al. [55] found significantly lower levels of plasma Aβ40 and Aβ42 among self-identified Black compared to White individuals.In sum, results of this study add to a growing body of findings suggesting that there are important racial and ancestral differences in age-and AD-related biomarkers that may be relevant for advancing understanding of racial differences in the rates of ADRD reported in large-scale epidemiological and cohort studies [3,56].
We had hoped that this study would shed new light on possible mechanisms by which PTSD confers risk for dementia but hypotheses for our primary variables of interest, APOE ε4, PTSD (and their interaction) were not supported.Specifically, none of the direct effects of APOE ε4, PTSD, or the APOE ε4 x PTSD interaction term on the Simoa factors met our SEM criterion for statistical significance (p < 0.001).Rather, and contrary to our hypothesis that PTSD would be associated with elevated levels of ATN biomarkers, the SEM revealed a negative correlation between PTSD and Factor TN that fell just short of our multiple testing threshold (β = − 0.140, p = 0.003) controlling for age, sex, number of lifetime traumas, APOE ε4, the APOE ε4 x PTSD interaction term, the ancestry PCs, a DNAm-based smoking score, and effects of the four EWAS-significant probes.The simple bivariate correlation between PTSD severity and the TN factor scores showed a similar association (r = − 0.133, p < 0.001) driven primarily by reduced levels of GFAP in individuals with more severe PTSD symptoms (r = − 0.128, p < 0.001).We also noted that antipsychotic medication use was associated with reduced levels of both Simoa factors, but our sensitivity analyses showed that the negative PTSD effect remained significant and unchanged with medication use in the model.
Though unexpected and opposite in direction of what we hypothesized, the finding of reduced GFAP in association with PTSD is not without precedent.Pierce et al. [57] examined a panel of ATN plasma biomarkers in a younger cohort of 550 post-9/11 veterans (from the same VA medical center, but independent of the participants evaluated in this study) and also reported a modest negative correlation between PTSD severity and GFAP controlling for age and sex (r = − 0.10, p < 0.05).Kulbe et al. [58] conducted a prospective observational study of 1,143 emergency department patients with mild TBI evaluated within 24 h of injury then re-assessed 6 months later and found that day-of-injury plasma GFAP levels were significantly lower among those with a PTSD diagnosis at follow-up.Finally, Natale et al. [59] examined a sample of 1,520 World Trade Center responders and also found that PTSD was associated with reduced levels of plasma GFAP.These authors offered several hypothetical explanations for this finding and while the association appears worthy of future investigation, its biological significance remains unclear.
More generally, previous studies on the association between PTSD and ATN biomarkers have shown mixed and largely null results.For example, in arguably the most methodologically rigorous prior study on this topic, Weiner et al. [60] examined 289 non-demented Vietnam-era veterans with traumatic brain injury (TBI) and/or PTSD who underwent cognitive testing, cerebrospinal fluid collection, and Aβ and tau PET scans.Results showed that compared to controls, veterans with histories of TBI and PTSD were more likely to have mild cognitive impairment and lower mental status scores but there were no differences between groups in any of the ATN biomarkers examined.The apparent discrepancy between findings from large cohort and epidemiological studies on PTSD and risk for ADRD, and studies of the association between PTSD and ATN biomarkers, may be attributable to a variety of issues including (a) the possibility that the PTSD-dementia association is mediated by medical comorbidities such as cardiovascular disease and diabetes, (b) that this association is driven by non-AD specific neuropathology such as vascular or other forms of dementia, and/or (c) inherent differences between the clinical diagnosis of dementia in medical records versus the biological processes indexed by specific ATN biomarkers.
The findings of this study should be evaluated in the context of several limitations.Most notably, the study was based on cross-sectional archival data from projects that did not include neurocognitive measures or neuroimaging data and most of our participants were well below the age at which dementia is normally manifested.It is conceivable that significant effects of PTSD on brain age-and AD-associated biomarkers would be observed in older cohorts and/or individuals with PTSD and mild cognitive impairment or dementia.On the other hand, the study featured a large and ancestrally diverse sample with genome-wide SNP and DNAm data, and 5 Simoa markers indexing essential components of the ATN biomarker framework.Findings identified (a) novel and biologically plausible epigenetic associations with the factors that underlie the covariation of these markers which should inform future efforts to identify epigenetic loci reliably associated with ADRD and its biomarkers, and (b) robust age and race/ancestral associations that will be essential to consider in future efforts to develop the clinical and diagnostic applications of these tests.
Fig. 1
Fig. 1 Manhattan plot of the Factor A EWAS.The x-axis depicts chromosomes and the location of each CpG site across the genome.The y-axis is the -log10 of the p value for the association with levels of Factor A Simoa markers defined by Aβ40 and Aβ42.Each dot represents a CpG site.The red line indicates the level for epigenome-wide statistical significance (p = 1 × 10 -7 ) and the blue line the level for suggestive significance (p = 1 × 10 -5
Fig. 2
Fig. 2 Manhattan plot of the Factor TN EWAS with the three EWAS-significant loci highlighted in green
Fig. 3
Fig.3Diagram depicting the significant direct paths (ps < 0.001) and associated variables from the SEM results.The Simoa latent variables are represented as circles with the loadings of each indicator shown as well as the correlation between the two factors.Age was the only variable in the model that showed significant (p < 0.001) direct effects on both variables.PC1 was positively associated with Factor A. The EWAS-significant CpG sites were evaluated as mediators of the association between the psychiatric and demographic variables and the latent variables.Significant indirect effects were found for a the effect of age on Factor TN via cg15356923, b the effect of sex on Factor TN via cg26033520, and c the effect of lifetime trauma count on Factor A via cg130534081
Table 1
Sample descriptive statistics and bivariate correlations with the Simoa factor scores
Table 2
Descriptive statistics and correlations between the Simoa markers n = 816 due to some pairwise comparisons having missing data.All p values < 0.001 Bottom row lists the means and (SDs) for each marker in pg/ml units
Table 3
Model fit of the exploratory and confirmatory factor analyses n = 849EFA exploratory factor analysis; CFA confirmatory factor analysis; RMSEA root mean square error of approximation; SRMR standardized root mean square residual; BIC Bayesian information criterion; CFI comparative fit index; TLI Tucker-Lewis Index
Table 4
Epigenome-wide significant CpG sites by factor n = 704 (lower due to samples failing the GWAS, EWAS or SIMOA QC pipeline). | 2024-03-04T05:04:36.447Z | 2024-03-02T00:00:00.000 | {
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203402730 | pes2o/s2orc | v3-fos-license | Foraging ecology drives social information reliance in an avian eavesdropping community
Abstract Vertebrates obtain social information about predation risk by eavesdropping on the alarm calls of sympatric species. In the Holarctic, birds in the family Paridae function as sentinel species; however, factors shaping eavesdroppers' reliance on their alarm calls are unknown. We compared three hypothesized drivers of eavesdropper reliance: (a) foraging ecology, (b) degree of sociality, and (c) call relevance (caller‐to‐eavesdropper body‐size difference). In a rigorous causal‐comparative design, we presented Tufted Titmouse (Baeolophus bicolor) alarm calls to 242 individuals of 31 ecologically diverse bird species in Florida forests and recorded presence/absence and type (diving for cover or freezing in place) of response. Playback response was near universal, as individuals responded to 87% of presentations (N = 211). As an exception to this trend, the sit‐and‐wait flycatcher Eastern Phoebe (Sayornis phoebe) represented 48% of the nonresponses. We tested 12 predictor variables representing measures relevant to the three hypothesized drivers, distance to playback speaker, and vulnerability at time of playback (eavesdropper's microhabitat when alarm call is detected). Using model‐averaged generalized linear models, we determined that foraging ecology best predicted playback response, with aerial foragers responding less often. Foraging ecology (distance from trunk) and microhabitat occupied during playback (distance to escape cover) best predicted escape behavior type. We encountered a sparsity of sit‐and‐wait flycatchers (3 spp.), yet their contrasting responses relative to other foraging behaviors clearly identified foraging ecology as a driver of species‐specific antipredator escape behavior. Our findings align well with known links between the exceptional visual acuity and other phenotypic traits of flycatchers that allow them to rely more heavily on personal rather than social information while foraging. Our results suggest that foraging ecology drives species‐specific antipredator behavior based on the availability and type of escape cover.
| Animal information networks
Vertebrates must constantly seek information about their surroundings to reduce uncertainty and make adaptive behavioral choices (Dall, Giraldeau, Olsson, McNamara, & Stephens, 2005;Danchin, Giraldeau, Valone, & Wagner, 2004;Schmidt, Dall, & van Gils, 2010;Seppänen, Forsman, Mönkkönen, & Thomson, 2007). Information from direct interaction with the environment (personal information) is combined with cues or signals obtained from other individuals (social information; Danchin et al., 2004) of same or different species. Social information often comes from heterospecifics (Goodale, Beauchamp, Magrath, Nieh, & Ruxton, 2010) because ecologically similar species that share predators and diet items are both collectively more abundant than conspecifics and better able to detect relevant threats and opportunities (Goodale & Kotagama, 2005a).
The production and consumption of social information in a community constitutes an "information network" (Goodale et al., 2010;Schmidt et al., 2010;Seppänen et al., 2007). Information networks are often asymmetrical in nature: A vocally complex "informationproducing" species serves a diverse audience of heterospecific eavesdroppers (Contreras & Sieving, 2011;Goodale et al., 2010;Schmidt et al., 2010). Knowing which species eavesdrop, and the relative value of the social information provided to different eavesdroppers in a network, is fundamental to defining the role of information sharing at the community level (Goodale & Kotagama, 2008;Magrath, Pitcher, & Gardner, 2009;Martínez & Zenil, 2012). Where eavesdropping species rely on antipredator cues provided by heterospecifics, social information provides a mechanism for large-scale facilitation (Contreras & Sieving, 2011;Hetrick & Sieving, 2012;Szymkowiack, 2013) and an important benefit from participation in mixed-species foraging groups (Martínez, Parra, Muellerklein, & Vredenburg, 2018;Pagani-Núñez et al., 2018). There is therefore a need to quantify how reliance on social information may vary among species.
| Factors determining the value of social information to eavesdroppers
Species traits and environmental factors both influence whether a species can personally collect all necessary information or must rely on social information (Parejo & Aviles, 2016;Seppänen et al., 2007). Some species are better at detecting threats by virtue of their foraging ecology (Goodale et al., 2010): Species that forage on substrates (substrate-based foragers) suffer from visual occlusion by foliage, while aerially foraging species (salliers) can scan for prey items and predators simultaneously. Among forest birds, substrate-based foragers respond more readily to heterospecific alarm calls than salliers, indicating greater reliance on social information (Goodale & Kotagama, 2008;Martínez, Gomez, Ponciano, & Robinson, 2016;Martínez & Zenil, 2012). Species with similar foraging behaviors convergently evolve similar morphological and physiological structures known as ecomorphs (Botero-Delgadillo & Bayly, 2012;Corbin, 2008); for instance, eye morphology differs significantly between bird species in different foraging guilds (Lisney et al., 2013;Moore, Doppler, Young, & Fernández-Juricic, 2013). These suites of physiological adaptations to foraging behaviors may result in similar physiological limitations on detection capability, and hence similar degrees of reliance on social information. We therefore predict that species which forage in more open microhabitats and employ more aerial foraging maneuvers will be less reliant on social information.
Alternatively, intraspecific sociality may play a key role in determining reliance on social information. Highly gregarious species living in groups may obtain most of their social information from group members, while solitary species must depend on heterospecifics.
Social species are more likely to give alarm calls than solitary ones in order to warn group members or kin about predation risk (Sridhar, Beauchamp, & Shanker, 2009), whereas some solitary lizards, for example, lack alarm calls entirely (Fuong, Keeley, Bulut, & Blumstein, 2014). Social species also employ complex vigilance behaviors such as the sentinel systems which can accurately assess ambient predation risk through alarm calls (Ridley & Raihani, 2007;Ridley, Raihani, & Bell, 2010). Both birds and social primates only make use of heterospecific social information when in small conspecific groups, switching to conspecific social information in larger groups (Bshary & Noë, 1997;Ridley & Raihani, 2007). Solitary species, by contrast, often respond to heterospecific alarm calls of social species (Lea, Barrera, Tom, & Blumstein, 2008;Ridley, Wiley, & Thompson, 2014).
We would predict, therefore, that intraspecifically social species will rely less on social information than solitary species.
Response to heterospecific alarm calls is also influenced by call relevance, or the proportion of instances in which the predator eliciting the alarm call represents a physical threat to the eavesdropper (Hua et al., 2016;Magrath et al., 2015). For example, arboreal hornbills are vulnerable to eagles but not leopards and only respond to the "eagle" alarm calls of a sympatric monkey species (Rainey, Zuberbuhler, & Slater, 2004a, 2004b. Similarly, New Holland honeyeaters (Phylidonyris novaehollandiae) respond to the alarm calls of white-browed scrub-wren (Sericornis frontalis; 18% of alarms given to nonshared predators), but not to those of superb fairy-wrens (Malurus cyaneus; 52% of alarms given to nonshared predators, Magrath et al., 2009). Because the success and likelihood of attacks by predators are strongly influenced by predator-prey body-size ratios, prey of similar body sizes will be vulnerable to the same predators (Rodgers, Downing, & Morrell, 2015). Therefore, the alarm-caller-to-eavesdropper body-size difference can serve as a proxy for the relevance of the call to the eavesdropper, and we would predict that species which are more similar in body size to the alarm caller will be more responsive to its alarm call.
The local context of an individual when an alarm is heard can also influence response to an alarm call because different foraging microhabitats have different associated predation risks (Brown & Kotler, 2004). The predators that prey on small forest passerines attack from above and target prey further from the trunk (Kullberg, 1995), so we predict that microsites will interact with species traits to define prey responses to simulated alarm calls. Density of vegetation and distance from trunk (Brotons, Orell, Lahti, & Koivula, 2000;Desrochers, 1989;Suhonen, 1993), height from ground (Carrascal & Alonso, 2006;Lee, Kuo, & Bollinger, 2005;Suhonen, 1993), and proximity to escape cover (Carrascal & Alonso, 2006;Lee et al., 2005) may all affect the perceived predation risk of forest passerines, and therefore, alarm call response might be greatest farther from escape cover and the trunk and closer to the ground. Perceived predation risk by small forest birds may also be higher in edge habitat than forest interior (Rodríguez, Andrén, & Jansson, 2001). Therefore, controlling for local microsite effects is important when attempting to make inferences from species-level traits.
While the above factors have all been shown to influence response to alarm calls in isolation, their relative importance has never been simultaneously assessed in one community. In this study, we therefore present a common heterospecific alarm call from a sentinel species to a winter community of forest birds to elucidate the drivers of reliance on social information in a vertebrate eavesdropping network. We conduct a comparative test of the role of three species-level ecological hypotheses (foraging ecology, sociality, and call relevance) in determining the degree of reliance on eavesdropping, while also controlling for local microhabitat effects. (29°40′58″N 82°13′29″W). We selected field sites only in upland hardwood forest, which has the most species-rich winter forest bird community in Florida (Engstrom, 1993). These mesic, upland hard- Florida hosts a diverse winter bird community due to the presence of numerous short-and long-distance migrants as well as resident species which vary widely in terms of foraging ecology, winter sociality, and body size (Kale & Maehr, 1990). It therefore offers an opportunity to test the influences of species traits over an extreme range of their values, yielding strong causal inference for each of our hypotheses (James & McCulloch, 1995). In this system, the information-producing species is the Tufted Titmouse (Baeolophus bicolor, hereafter titmouse), an abundant, year-round-resident. It acts as a sentinel species through high vigilance combined with aggressive predator mobbing (Sieving, Contreras, & Maute, 2004) and alarm calling (Gaddis, 1980;Morse, 1970). Titmice produce complex alarm calls that accurately and reliably encode the size and threat level of a predator (Sieving, Hetrick, & Avery, 2010;Templeton et al., 2005) and thus have a community-wide audience of eavesdroppers (Langham, Contreras, & Sieving, 2006;Sieving et al., 2004). Titmice also act as nuclear species for mixed-species foraging flocks of birds (Contreras & Sieving, 2011) which form around small family groups that hold stable winter territories (Brawn & Samson, 1983). These foraging flocks are joined by many species of small forest passerines in winter that follow titmouse groups and forage with them (Farley, Sieving, & Contreras, 2008;Gaddis, 1983).
| Characterizing foraging behavior
We obtained local data on the foraging ecology of the full winter community through focal individual field observations conducted during both winters at the same sites at which we conducted alarm call playbacks. Full methods are given in Jones, Sieving, and Robinson (2018); briefly, a single observer (HHJ) walked transects and trails at each site and recorded sequences of foraging behavior for each species encountered. We recorded sequences of foraging maneuvers until the focal individual was lost from view. For each foraging maneuver, we recorded the foraging height (estimated in meters using a laser rangefinder), the foraging maneuver type, the foraging substrate, the distance category from the trunk (near, medium, or far), and the foliage density at the microsite where the prey item was attacked (measured on a 0-5 scale). Foraging data were recorded in the field using a voice recorder, and later transcribed into a spreadsheet for analysis. Substrate and attack maneuver nomenclature follows Robinson and Remsen (1990), see Tables S5-S8 in Appendix S1 for proportional use of attack maneuver and microhabitat categories for each species. We performed foraging observations along a transect or trail only once per winter to avoid repeat foraging observations of the same individuals. For the same reason, when we encountered a mixed-species foraging flock, we only recorded foraging observations of a single individual of each species in the flock.
| Describing winter sociality and call relevance
Data on winter sociality and body mass were obtained from the literature. For the sociality data, we used the average maximum number of individuals of a species encountered in paired mixed-species flock and point count surveys conducted at our field sites by Farley et al. (2008). Measuring winter sociality is challenging at our sites because species participate in mixed-species foraging flocks, join single-species foraging groups, or are solitary (Farley et al., 2008;Jones et al., 2018;Jones, Walters, & Robinson, 2019). Ten of 17 commonly occurring species at our study sites spend upwards of 80% of their time in mixed-species flocks (Jones et al., 2019), so it is important to account for these foraging associations when quantifying winter sociality. Farley et al. (2008) We used the absolute value of the difference in mass between the titmouse and each focal species as a proxy for the degree of overlap in predator suite, as the bird-eating hawk species in our system (Accipiter spp.) preferentially prey on statistically different size classes of birds (Opdam, 1975;Reynolds & Meslow, 1984). Because body mass in birds affects vertical escape flight performance and other aspects of foraging and social behavior (Dial, Greene, & Irschick, 2008), difference in body mass should serve as a proxy for the difference in predation risk posed by a shared predator. Empirical data on Accipiter prey preferences from Europe support this assumption by suggesting that they prey less on species with very small and very large body masses in forest communities (Götmark & Post, 1996). Given the lack of published empirical data on relative prey preferences for North American Accipiter species on our focal community, we believe our measure represents our best estimate of the number of shared predators, with the relevance of the alarm call decreasing as the difference in mass increases. We obtained body mass estimates in grams from Sibley (2014).
| Alarm call playback procedures
We conducted playback presentations from December 2014 to February 2015 (winter 1) and from November 2015 to January 2016 (winter 2) at the same field sites as foraging observations, though on different days. We used response to presentation of the titmouse Z call stimulus, an alarm call given by titmice in the presence of attacking hawks (Gaddis, 1980;Morse, 1970;Sieving et al., 2010), as a measure of reliance on social information. The call was presented in the absence of a predator, so species with complete personal information could "know" there was no predator, but species with limited personal information would be expected to respond. We did not present the alarm call with a predator model (e.g., taxidermied mount) because our methodology relied on presenting a "false" alarm call to the focal individual. Responding to false alarms is costly in terms of lost foraging efficiency (Bradbury & Vehrencamp, 2011), so we would expect for species to not respond when their personal information indicates that there is no predator. We selected our stimulus because it is a high-urgency call, associated with the highest responsiveness by eavesdroppers (Fallow & Magrath, 2010). Even migratory species should be familiar with this stimulus because (a) most breeding ranges overlap with that of the titmouse (Sibley, 2014) and (b) birds can quickly learn novel alarm calls through acoustic association (Potvin, Ratnayake, Radford, & Magrath, 2018).
We presented free-living, wild individuals of each bird species with the Z call, walking trails or transects until encountering a focal individual. We went into the field each day with a prioritized list of species needing more sampling, but otherwise, our sampling was opportunistic; we presented ten or more playbacks to each commonly encountered species in our nonbreeding community (sample sizes in Table 2). We only observed the response of a single focal bird, and in all cases, the observer remained at least 30 m from the focal bird to not influence behavior. The primary observer followed the focal bird with binoculars while a second observer set up and played the recording. Our recordings used known-context alarm calls recorded during predator presentations to titmice in aviaries (Hetrick & Sieving, 2012). We created 30-s playback recordings from natural calls (N = 5 exemplars) by repeating each natural recording with silence in between. We selected a random exemplar for each trial and played it at a standardized volume (~76 dBA at 1 m) from a speaker (Ultimate Ears BOOM) attached to an extension pole leaned on a tree (3.6 m height). The amplitude of the experimental exemplars was measured post hoc at 1 m in a similar habitat and using the same speaker and speaker settings (Table S12 in Appendix S1). Amplitude was measured as the maximum amplitude during the 30-s recording in A-weighted decibels using a digital sound level meter (B&K Precision 732A) on a fast setting. While the amplitude of natural Z calls is unknown, free-living birds have been shown to respond to the same stimulus in the same habitat when given at ~50 dBA at 1 m (Grade & Sieving, 2016), so we feel confident that birds could hear our stimulus. We recorded the exemplar during the second winter only and therefore could not include it in our statistical analyses.
However, this is a stereotyped alarm call only used in high-risk contexts (Sieving et al., 2010), and we found no difference in response rates between exemplars (see Section 3; Tables S10-S11 in Appendix S1). Alarm call attenuation over greater signaler-receiver distances affects heterospecific response (Murray & Magrath, 2015), so we only used focal individuals within 30 m of the speaker (mean ± SD; 16.97 ± 6.01 m), as recorded by laser rangefinder prior to playback.
To maintain sample independence and minimize pseudo-replication, we recorded a GPS point for each playback and separated all playbacks for each species, whether they were conducted on the same or different days, by at least 200 m. Playbacks conducted 200 m apart were also acoustically independent because the signal-to-noise ratio of Z calls degrades to 0 within 60 to 70 m of sound source in hardwood forests of the study region (K. E. Sieving, unpublished data). For each playback, we recorded three response variables: yes/no overall response, type of response (freezing in place or diving for cover), and length of freezing time if the bird froze. We scored a focal individual as responding if they immediately ceased baseline activity and adopted antipredatory behavior, while a response was scored as a no if the specific antipredatory behavior was not observed. Individuals respond to Z calls by either diving for cover or freezing in place and remaining motionless for two or more minutes (Gaddis, 1980;Hetrick & Sieving, 2012;Morse, 1970). Because response was immediate and behavioral changes were obvious and extended, it could never be confused with baseline behavior. Even stationary sit-and-wait flycatchers make noticeable and frequent head and body movements while scanning for prey while remaining on the same perch for extended periods of time. Therefore, we are confident that freezing behavior was not confused with even the most lethargic baseline behaviors exhibited by sit-and-wait flycatchers.
To describe the microsite of the focal individual at the time of playback, we recorded the density of vegetation (measured as the Note: Species codes are described in Table S9 in Appendix S1. Sample size = the number of Z call playbacks presented to each species. Overall Response = the proportion of individuals that responded by freezing or diving (vs. no change in behavior) to the playback stimulus. Freezing Proportion = the ratio of individuals for each species that froze versus dove (given a response). Mean Freeze Time = the mean number of seconds each species remained motionless (minimum of 2 responses to playback). Difference in Mass = absolute value of the difference in mass from the Tufted Titmouse (data from Sibley, 2014). Mean Local Abundance = a measure of the nonbreeding sociality of a species (calculated in Farley et al., 2008) Foraging Guild = foraging maneuver assigned to species based on field observations or data from the literature. The last three variables were used as predictor variables in the GLMs (see Table 2; Table S3 in Appendix S1).
proportion of vegetation within a 1-m radius sphere around the focal individuals; see Robinson & Remsen, 1990), distance from trunk (near, medium, or far), height from ground, and distance to escape cover (both estimated in meters using a rangefinder) before each playback.
We classified playback locations based on whether they were located within 50 m of a forest edge (e.g., clear cut, pond edge) or not.
Finally, we determined approximate temperature at time of playback post hoc using hourly averages at 10 m elevation for Gainesville from the Florida Automated Weather Network; temperature can influence vigilance levels in Holarctic parid-led flocks (Brotons et al., 2000). To determine whether merely the presentation of any sound at the height of our speaker would startle birds into antipredator behavior, we performed a procedural control during the second winter using playback of the call of the spring peeper (Pseudacris crucifer).
Procedural playbacks followed the same protocol and projected a call that sounded natural to us within 30 m (dBA at 1 m = ~80; Table S12 in Appendix S1). This small frog is a common resident of hardwood habitat and gives a somewhat similar high pitched, repeated call during its breeding season from November to March (Conant & Collins, 1998). As such, this represents a familiar, nonthreatening stimulus with similar acoustic qualities to the Z call.
| Data reduction of foraging and microhabitat variables
All statistical analyses were performed in R (version 3.5.1). dissimilarity index (Gower, 1971) to create a dissimilarity matrix. We selected axes to retain for further analyses (see Results in Appendix S2) by consulting a scree plot and retaining only interpretable axes.
| Hypothesis evaluation using generalized linear models
We ran three generalized linear model (GLM; glm function, stats package) analyses of response to alarm call playback. First, we modeled (1) the overall (Y/N) response and (2) the response type-dependent variables, using logit and log link GLMs, respectively. We did not model length of freezing response because we believe that resumption of baseline behavior is based on an "all clear" stimulus from the titmouse rather than species-specific traits. Models of response type used the subset of the data in which the focal bird responded to the playback (N = 182 trials). We included eleven predictor variables ( Table A1 in Appendix 1), encompassing both species-level traits obtained from foraging observations and in the literature as well as local microhabitat data recorded before each playback. We included only complete cases in our analyses (N = 205 playback presentations).
In order to include a greater diversity of species in our analysis of playback response, we then ran (3) a second GLM model of response in which we included all species that received playbacks (N = 31 species, 238 playbacks), and a greater diversity of foraging behaviors and body masses. These additional species represent late migrants or rare overwintering species at our study sites, and since we did not have detailed foraging and sociality data for these taxa, we did not include them in the first analysis. We included seven predictor variables in the second model, including the local microhabitat variables, distance to speaker, difference in mass to the titmouse (e.g., call relevance), temperature, and foraging maneuver (see Table S3 in Appendix S1). Because foraging maneuver was the only important predictor variable for response in our first model, we classified it categorically by most common maneuver in the second model. Sociality was not included in the second model because it was not significant in the first model, and because we did not have local sociality data for the additional species. Foraging maneuver categories selected were either the most commonly observed maneuvers in foraging observations (Table S8 in Appendix S1) or the most common maneuver identified in the literature (see Table 1 for foraging maneuver categorization).
We did not consider interactions in our models because main parameter estimates can be biased by interaction terms when model averaging (Richards, Whittingham, & Stephens, 2011).
We used an information theoretic approach (Burnham & Anderson, 2002) to evaluate our generalized linear models and determine the best models for each of our two response variables, using the Akaike information criterion modified for small sample sizes (AIC c ), recommended for small datasets (Symonds & Moussalli, 2011). We calculated AIC c scores and model weights for the full model set using the dredge function of the MuMIn package. Because there was no best model (model with a ΔAIC c of 2 or greater over the second-best model), we performed full model averaging over a candidate set of models (model.avg function, MuMIn). Because model weights were low, the 95% confidence set of models contained over 500 models. As such, we selected candidate sets with ΔAIC c of 2 or less (Tables S1, S2, and S4 in Appendix S1) because these models are considered to be as good as the best model (Burnham & Anderson, 2002). The goodness of fit of models was assessed by the pseudo-r 2 value calculated in the dredge function.
| Foraging observations
Over two winters of observations, we observed 1,242 foraging maneuvers of 327 foraging individuals belonging to 25 species. Of these, 19 species had greater than 5 independent observations of foraging individuals (Table S8 in Appendix S1; full species-level foraging data available in Tables S5-S7 in Appendix S1). (Table S8 in Appendix S1).
| Playback experiment
We presented the alarm call stimulus to 242 individuals of 31 bird species, representing the entire winter bird community, and all species for which foraging data were collected (Table 1; full species names in Table S9 in Appendix S1). Of these, 16 species had nine or more playbacks, with the rest representing late southward migrants or rare species in the hardwood habitat. Individuals responded to 87% of presentations (N = 211), and 20 out of 31 species sampled (65%) responded to all stimuli. The exception was the Eastern Phoebe (Sayornis phoebe), a sit-and-wait flycatcher that rarely responded (6% response rate, N = 1 response) and represented 48% of the nonresponses to playback. A small subset of species responded primarily by diving, but the freezing response represented 152 of 211 playback responses (72%; Table 1). Length of freezing response, by contrast, showed strong intraspecific variation and weak interspecific variation, with averages ranging from 100 to 300 s (Table 1).
For our procedural control, we performed 20 frog call playbacks to 11 species over the second winter, with no responses. We recorded the playback exemplar for 125 of 242 total trials (52%), all in the second winter. While the five exemplars were used at different frequencies (ANOVA, F = 2.67, df = 4, p = .04; Table S10 in Appendix S1), we found no statistical difference in response rates between exemplars when broken down by species (ANOVA, F = 0.72, df = 4, p = .58; Table S11 in Appendix S1).
| Modeling determinants of reliance on social information
Our model averaging results for overall playback response provide strong support for the foraging ecology hypothesis, as model averaging yielded a single significant foraging ecology predictor, the degree of aerial foraging (Aerial-F, p = <.001, beta = −33.73; Table 2). The height at which a species forages also had a high beta estimate and was near significant (Height-F, p = .095, beta = 11.18). By contrast, we found poor support for the sociality hypothesis (Sociality, p = .29, beta = 0.48) and our proxy for call relevance, difference in body mass, also had poor explanatory power (Difference in Mass, p = .13, beta = −0.07).
When we modeled response using the expanded-species pool, foraging ecology was similarly important, with sallying foragers showing a significant nonresponse when compared to gleaning species (Sally vs. Glean, p = <.001, beta = −6.22; Figure 1b and Table S3 in Appendix S1). However, difference in body mass also became a significant predictor once a larger sample size of body masses was included in the analysis (Difference in Mass, p = .003, beta = −0.62). Broadly, species that forage using aerial, sallying maneuvers are less likely to respond to the alarm call playback, and there was decreased responsiveness as body size increasingly differed from the titmouse. Our information theoretic approach yielded 20 candidate models for overall response with an average pseudo r 2 of 0.55 ± 0.01 (Table S1 in Appendix S1); the expanded-species model set included 8 candidate models with an average pseudo r 2 of 0.62 ± 0.01 (Table S4 in Appendix S1).
For our models of response type, model averaging produced a single-species-level predictor of response type, the distance from the trunk at which a species forages (Trunk-F; p = <.001, beta = 9.780; Table 2). However, local factors also affected response type, with the availability of escape cover for the focal individual (Escape-MH; p = .01, beta = −5.03) and the distance of the focal individual from the playback speaker (Distance to Speaker; p = .04, beta = 0.07) also significant, though the effect size of distance to speaker was extremely small. Species that forage farther from the trunk were more likely to dive, whereas trunk-foraging species were more likely to freeze (Figure 2a). Individuals located in exposed microhabitats that were farther from cover at the time of playback were more likely to dive than those located in sites with denser vegetation and closer to escape cover (Figure 2b).
Finally, individuals were statistically more likely to freeze in place when they were located farther from the alarm call stimulus ( Figure 2c). Our candidate set of models for response type consists of 15 models with an average pseudo-r 2 of 0.21 ± 0.02 (Table S2 in Appendix S1).
| Sit-and-wait salliers do not rely on social information
We determined that foraging ecology was the best of the three, species-level hypotheses of the determinants of reliance on social information. The percentage of aerial foraging maneuvers (Aerial-F) was the sole significant variable in explaining overall response in our first model (Table 2). While this trend was strongly driven by the Eastern Phoebe, we found a significant nonresponse by all sallying species when we expanded our model of playback response to include all species to which we performed playbacks (Figure 1a).
The other two tyrannid flycatchers for which playback elicited no response (Acadian Flycatcher, Empidonax virescens; Eastern Wood Pewee, Contopus virens; Table 1) forage similarly using aerial sallies F I G U R E 1 Importance of aerial foraging in determining playback response. Model results of overall response obtained from all species to which playback was presented (N = 31 species, 238 playbacks) are obtained from full model averaging of a candidate set of 8 generalized linear models (Table S4 in Appendix S1). (a) Species that forage more frequently using aerial sallying maneuvers responded to playback less often. Bolded line shows median response rate of all species in a foraging guild, and sample size indicates number of species. (b) Overall response rate to Z call playback is significantly lower for the sallying foraging maneuver (beta = −6.22, p = <.001) and at greater difference in mass from the titmouse (beta = −0.62, p = .003). Foraging maneuvers were assigned to species based on the most frequently observed foraging maneuver in foraging observations (Table S8 in Appendix S1) or based on values from the literature (Table 1). We did not include sociality in this model because it was not shown to be significant in the first model of overall response (Table 2). Full model results are shown in Table S3 in Appendix S1
Probability of response
Response proportion (Table S3 in Appendix S1). While all nonresponding flycatchers are suboscines, we suggest that our findings highlight the uniqueness of aerial foraging behavior rather than an effect of phylogeny. Even in suboscine-dominated Amazonian eavesdropping networks, substrate-based foragers are more responsive to alarm calls than aerially foraging species, suggesting a unique nonresponse by aerial foragers (Martínez et al., 2016;Martínez & Zenil, 2012).
The nonresponse by sit-and-wait flycatchers may be explained by high visual acuity associated with the sit-and-wait sallying ecomorph. Inspection of wholemount retinas of sit-and-wait flycatchers (Tyrannidae) reveals high foveal neuron densities (Tyrrell & Fernández-Juricic, 2017), as well as a cohort of giant retinal ganglion cells which are thought to be involved in movement detection (Coimbra, Luiza Videira Marceliano, Lara da Silveira Andrade-da-Costa, & Yamada, 2006). Such adaptations allow for high spatial resolution and visual acuity, enabling greater probability of predator detection and greater maximum and average detection distances (Tyrell & Fernández-Juricic, 2015). Sallying species are known to act as sentinel species in both Neotropical (Thamnomanes antshrikes; Munn, 1986)
| Species-and microhabitat-specific antipredator behavior
Species-level traits were more important than local microhabitat factors in determining reliance on social information. Local social and microhabitat factors were not significant predictors in either model for overall response, though the availability of escape cover (Escape-MH) and distance from playback stimulus were significantly correlated with response type, suggesting that these factors influence how individuals respond. Small changes in microhabitat can greatly affect the cost of predation imposed on foraging prey species such as small forest passerines (Brown & Kotler, 2004), and shifts in microhabitat use have been documented under changing predation regimes in both time and space (Rodríguez et al., 2001;Suhonen, 1993). However, because foraging and vigilance behaviors vary from mutually exclusive in substrate-based foragers to simultaneous activities in aerial foragers (Lima & Dill, 1990), the physiological trade-offs between foraging mode and specialized eye morphology may be more important in driving reliance on social information (Guillemain, Martin, & Fritz, 2002).
F I G U R E 2 Fitted values for the significant predictors of response type (diving vs. freezing response). Significant predictors are obtained from model averaging of a candidate set of 15 generalized linear models with response type as the dependent variable (Table S2 in Appendix S1). We only analyzed cases in which the focal individual responded (N = 182 playbacks). Solid lines show probability of freezing response calculated by imputing values for the predictor variable of interest into the logistic regression equation for the full model (11 predictor variables; Table 3 in Appendix 1) and using the parameter estimate and intercept values from our model averaging (see Table 2). All other predictor variables were set to mean values. (a) Species which forage further from the trunk were more likely to dive than those that forage on or near the trunk. (b) Individuals foraging in more exposed microhabitats were more likely to dive than those closer to cover. (c) Individuals were more likely to dive for cover when located closer to the playback stimulus Far from cover Near cover The type of escape behavior adopted by responding individuals was species-specific and dependent upon foraging microhabitat. Distance from trunk of the foraging microhabitat was the main driver: Trunk-foraging species were more likely to freeze, while those foraging in the outer branches were more likely to dive. Outerbranch-foraging species need to dive for escape refugia to escape an attack (Lima, 1993), as microhabitats farther from the trunk are more exposed to small raptors (Kullberg, 1995). By contrast, trunkforaging species are less exposed to direct attack and can freeze to avoid detection. The feeding substrate also acts as a refuge by shielding the individual from an attacking predator (Lima, 1992): Woodpeckers typically freeze against the trunk and place themselves on the opposite side of the trunk from the playback speaker (Sullivan, 1984;H. H. Jones, personal observation). This mirrors the previously reported escape behaviors of foliage-gleaning and barkforaging species (Lima, 1993) and provides more evidence that antipredator behavior is highly species-specific and adapted to foraging microhabitat (Lima, 1992;Vanhooydonck & Van Damme, 2003). In birds, escape behavior may be taxonomically stereotyped-at least some species appear to lack the plasticity to adjust it in novel microhabitats (Koivula & Rönkä, 1998).
| "Call relevance" influences response, sociality does not
We found empirical support for the call relevance hypothesis within our system, as measured by difference in body size between each focal species and the titmouse, when we modeled response across a larger range of difference in body masses (0.5-55.5 g, Table 1). This result mirrors trends observed in Australian and Sumatran eavesdropping networks, where similar-sized species were also more responsive to the sentinel species' alarm call (Hua et al., 2016;Magrath et al., 2009). We only detected this effect when including larger-bodied species in our analysis, though the importance of call relevance in our system might be mediated by the information content of alarm calls, which can differ significantly between nuclear species (Goodale & Kotagama, 2005a).
The greater information content of parid alarm calls, which encode more information about the size and threat level of a predator than those of heterospecifics (Sieving et al., 2010;Templeton et al., 2005), may increase the eavesdropping audience because this information can be relevant even to species that share fewer predators. Alternatively, our proxy measure of call relevance may be failing to account for true overlap in the predation risk. Accipiter hawks in Europe, for example, select prey in greater proportion to abundance based on habitat preferences and migratory habit (Götmark & Post, 1996), which we do not measure.
Sociality was not an important factor in determining degree of reliance on social information in our Florida winter community, in contrast to findings from tropical Africa (Radford & Ridley, 2007;Ridley & Raihani, 2007;Ridley et al., 2014). However, these findings come from social systems that comprise kin groups, such as many African primates and the Pied Babbler (Turdoides bicolor). Delayed dispersal and cooperative breeding is more common in the tropics (Brown, 1987), and the resulting family groups often become leaders of mixed-species foraging flocks-possibly because of their alarm call systems (Sridhar & Shanker, 2014). By contrast, the species that form single-species flocks in Florida (e.g., American Goldfinch, Spinus tristis; Yellow-rumped Warbler, Setophaga coronata) are short-distance migrants that form seasonal and temporary groups of nonkin individuals in winter (Hunt & Flaspohler, 1998;Prescott & Middleton, 1990). Thus, these species likely lack sentinel individuals and complex alarm call systems, reducing access to conspecific social information.
| Parids as community informants
In sum, we found a strikingly near-universal response to the Z call, highlighting the important role of the titmouse as an antipredator sentinel. Responsive species exhibit various migratory strategies and flocking propensities, and, in all, 28 species responded of the 31 tested. To our knowledge, this is the first empirical documentation of community-wide responsiveness to the alarm call of a single sentinel species. Our results agree with previous findings of community-wide responsiveness to the antipredator mobbing calls of species of the family Paridae (Gunn, Desrochers, Villard, Bourque, & Ibarzabal, 2000;Hurd, 1996;Langham et al., 2006;Sieving et al., 2004). Taken together, this reliance of the avian community on the social information of one species suggests a keystone role for the titmouse (sensu Kotliar, Baker, Whicker, & Plumb, 1999), likely because it provides highly reliable and complex information about predation risk (Hetrick & Sieving, 2012;Sieving et al., 2010;Templeton et al., 2005). Our results therefore support the idea that titmice play a keystone "community informant" role (Szymkowiack, 2013).
ACK N OWLED G M ENTS
We thank the many field assistants without whom this project would not be possible. Special thanks go to Drs. Scott Robinson and Ben Baiser for their suggestions on methods and Drs. Eben Goodale and Ari Martínez, as well as three anonymous reviewers, for their comments on the manuscript. We also thank Drs. Kristen Malone and Andrea Larissa Boesing for their help, as well as the managers at San Felasco and Paynes Prairie for graciously providing access to the study sites. The National Science Foundation (IOS award #3351308 to KES) funded HHJ on a research assistantship. All work was conducted in compliance with animal use standards of the University of Florida IACUC Committee (permit # 201408466).
CO N FLI C T O F I NTE R E S T
Both authors gave final approval for publication and have no competing interests.
AUTH O R S ' CO NTR I B UTI O N S
HHJ conceived the study, helped design the study, carried out the field work and statistical analyses, and drafted the manuscript. KES helped with the study design, statistical analyses, and manuscript drafting.
DATA AVA I L A B I L I T Y S TAT E M E N T
The original datasets of Z call playbacks and winter foraging obser- (2014) -Temperature Hourly temperature average at 10 m Florida Automated Weather Network Note: The first four variables represent principal coordinate axes obtained from ordination of field observations of foraging behavior (37 variables; Table S3 in Appendix S2) while the next three variables similarly represent principal coordinate axes obtained from an ordination of six microhabitat variables associated with the target individual recorded just before each playback (Table S4 in Appendix S2). Abbreviations: F, foraging; MH, microhabitat. | 2019-09-19T09:08:57.945Z | 2019-09-14T00:00:00.000 | {
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229351220 | pes2o/s2orc | v3-fos-license | Characteristics of the Tumor Microenvironment That Influence Immune Cell Functions: Hypoxia, Oxidative Stress, Metabolic Alterations
Simple Summary For decades nearly all cancer patients were treated with cytotoxic chemotherapy, killing any rapidly dividing cell in the body. More recently, researchers have been studying the immune system’s response to cancer and have developed a novel class of drugs that stimulate the body’s own response to tumors. This class of immunotherapy drugs primarily involve T-cells, immune cells that target and destroy cancer cells. While these drugs can lead to remarkable and sustained response, most patients do not respond. Understanding the resistance mechanisms in non-responding tumors is now an active area of investigation. In this review, we explore a host of factors in the tumor microenvironment, the cellular and molecular space within tumor tissue, to identify possible culprits of immunotherapy resistance. Specifically, the downstream effects of low oxygen and metabolic byproducts in the tumor microenvironment have been associated with immune cell dysfunction. Importantly, targeting these pathways may offer promising therapies to improve the response to current immunotherapy. Abstract Immunotherapy (IMT) is now a core component of cancer treatment, however, many patients do not respond to these novel therapies. Investigating the resistance mechanisms behind this differential response is now a critical area of research. Immune-based therapies, particularly immune checkpoint inhibitors (ICI), rely on a robust infiltration of T-cells into the tumor microenvironment (TME) for an effective response. While early efforts relied on quantifying tumor infiltrating lymphocytes (TIL) in the TME, characterizing the functional quality and degree of TIL exhaustion correlates more strongly with ICI response. Even with sufficient TME infiltration, immune cells face a harsh metabolic environment that can significantly impair effector function. These tumor-mediated metabolic perturbations include hypoxia, oxidative stress, and metabolites of cellular energetics. Primarily through HIF-1-dependent processes, hypoxia invokes an immunosuppressive phenotype via altered molecular markers, immune cell trafficking, and angiogenesis. Additionally, oxidative stress can promote lipid peroxidation, ER stress, and Treg dysfunction, all associated with immune dysregulation. Finally, the metabolic byproducts of lipids, amino acids, glucose, and cellular energetics are associated with immunosuppression and ICI resistance. This review will explore these biochemical pathways linked to immune cell dysfunction in the TME and highlight potential adjunctive therapies to be used alongside current IMT.
Introduction
Metabolism serves as a unifying theme to the complex set of cells and cellular processes of the heterogeneous tumor microenvironment (TME). Indeed, all cells require fuel to perform their diverse functions, and the metabolic by-products of cellular energetics are a key component of the TME [1]. As the field of immunotherapy continues to grow, our understanding of this metabolic meshwork has become a critical factor in optimizing immune cell function [2]. Although immune checkpoint inhibitors (ICI) and other novel immune-based therapies have revolutionized the current treatment paradigm of many cancers, only a minority of patients show sustained response [3]. Thus the burgeoning field of immunometabolism is now being harnessed to highlight the interconnecting elements of the TME that may lead to a dysfunctional immune state [2].
The widespread metabolic dysregulation found in the TME creates numerous hurdles but also provides key insight into more effective combinatorial approaches with ICI. Given the plasticity and dynamic nature of the TME, reversion to an immune-responsive state will require a more detailed schema into the timing and precise nature of combination therapies [4,5]. Furthermore, the signaling pathways of immune suppressor cells (e.g., Treg, MDSC) can be hijacked by metabolic mediators and further disrupt cytotoxic T cell activity in the setting of ICI [6]. Based on many more examples that will be described below, it is clear that the harsh metabolic milieu of the TME requires reversal with novel therapies to unleash the full benefit of ICI and other immune-based therapies, and extend the life saving benefits of immunotherapy to a higher number of patients.
We have chosen to categorize the primary immune components of the TME based on the most evidence-based pre-clinical models, and the potential implications for clinical translation. These three categories include: hypoxia, oxidative stress, and metabolic alterations. We will highlight several pioneering discoveries from the last decade that will have broad implications for the field of immunotherapy in the coming years.
Hypoxia
Hypoxia is perhaps the most well-studied perturbation found in the TME. The translational ramifications of tumor hypoxia were described in detail over 60 years ago, when it was discovered that hypoxic tumor cells became resistant to radiation therapy [7]. Over the ensuing years, hypoxia became a central theme in the pathophysiology of tumor growth, especially as it related to the evasion of cytotoxic therapies [8]. The original "Hallmarks of Cancer," though focused on genetic perturbations leading to unrestrained proliferation, did highlight hypoxia as a key signal in promoting VEGF-mediated angiogenesis [9]. In the revolutionary era of immunotherapy, hypoxia has been recognized as a fundamental link between the emerging hallmarks of "tumor-promoting inflammation" and "deregulated cellular energetics" [10]. As the resistance to ICI-and how to overcome it-has become a primary focus in oncology, hypoxia and its relation to several components of the TME is now a key target for the next phase of immune-based therapies. As we explore these novel therapies, it is first essential to understand the pathophysiology of hypoxia in the TME: in this section we will review hypoxia as it relates to (a) molecular markers, (b) immune cell dysfunction, (c) tumor and stromal cell interaction, and (d) angiogenesis in the context of a tumor-induced immunosuppressive phenotype.
Although the implications of tumor hypoxia are well-documented, the exact etiology behind low oxygen levels in the TME are perhaps less well understood. The unregulated and explosive growth of cancer leads to a chaotic conglomeration of tumor and stromal cells with a dysfunctional vascular supply and poor access to nutrients [11]. Additionally, as will be described below, the metabolism and energy production of aggressive tumors can shift towards a highly oxidative state leading to hypoxic conditions for the remainder of the TME [12].
Molecular Markers
The hypoxia inducible factor (HIF) proteins are a set of transcription factors that are responsible for many of the phenotypic characteristics of the hypoxic TME. HIF-1α forms a heterodimer with HIF-1β; this complex binds to the hypoxia-responsive element (HRE) to induce expression of an assortment of genes used for normal tissue adaptation to low oxygen levels but also the aforementioned immunosuppressive phenotype of the TME [13]. As previously described by Noman et al., a variety of immune-related molecular markers are upregulated in response to low oxygen levels in the TME [14]. The first, and perhaps most relevant to current ICI, involves PD-L1, a ligand found on the surface of antigen-presenting cells and many tumor cells that binds to the PD-1 receptor on activated T cells [15]. HIF-1α, activated under hypoxic conditions, has been shown to directly upregulate expression of PD-L1, a process that inhibits further T-cell activation and signaling upon contact with PD-1 [16]. Another checkpoint found on myeloid derived suppressor cells (MDSC), V-Domain Ig Suppressor of T-Cell Activation (VISTA) was also shown to be upregulated in hypoxic regions of a colon cancer (CT26) model via HIF-1α [17]. Finally, the immune checkpoint CD47 has also been shown to be more highly expressed under hypoxic conditions. This protein found on the surface of tumor cells binds to macrophage and dendritic cells preventing phagocytosis, a defense against the innate immune system. While CD47 has been a more well-established biomarker and negative predictor in hematologic malignancies, recent studies with breast and pancreatic cancer have also revealed an association between CD47 expression with HIF-1α [18,19]. Finally, a non-classical MHC-I variant, HLA-G, has been shown to have several HREs in its promoter region and induced expression under hypoxic conditions. HLA-G presents peptide fragments on the surface of tumor cells, binds to several different proteins expressed by both lymphocytes and innate immune cells, and has well-documented associations with immune suppression and poor prognosis in a variety of tumor types [20].
Immune Cells
There are a variety of innate and adaptive immune cells that functionally mediate the immunosuppressive phenotype of the TME. The work of Chouaib et al. has highlighted many of the immune cells affected by the hypoxic conditions in cancer-namely, tumor-associated macrophages (TAM), MDSCs, regulatory T cells (Treg), and of course, cytotoxic T cells (CTL) [6]. A variety of TAM subtypes exist in the TME, with the M2 type exhibiting immunosuppressive characteristics. Data from hepatocellular carcinoma (HCC) models have shown significant correlation between HIF-1α expression and an increased proportion of M2 macrophages [21]. TAMs have also been associated with increased matrix metalloproteinase-7 (MMP-7) expression in hypoxic regions of the TME; MMP-7 inhibits tumor cell lysis via cleavage of the Fas ligand, a transmembrane protein involved in apoptosis [22,23]. Another cell line from the myeloid lineage, MDSCs, have even more data tied to hypoxia and immunosuppression. MDSCs were first called "natural suppressor" cells upon recognition of their potent inhibition of proinflammatory cytokines and both CD8+ and CD4+ T-cell activation [24]. The discovery of increased peripheral circulation of MDSC in tumor models led to their further characterization as detrimental "immune suppressor" cells in the TME [25][26][27]. Hypoxic conditions have been shown to further stimulate the immunosuppressive function of these cells; MDSC PD-L1 expression is also strongly upregulated in response to HIF-1α [28]. MDSCs in hypoxic tumors suppress both antigen-specific and antigen non-specific T cells, contributing to a more depleted TIL state [29]. HIF-1α has also been shown to increase differentiation of MDSCs into TAMs, with functional ramifications [30]. Moving to the lymphoid lineage, Tregs are likely the most recognized immunosuppressive cells found in the TME. Derived from CD4+ T cells, Tregs inhibit innate and adaptive immune function via cytokine signaling (TGF-β, IL-10) and checkpoint interactions (CTLA-4, LAG3) among other mechanisms [31]. The expression of FOXP3, a differentiating biomarker of CD4+ to Treg morphology, is directly upregulated via HIF-1α [32]. Various chemokines (CCL-17, CCL-28, TGF-β1 itself) secreted by hypoxic tumors also attract Tregs to the TME [33]. As a functional example, Hasmim et al. sought to inhibit the chemoattractant TGF-β1 by blocking one of its transcription factors, NANOG; this led to a significant decrease in Treg infiltration in a B16-F10 melanoma tumor bed [34]. Finally, in describing the effects of hypoxia on CD8+ T cells, the picture is less clear. As described by Vuillefroy de Silly et al., a dichotomy of both in vitro and in vivo CD8+ T cells under a varied range of normoxic and hypoxic conditions has made experimental results difficult to summarize [35]. On one hand, T-cell expansion is diminished under hypoxic conditions, possibly due to disrupted T-cell receptor (TCR) signaling. On the other hand, no compelling evidence has linked HIF-1α expression with decreased CTL function in the TME. Furthermore, the effects of hypoxia on CTL cytolytic capabilities have been mixed [35], although our work with antigen-specific T cells revealed lower IFN-γ levels and depressed cytolytic activity under acutely hypoxic conditions [36]. While more research is needed to further characterize the effects of hypoxia on CTL function, these crucial cells have clearly evolved to adapt to a wide range of harsh environments. In general, the detrimental effects of hypoxia are spread amongst most types of immune cells in the TME.
Tumor and Stromal Cells
While immune cells are of significant interest in the era of immunotherapy (IMT), tumor cells themselves along with the tumor-stromal interaction create a climate of immune resistance that is enhanced by hypoxia. Autophagy is an oft-cited resistance mechanism of tumor cells in which altered endosomes degrade cytolytic proteins (e.g., granzyme B, perforin) released by NK and CTLs [37]. Recent work has demonstrated that hypoxia, via a HIF-1α-and pSTAT3-dependent mechanism, upregulates this process leading to selective degradation of granzyme B and depressed cytolysis [38]. Cancer-associated fibroblasts (CAF) are one of the main inducers of extracellular matrix (ECM) remodeling, a deleterious process that enhances tumor growth, immune resistance, and migration [39]. Under hypoxic conditions, CAFs have been shown to induce tumor progression in a pancreatic cancer model via arginase II secretion, a mechanism that leads to T cell anergy and a depressed immune state [40]. Finally, the unique metabolic interaction between CAFs and tumor cells can lead to a "corrupted" nutritional state in the TME that further disrupts immune cell function [41]. Stromal cells populating the most hypoxic conditions of the tumor will rely on glycolysis, produce large amounts of lactate/pyruvate intermediates, and "feed" neighboring tumor cells that can participate in the more valuable oxidative process to stimulate hyperproliferation [42]. In this "reverse Warburg effect" model, oxygen is a rare and important cofactor in the TME with repercussions on immune cell function, as previously described [42]. Further ramifications of this model and other metabolic alterations will be described in the upcoming sections.
Angiogenesis
Another element interconnecting hypoxia and the immune state is the blood vessels that carry oxygen to-and waste products out of-the TME. The role of angiogenesis contributing to a pathological TME has been studied for nearly fifty years; tortuous, chaotic, leaky vessels lead to elevated interstitial pressure, disorganized oxygen delivery, and inefficient nutrient transfer [43]. Indeed, the critical role of angiogenesis in cancer biology has long been appreciated, and anti-angiogenic therapies have been successfully used in several cancer types, including renal cell carcinoma and colorectal cancer. While tumor cells are constantly mutating and adapting to their harsh climate, surrounding "normal" cells and tumor infiltrating immune cells struggle to compete. Tumor angiogenesis is primarily mediated through a set of VEGF growth factors and their family of receptors (VEGFR); angiopoietin and various adhesion molecules are also involved [44]. Disrupting angiogenesis via VEGF inhibitors initially held promise as a means of nutrient and oxygen blockade to tumor cells, however, most do not respond to monotherapy [45]. Further disruption of already dysfunctional vessels has been shown to increase intratumoral hypoxia; a vicious cycle that upregulates HIF-1α, exacerbating many of the resistant mechanisms already described [46]. Indeed, while anti-angiogenic therapy was first thought to "starve" the tumor by cutting off blood supply, it is now appreciated that oxygen tension and immune infiltration are critical to maintain antitumor activity. This is especially important when considering partnering an anti-angiogenic with an immune-based therapy. More recent investigations have sought to "normalize" the vasculature of the TME via transient VEGF blockade to stabilize the endothelium, increase oxygen and drug delivery, and reduce vascular permeability with associated metastatic potential [47]. As explained by Ramjiawan et al., prudent timing of both anti-angiogenic and ICI therapies could reprogram and stabilize the TME and allow infiltrating lymphocytes to properly function before resistant mechanisms (many secondary to hypoxia) develop [43]. As of 2020, there are now several early phase clinical trials looking into various combinations of VEGF(R) + PD1/L1 blockade in colorectal, hepatocellular, prostate, melanoma, and other cancers [48][49][50][51][52][53]. Additionally, in advanced renal cell carcinoma (RCC) where VEGF inhibition has already been standard therapy, a phase III trial (KEYNOTE-426) showed improved outcomes with combination PD1 + VEGF therapy as compared to VEGF inhibition alone [54].
Targeting Hypoxia
When considering drug therapy related to hypoxia and the TME, it is important to first recognize the heterogenous state of intratumoral immune infiltration and the limitations of ICI monotherapy. Because of the dichotomous results seen in early clinical trials with ICI, a spectrum of tumor histologic subtypes were explored and characterized to better explain these differential outcomes [55]. Tumors with favorable ICI response, high density of IFN-γ producing CTLs, high tumor mutation burden or microsatellite instability (i.e., neoantigen production) can be categorized as "inflamed" TMEs with pre-existing immunity. The other end of the spectrum includes "noninflamed" TMEs with diminished CTL infiltration, low PD-L1 expression, and low mutational burden (i.e., immunologically ignorant) [55]. Tumors between these extremes are characterized by moderate CTL infiltration and neoantigen production but with hampered immune activity secondary to dysfunctional angiogenesis, hypoxia, and suppressive immune cells, cytokines, and metabolic cofactors. This "middle" group of tumors has the greatest potential to benefit from combinatorial therapy with ICI, with the aim of reversing some of the detrimental immunosuppressive effects of hypoxia (among others) and restoring a more functional "inflamed" TME [56,57].
Given the intense focus on hypoxia, one would surmise that blocking the primary transcriptional regulator (i.e., HIF-1α) would be a key opportunity to correct the immunosuppressive climate of the TME. However, given the ubiquity of this transcription factor and its role in controlling multiple cellular processes, the lack of specificity and risk of toxicity have mired the investigations of direct HIF-1α inhibitors [14]. Thus, indirect inhibitors of HIF-1α and blockade of downstream effector molecules have become a more pertinent area of research [58]. A host of agents have been discovered with incidental HIF-1α modulation; these include antisense oligonucleotides, the translational inhibitor and microtubule-targeting metabolite 2-methoxyestradiol (2ME2), heat shock protein inhibitors, histone deacetylase inhibitors, and even topotecan (a topoisomerase I inhibitor) among others [59]. Many of these drugs are under phase I clinical trial investigation in combination with various chemotherapies, although there are limited data on ICI combination. Over the last twenty years, a novel approach involving oxidation-reduction (redox) reactions has led to the development of hypoxia pro-drugs [60]. These prodrugs are activated via redox under extremely hypoxic conditions (<0.5% O 2 ) with most active drugs leading to cytotoxic activity via DNA cross-linking [61]. After early promise [62], a phase III clinical trial (MAESTRO) is now underway, studying the effects of the hypoxia prodrug evofosfamide with gemcitabine in patients with pancreatic adenocarcinoma [63]. This same prodrug has also been studied pre-clinically in combination with anti-CTLA4 and anti-PD1 antibodies in a murine prostate cancer model [64]. Dual therapy led to a significant reduction in tumor growth with an elevated CTL to MDSC ratio as compared to either evofosfamide or ICI alone [64]. Finally, antagonists of two hypoxia-driven elements, CAIX and A2AR, are also under investigation with the goal of combating immunosuppression in the TME. The carbonic anhydrase enzyme, CAIX, is upregulated via HIF-1α and a significant prognostic marker in RCC; a preclinical study of anti-CAIX antibodies (girentuximab) was shown to stimulate both innate and adaptive immune-mediated killing of tumor cells [65]. The phase III ARISER clinical trial showed no significant survival benefit between monotherapy girentuximab and placebo in the adjuvant setting [66], though this has yet to be evaluated in the metastatic setting. The adenosine A2A receptor (A2AR) has been associated with advanced pathological grade along with HIF-1α expression in head and neck squamous cell carcinoma (HNSCC) [67]. An A2AR antagonist, SCH58261, was shown to not only decrease tumor growth but also increase the ratio of CTL to Treg cells in a murine HNSCC model [67]. Based on these preclinical data, multiple phase I clinical trials evaluating A2AR blockade plus anti-PD(L)1 are underway in advanced solid tumors [68,69]. In general, modulators of HIF-1 and its downstream elements have shown strong potential to reverse immunosuppressive factors in the TME; future clinical trials should be aimed at combinatory methods with ICI and other immune-based therapies (see Table 1) [70].
Oxidative Stress
The role of oxygen is two-fold in regards to TME immunosuppression. While hypoxia may be one of the most studied elements, evidence also points to oxidative stress as an important byproduct leading to a dysregulated immune state [71]. Originally studied in the field of chronic inflammation secondary to autoimmune and infectious diseases, lipid peroxidation has more recently been linked to several carcinogenic effects. Reactive oxygen species (ROS) and free radicals can preferentially oxidize fatty acids through lipoxidation, a process that leads to reactive aldehydes with numerous downstream consequences [72]. As detailed by Martin-Sierra et al., a specific aldehyde, HNE, has demonstrated both anti-tumorigenic effects via NFkB inhibition as well as immunosuppression depending on the relative concentration and specific HNE adduction [73]. One of these protein-HNE adducts has been shown to inhibit the phagocytic properties of innate cells in the TME [74]. Other HNE reactions have been associated with altered cytokine signaling in macrophages [73]. Thus, while more work is needed to summarize the effects of lipoxidation on downstream immune functions, harnessing this important redox process could hold promising therapeutic options in the future.
The endoplasmic reticulum (ER), the protein-folding workhorse of the cell, is also significantly affected by oxidative stress; this state of ER stress has been linked to various pro-tumorigenic effects, including immunosuppression [75]. A cervical cancer murine model was found to have increased tumor-infiltrating MDSCs with enhanced immunosuppressive capacity upon administration of thapsigargin, a specific ER stress inducer [76]. Furthermore, XBP1, a multimodal transcription factor associated with MHC II regulation along with the ER stress response was found to impede tumor immunogenic cell death in a colorectal cancer model [77]. While these examples have been specific to the tumor cells themselves, immune cells are also affected by ER stress. Early in vitro trials showed that ER stress led to dysfunctional antigen-presenting dendritic cells and an increase in immunosuppressive signaling molecules (e.g., arginase) [78]. Additionally, Cubillos-Ruiz et al. analyzed tumor-infiltrating dendritic cells within ovarian tumors-elevated ROS levels led to increased lipid peroxidation, HNE generation, and resultant ER stress [79]. Given these findings, several therapeutic options have been proposed: antioxidants to control ROS production, lipid uptake blockade to prevent peroxidation and HNE formation, and conditional knockout of XBP1 in specific immune cells [75]. Per the latter, XBP1 deletion in dendritic cells was found to stimulate CTL activity and delay tumor progression in multiple ovarian tumor models [79]. Again, further validation in both preclinical and clinical settings are warranted, but the effects of ER stress on TME immune activity may provide a window of therapeutic opportunities. Furthermore, lipid peroxidation and HNE could serve as important biomarkers for an immunosuppressed phenotype [75,80].
The role of Tregs in the TME has been thoroughly investigated, but more recent evidence points to a novel mechanism of immunosuppression related to oxidative stress. Maj et al. studied the role of oxidative stress-induced apoptotic Treg cells in the TME [81]. Surprisingly, the apoptotic cells were shown to have superior immunosuppressive qualities as compared to the live counterparts. Specifically, both the functional secretion and expression levels of CTL IL-2 and IFN-γ were significantly lower when exposed to apoptotic Treg cells. While immunoblotting revealed high levels of PD-L1, CTLA-4, CD39, and CD73 in the apoptotic cells, PD-L1 and CTLA-4 blockade did not mitigate CTL suppression. Thus, supernatant analysis was performed and showed that novel, small molecules were responsible for this increased suppression rather than well-characterized cytokine proteins (e.g., TGF-β, IL-35, IL-10). Further assays revealed that adenosine (via its A2A receptor, a known immunosuppressive mediator) was one of these culprit molecular factors [67]. Finally, researchers compared the results of oxidative stress on Treg versus CTL cells (via H 2 O 2 administration); expression of the NRF2 antioxidant gene family was significantly lower in the Treg cells and likely the responsible factor for the high rate of apoptosis [81]. This experiment not only characterizes a unique functional mechanism of Treg immunosuppression, it also highlights oxidative stress as another key mediator of ICI resistance.
Metabolic Alterations
Thus far, we have detailed the important effects of both hypoxia and oxidative stress on the immune state of the TME. However, much of the basis for these and other immunosuppressive factors derives from deregulated energy metabolism, an "emerging hallmark" of cancer as described in Weinberg's 2011 landmark paper [10]. This overarching metabolic category is flanked by two additional hallmarks, "avoiding immune destruction" and "tumor-promoting inflammation," a collective foreshadowing into the pioneering research of immunometabolism.
Glycolysis and Oxidative Phosphorylation
The oft-touted Warburg hypothesis has shaped the metabolic vernacular of carcinogenesis for the last century [82]; however, this primary focus on aerobic glycolysis is a limited and archaic vantage point for understanding cellular energetics in the TME. An increase in glycolytic flux has certainly been demonstrated in many cancers, although glycolysis alone is not sufficient for energy production or tumor progression [83]. Indeed, several activating mutations within the PI3K-AKT-mTOR pathway are linked to higher glycolytic rates, yet the competitive advantage is likely tied to enhanced production of glycolytic intermediates rather than ATP generation [84]. These glycolytic intermediates are shunted into various anabolic pathways for nucleotide, protein, and lipid synthesis as well as oxidative metabolism for enhanced ATP production in less hypoxic regions of the TME. This symbiotic transfer of metabolites has been neatly described as the "reverse Warburg" effect [85], however, the heterogenous nature of each cancer precludes an assumption of any one consistent model. Regardless, increasing evidence points to an equal if not more critical dependence on mitochondrial metabolism as compared to glycolysis in regards to tumor progression and immunosuppression [86]. For example, in addition to studying the oncogenic PI3K/AKT pathway, loss of the p53 tumor suppressor has also been shown to increase the flux of glycolytic intermediates [87]. However, mitochondrial inhibition (via metformin) of p53 (−/−) colon cancer xenografts selectively leads to significant growth suppression, demonstrating that increased glycolysis alone does not explain the unchecked pattern of tumor proliferation [88].
To further characterize the role of glycolytic and oxidative tumor metabolism, oxygen as a critical metabolite, and the collective effects on immunotherapy, we developed a series of experiments modelling TIL function in the TME. Scharping et al. analyzed the rates of glycolysis and oxidative phosphorylation (OxPhos) using a Seahorse Bioanalyzer, which measures extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively [36]. Both melanoma and colon cancer tumor cells were measured, and although there was no difference in ECAR, the melanoma model had significantly higher OCR. Furthermore, TILs derived from the melanoma tumor demonstrated increased levels of hypoxia along with lower IFN-γ secretion and decreased cytolytic activity. Finally, complex I inhibition via metformin reversed both hypoxia and TIL dysfunction in the previously high OCR melanoma model. Not only are these results consistent with the previous discussion of hypoxia and its detrimental effects on TIL function, but it links these effects to a core metabolic driver-oxidative metabolism. In further work, we developed murine melanoma models with either Glut1 (glycolytic) or Ndufs4 (OxPhos) knockdown tumors. OxPhos-deficient tumors showed less hypoxia and more functional TIL activity as compared to control and Glut1 knockdown tumor samples [89]. Additionally, only the OxPhos-deficient tumors showed response to anti-PD1 treatment, highlighting the impact of OxPhos-mediated hypoxia on TIL function and ICI efficacy. As a clinical correlate, tumors from 19 patients with melanoma were biopsied; higher OxPhos was significantly associated with decreased TIL function and higher TIL exhaustion, non-response to anti-PD1 therapy, and also decreased progression free and overall survival [89]. Based on these preclinical data, the phase I clinical trial investigating pembrolizumab plus metformin in advanced melanoma is now underway (NCT 03311308) [90]. An additional analysis of melanoma brain metastases (MBM) revealed a significant increase in OxPhos levels, both via genetic expression and metabolite profiling [91]. These highly oxidative tumors correlated with increased immunosuppression and reduced survival in murine MBM models; reversal of these effects was achieved using a novel OxPhos inhibitor, IACS-010795, a drug currently being investigated in multiple phase I clinical trials [92,93]. Understanding the impact of tumor cell metabolism on response to ICI has broad clinical implications, and will need to be evaluated across various tumor types. These results suggest that oxidative metabolism may serve as a predictive marker for response to ICI and other TIL-based therapies.
Amino Acids
While the interplay of glycolysis and OxPhos have distinct implications for the immune state of the TME, a number of other metabolic alterations also affect CTL function. For example, amino acids play a large role in tumor-mediated anabolic production, and competition between cancer cells and T cells can be a prime source of immunosuppression [94]. Yang et al. describe a "glutamine loop" in an ovarian cancer model in which CAFs convert the metabolic byproducts glutamate and lactate to glutamine; amino acid transporters upregulated in tumor cells rapidly import glutamine and re-export glutamate and lactate into the extracellular space to repeat the process [95]. While all amino acids are critical for cellular proliferation and protein biosynthesis, glutamine in particular has been shown to correlate with T-cell activation; in conjunction, decreased glutamine concentration has been tied to dysfunctional TIL activity [96]. In similar competitive fashion, arginine is preferentially imported by proliferating tumor cells; increased intracellular concentration has also been tied to elevated TAM infiltration in the TME, an immunosuppressive macrophage as described above [97]. As with glutamine, arginine has also been shown to be a necessary cofactor for T cell survival and anti-tumor activity [98]. Finally, a catabolic enzyme within the tryptophan pathway, indoleamine 2,3-dioxygenase-1 (IDO1), has gained recent interest due to its significant immunosuppressive effects. IDO1 was first studied due to associations between expression level and poor prognosis in a variety of cancers [99]. Subsequently, tryptophan catabolites (within the Trp-Kyn-AhR pathway) and IDO1 were both found to promote MDSC and Treg trafficking in addition to direct T cell anergy [100,101]. Because PD-1/PD-L1 interaction was found to induce IDO1 expression in dendritic cells [102], large-scale investigations into combined IDO1 blockade plus ICI were initiated [103]. While initial results were promising [103], the phase III clinical trial of epacadostat plus pembrolizumab vs. pembrolizumab alone did not show improved survival outcomes [104]. Despite these results, efforts to identify additional targets within the immunosuppressive Trp-Kyn-AhR pathway are still ongoing [105].
Lipids
Lipid metabolism is another area of active research as fatty acids (FA) play a significant role in tumor and immune cell function. Cancer cells upregulate various enzymes to both synthesize FAs de novo for proliferative purposes as well as catabolize FAs from neighboring cells for enhanced TCA throughput [106]. At the center of lipid metabolism, inflammation, and immune function lies prostaglandin E2 (PGE2), a lipid derivative. Researchers have demonstrated that tumor-induced PGE2 leads to MDSC and CAF activation in breast cancer models, differentiation of monocytes into TAMs in cervical cancer, and can also lead to COX-2 mediated angiogenesis in pancreatic cancer [107][108][109][110]. Additionally, as described by Herber et al., tumors with increased lipid content contain dendritic cells with dysfunctional antigen-presenting capabilities, likely secondary to impaired MHC-I trafficking [111,112]. Furthermore, inhibition of acetyl-CoA carboxylase led to normalization of lipid content and consequently restored dendritic cell function in a murine lymphoma model [111]. Overall, higher lipid content in tumors has been associated with increased metastatic potential and resistance to chemotherapy [113,114]. Additional evidence now points to the immunosuppressive characteristics of altered lipid metabolism; targeting these dysregulated pathways may be a viable combinatorial approach with current immune based therapies. As an example, Colacios et al. found that dysregulated sphingolipid metabolism, via sphingosine kinase 1 (SK1) and sphingomyelin synthase 1 (SMS1), in melanoma cells was associated with increased immunosuppressive markers and a more aggressive phenotype [115,116]. SK1-silenced murine models led to enhanced response to PD-1 and CTLA-4 blockade, promoting SK1 as a "checkpoint lipid kinase" that could modulate the immune state of the TME [115].
Lactate
With enhanced glycolytic flux as described in such models as the reverse Warburg effect, increased lactate production leads to another important metabolic byproduct in the TME. While extracellular lactate can be shunted to normoxic tumor cells for enhanced proliferation, its effect on immune infiltration is more detrimental [94,117]. For example, increased lactate concentration can induce M2-polarization of TAMs as well as suppress NK function via enhanced MDSC trafficking in the TME [118,119]. Additionally, lactate has been shown to stimulate angiogenesis via a HIF-1-dependent process [120]. Peppicelli et al. studied the effects of lactate-mediated acidity in melanoma tumors; an increase in epithelial-to-mesenchymal transition was found (marker of aggressive phenotype) in addition to enhanced oxidative metabolism [121]. The OxPhos inhibitor, metformin, was applied to these tumors and resulted in a striking decrease in proliferative capacity in this acidic tumor subset [121]. Given the relevance of lactate flux throughout the TME, lactate transport (MCT) inhibitors have been proposed as a viable anti-cancer agent. A current MCT1 inhibitor under clinical investigation (AZD3965) has been shown to inhibit tumor growth via altered lipogenic enzyme activity; MCT1 blockade also led to enhanced dendritic and NK cell infiltration in this lymphoma xenograft model, highlighting the immunosuppressive effects of lactate [122]. Finally, an intriguing experiment by Benjamin et al. combined an MCT1/MCT4 inhibitor with metformin in a murine liver tumor model; this combined approach led to impaired NAD+ regeneration, a form of synthetic lethality secondary to intratumoral redox cessation [123]. Identifying efficacious drug regimens to combine with current IMT is certainly attainable (see Figure 1), however, this latter example highlights the more toxic ramifications that may preclude widespread applicability.
Cancers 2020, 12, x 10 of 17 inhibitors have been proposed as a viable anti-cancer agent. A current MCT1 inhibitor under clinical investigation (AZD3965) has been shown to inhibit tumor growth via altered lipogenic enzyme activity; MCT1 blockade also led to enhanced dendritic and NK cell infiltration in this lymphoma xenograft model, highlighting the immunosuppressive effects of lactate [122]. Finally, an intriguing experiment by Benjamin et al. combined an MCT1/MCT4 inhibitor with metformin in a murine liver tumor model; this combined approach led to impaired NAD+ regeneration, a form of synthetic lethality secondary to intratumoral redox cessation [123]. Identifying efficacious drug regimens to combine with current IMT is certainly attainable (see Figure 1), however, this latter example highlights the more toxic ramifications that may preclude widespread applicability.
Conclusions
Checkpoint blockade and other immune-based therapies are now the standard of care for many cancers. However, given the relatively low and stagnant response rates seen with monotherapy, critical focus will be placed on improving the efficacy of these drugs over the next decade, by overcoming both primary and secondary resistance. While heterogenous and complex, characterizing the immune state of the TME is a key factor in this endeavor, and it is now clear that tumor-mediated metabolic perturbations are a primary driver of immune dysfunction. Hypoxia, oxidative stress, and other metabolic alterations can have a wide range of detrimental effects on T cell function, but further characterization also leads to important translational implications. Hypoxia, primarily through HIF-1-dependent processes, is strongly linked to an immunosuppressive phenotype via altered molecular markers, immune cell trafficking, and angiogenesis. Similarly, lipid peroxidation, ER stress, and Treg function can lead to immune dysregulation secondary to oxidative stress. Finally, a host of metabolic byproducts related to lipids, amino acids, glucose, and cellular energetics have been linked to T-cell suppression and ICI resistance. Targeting these alterations may help reverse the metabolic insufficiency of the TME and provide promising adjunctive therapies
Conclusions
Checkpoint blockade and other immune-based therapies are now the standard of care for many cancers. However, given the relatively low and stagnant response rates seen with monotherapy, critical focus will be placed on improving the efficacy of these drugs over the next decade, by overcoming both primary and secondary resistance. While heterogenous and complex, characterizing the immune state of the TME is a key factor in this endeavor, and it is now clear that tumor-mediated metabolic perturbations are a primary driver of immune dysfunction. Hypoxia, oxidative stress, and other metabolic alterations can have a wide range of detrimental effects on T cell function, but further characterization also leads to important translational implications. Hypoxia, primarily through HIF-1-dependent processes, is strongly linked to an immunosuppressive phenotype via altered molecular markers, immune cell trafficking, and angiogenesis. Similarly, lipid peroxidation, ER stress, and Treg function can lead to immune dysregulation secondary to oxidative stress. Finally, a host of metabolic byproducts related to lipids, amino acids, glucose, and cellular energetics have been linked to T-cell suppression and ICI resistance. Targeting these alterations may help reverse the metabolic insufficiency of the TME and provide promising adjunctive therapies alongside current IMT. | 2020-12-23T06:16:38.259Z | 2020-12-01T00:00:00.000 | {
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250685094 | pes2o/s2orc | v3-fos-license | Inert Doublet Model and DAMA: Elastic and/or inelastic dark matter candidates
We investigate the compatibility of the DAMA results with the other direct detection experiments in the framework of a specific dark matter model, the Inert Doublet Model. In this model there are three different regimes for which the dark matter candidate is either light, heavy or in the middle mass range. We present our analysis for the light WIMPs cases with elastic interaction on nuclei. The heavy mass regime is instead an inelastic dark matter candidate, while in the middle mass range both interactions are considered. In the three regimes we exhibit regions of the parameter space in agreement with all the experiments, even though they appear to be significantly constrained.
Overview on the Inert Doublet Model
The Inert Doublet Model (IDM) is a simple extension of the Standard Model with dark matter (DM), consisting in two Higgs doublets and a Z 2 symmetry and it has been widely discussed in the literature. Here we focus our attention on the direct detection phenomenology in the light of the DAMA experiment.
The usual Higgs doublet is denoted by H 1 , while the extra, or inert doublet, H 2 , is the only field of the model that is odd under the Z 2 symmetry. This ensures the stability of the lightest member of H 2 , which will be a DM candidate, and prevents from flavor changing neutral currents [1]. We will assume that Z 2 is not spontaneously broken and that H 2 does not develop a vacuum expectation value. We write H 2 = (H + (H 0 + iA 0 )/ √ 2) T , similarly to the ordinary Higgs doublet, and The potential is written as After electroweak symmetry breaking, H 1 = v = −µ 2 1 /λ 1 = 246 GeV, the masses of the physical scalar fields are given by with λ Hc ≡ λ 3 /2 and λ H 0 ,A 0 ≡ (λ 3 + λ 4 ± λ 5 )/2. We will consider H 0 to be the DM candidate (i.e. λ 5 < 0) though the results would be exactly the same for A 0 changing the sign of λ 5 . In the next section we discuss two particular limits of the mass relations, leading to an elastic scenario with a light DM candidate, and to an inelastic scenario in the multi-TeV DM mass range. The solid (brown) curve is the total unmodulated rate for DAMA. The dashed (black) curve is the exclusions contour of CDMS-Ge, while the dotted (green) corresponds to XENON10, both at 99% CL. The velocity distribution is a standard Maxwellian distribution with escape velocity v esc = 650 km/s. The solid (red) lines delimit the WMAP bounds at 3 σ CL.
Direct detection analysis -IDM and DAMA
The former DAMA/NaI [2] and present DAMA/LIBRA [3] experiments are designed to detect the dark matter recoil off nuclei through the model independent annual modulation signature due to the motion of the Earth around the Sun. The experimental results obtained by DAMA/LIBRA combined with the ones of DAMA/NaI show a modulated signal with a confidence level (CL) of 8.2 σ [3]. The analysis of the DAMA data is based on a goodnessof-fit method and considers the full 36 bins. In addition we take into account the constraint on the total event rate from the unmodulated signal. All the other collaborations for direct searches have reported no positive detection of DM recoils, setting upper bounds on the allowed dark matter scattering cross-section on nucleon. The exclusion limits are calculated with the standard Poisson statistics. A detailed discussion on the analysis procedure and on the astrophysical factors can be found in [4] and references therein. [5]. The H 0 is a light DM candidate and its interaction on nucleon is through spin-independent (SI) elastic scattering exchanging an Higgs boson. In figure 1 we show the allowed regions compatible with the DAMA modulated signal, as a function of the model parameters |λ H 0 |, M H 0 . The strongest exclusion limits come from Xenon10 [6], CDMS-Ge [7] and the total unmodulated DAMA rate. Also shown is the prediction for the relic density; we underline the fact that no further adjustment of the parameters is needed to fix the relic abundance. The compatibility between DAMA and the other exclusion experiments, in this mass range, is ameliorated by the channelling on Iodine [8,9], even though the model appears severely constrained. We remark however that the nuclear and astrophysical uncertainties can play a role in opening the available parameter space, see [4].
High mass regime (M H 0
m W ) -inelastic dark matter The second limit arises from λ 5 → 0, in which case the neutral particles are degenerate [11] and the theory is invariant under a U (1) PQ symmetry. The presence of an enhanced symmetry allows to consider a small mass splitting δ = M The solid (brown) curve is the DAMA total unmodulated rate. The dashed (black) curve is the exclusion contour of CDMS-Ge, while the dot-dashed (blue) corresponds to CRESST-II, both at 99% CL. The velocity distribution is as figure 1, with escape velocity v esc = 600 km/s. The brown region (dark grey bottom) is excluded by LEPI measurements on the Z decay width, the grey region (top) denotes the unitarity limit [10,11].
The red (light grey bottom) region represents subdominant dark matter halo components.
O(10 −7 − 10 −5 )) between the neutral components in a technically natural way. This small mass splitting leads to inelastic dark matter [12], with a SI cross-section on nucleon mediated by the Z boson. The predictions for the model parameters δ and M H 0 are shown in figure 2. The most constraining exclusion limits come from Cresst-II [13] and CDMS-Ge, while the DAMA total unmodulated rate is less restrictive respect to the elastic scenario. There exists a whole range of candidates, between M H 0 ∼ 535 GeV and M H 0 ∼ 20 TeV, which are compatible with DAMA and all other direct detection experiments and which have the relic density within the WMAP [14] bounds. The viable candidates in the mass range 50 − 100 GeV (light red/grey bottom) have a cosmic abundance below the WMAP value. Either they can be considered as subdominant components of the dark matter halo either one can infer an asymmetry mechanism in the early universe, provided by the smallness of the λ 5 coupling, to account for the suitable relic density amount. More details on the inelastic candidate and on the asymmetry are discussed in [4]. | 2022-06-28T04:21:53.276Z | 2010-01-01T00:00:00.000 | {
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213151753 | pes2o/s2orc | v3-fos-license | Household Sector Innovation in China: Impacts of Income and Motivation
This research note reports upon the first survey of household sector innovation in China. Compared to previous survey studies we add two first-of-kind variables and related findings.<br><br>First, we include data on individual income, a resource-related antecedent of household sector innovation. We find that higher individual incomes are strongly associated with increased frequency of both household sector innovation and innovation diffusion. When we combine personal income effects with the positive impact of educational levels and technical training (both competence-related antecedents), it appears that increases in national development are associated with increases in household sector innovation - a very useful public policy finding.<br><br>Second, in this survey we included household sector innovations motivated by personal need and additional motivations related to learning, fun, helping others and selling/commercialization. This has a major impact on estimated household sector innovation frequencies - raising them by a factor of approximately 1.4. Reanalysis of data obtained in two earlier national surveys suggests that similar adjustment factors hold in those nations too. This finding shows that prior surveys have significantly underestimated household innovation. For many research purposes, such as national accounting, the total amount and value of household sector innovation is what is of interest, independent of motivations that may drive the activity.
Introduction and overview
A household sector innovation is defined as a functionally novel product, service or process developed by consumers at private cost (von Hippel, 2017: 1). In contrast to the business or government sectors, the household sector is the consuming population of the economy, in a word all of us, all consumers, "all resident households, with each household comprising one individual or a group of individuals" (OECD, 2013: 44). Household production entails the "production of goods and services by members of a household, for their own consumption, using their own capital and their own unpaid labor" (Ironmonger, 2000: 3). Household sector innovation, therefore, is a form of household production.
At the time of this writing, surveys of household sector innovation have been carried out in nine countries, showing that, in aggregate, tens of millions of individuals in these nations spend tens of billions of dollars annually developing and improving consumer products. In the study of household sector innovation in China we report upon here, we add two new important findings to the learnings from previous surveys and studies.
First, in the China survey we collect data on the income of household sector innovators. Previous studies have investigated competencerelated indicators of consumers' innovation ability, including education level and technical education and work experience (von Hippel, Ogawa and de Jong, 2011). Income, we suggest, adds an important resourcerelated dimension. Our analyses show that income is strongly related to levels of household sector innovation. Individuals at the highest income levels measured in China are 4 to 5 times more likely to innovate. In addition, we find that income is positively related to the likelihood of diffusion of household sector innovations. Specifically, at high incomes the odds of diffusion to peers more than double, while the odds of commercial diffusion are 15 times higher relative to those in the lowest income categories. When we combine the effect of income with previously-documented variables found significantly associated with household sector innovation (education level and technical work experience) a general picture emerges that the frequency and diffusion of household sector innovation is likely to increase along with global trends towards increased education and income.
https://doi.org/10. 1016/j.respol.2020.103931 Second, we remove a key restriction included in prior surveys (de Jong, 2016). All prior surveys only reported on household sector innovations that respondents said were motivated by personal need. In the present survey, we also include innovations induced by additional motives: fun, learning, helping others, and selling/commercialization. When we do this, we find that the result is to raise the documented level of household sector innovation by a factor of 1.4. To increase researcher confidence in the generalizability of this correction factor developed by analysis of China survey data, we also go back and reanalyze data from two previous national surveys and find approximately the same factor present. This increase will clearly be important for many economic and policymaking analyses. For many purposes, analysts do not care why a household sector innovation was developed -only that it was.
In the sections that follow, we first review relevant literature (Section 2). Next, we describe our survey and analytical methods (Section 3). Then, we present our analyses and findings (Section 4), and conclude with a discussion (Section 5).
Findings from prior household sector innovation surveys
At the time of this writing, nine national surveys have explored household sector innovation by individuals motivated to personally use what they develop. The surveys were carried out in the United Kingdom, the United States, Japan, Finland, Canada, South-Korea, Sweden, Russia and the United Arab Emirates (UAE) -see Table 1. All surveys utilized broad samples of individual end consumers. Estimates of population numbers were then generated by applying weighting procedures. Data were collected by means of a questionnaire administered by telephone interviewers in various countries (e.g., United Kingdom, Canada) and by means of internet surveys in other countries (e.g., United States, Japan, Finland). In one nation only (Russia) data was collected by face-to-face interviews. Questions asked in all studies included those asked in the initial survey of household sector innovation conducted in the UK. Later studies added some important additional questions such as those having to do with the motivation of household sector innovators. A basic survey script applied in the most recent studies can be found in de Jong (2016), and in von Hippel (2017).
Prior surveys demonstrate that household sector innovation is a major phenomenon. In aggregate across nations measured to date, tens of millions of consumers were found to spend billions of dollars per year developing and improving products. The percentage of populations innovating in the household sector differs quite significantly among nations surveyed. As can be seen in Table 1, the range was from 1.5% of the population in South Korea to 9.6% of the population in Russia.
To date, the significant variations in the percentage of consumers innovating among nations is not well understood. National surveys have only identified three major variables significantly associated with increased levels of household sector product innovation across all studies. These are higher levels of education, the presence of technical training or technical work experience, and male gender. By including the additional major variable of income in our data collection in China, we add a new dimension to the list of antecedents studied so far.
Income: Resources for household innovation
We hypothesize that income is positively related with individual household innovation. A range of studies about human behavior have shown that engagement in (any) volitional behavior partly depends on individual ability. For example, the theory of planned behavior offers perceived behavioral control as a key antecedent of individuals' decision to engage in volitional behaviors at work and in leisure (Ajzen, 1991). In the context of our study, theories of individual innovation have stressed ability-related antecedents as well. Examples include proactive motivation theory which distinguishes individuals' control beliefs in order to engage in proactive behaviors (Parker et al., 2010), and the compenential theory of creativity which includes creativy-related skills as a determinant of creative output (Amabile, 1983).
The survey studies of household sector innovation done so far did consider ability-related antecedents, but only related to individuals' personal competences to innovate. Thus, education level, technical training and technical work experience indicate personal innovation competence, or human capital relevant to household innovation. If a consumer encounters an unsatisfied personal need, seeks to learn, help others, make money, or simply enjoys the process of innovation, innovative behavior is more likely with better education, technical training and/or technical work experience.
We argue that income represents a different dimension of individual innovation ability. Controlling for education and technical training, income represents consumers' access to resources other than competence/human capital. In essence income reflects access to physical capital, that is, at higher income levels consumers are more likely to have access to technical resources (e.g., a workshop, development tools, software) and financial resources (to pay for out-of-pocket innovation expenses like materials, memberships, assistance). Moreover, high income indicates better opportunities to expand personal knowledge and abilities, e.g., by taking training. In this vein, studies of inventors (Meyer, 2005;Bell et al., 2017) have shown the importance of income for innovative behavior.
A second argument is that individuals with high incomes are usually more prosperous, and prosperity is known to make individuals pursue different life goals. Maslow's (1943) hierarchy of needs implies that at low prosperity individuals are concerned with subsistence, safety and belonging. In case such needs are met, which usually happens at higher prosperity levels, other needs become important: for instance esteem and self-actualization. Our reasoning is that at higher income levels consumers attach more value to life goals which can be accomplished by developing novel objects which do not yet exist. A potential counterargument is that consumers at low incomes are deprived of resources to secure subsistence and safety. Such needs are likely to be quite acute. In support of this argument studies have provided existence proofs of the poor engaging in innovation (e.g., Praceus and Herstatt, 2017;Gupta, 2013;Goeldner et al., 2017). On the other hand, we notice that none of these studies involved broad consumer samples, and did not analyze innovations develop by more prosperous individuals. We expect that, being short on subsistence and safety, individuals with very low incomes are not necessarily triggered to develop innovative objects with functional novelty. Rather, at low incomes 'do it yourself' production (of already existing objects) may be more likely. Also, we observe that many, such as Prahalad (2004;2012), have argued that the very poor want products different than those sought by those with more income -and that serving the "bottom of the pyramid" with commercial products uniquely suited to their needs was a market opportunity that producers had been neglecting. However, the argument made by these scholars was that producers should develop products suitable to the bottom of the pyramid -not that the innovations are developed by the poor.
We also anticipate that income will be positively related with the diffusion of household sector innovations. Again, income represent access to resources helpful for diffusion, and lowers the threshold to engage in diffusion behaviors (e.g., income enables individuals to spend money on diffusion, for example by sharing information about their innovation on the web) (Bell et al., 2012). Also, higher-order needs related to esteem and self-actualization, which are more prominent at higher incomes, make it more important to individuals to have their innovations become visible and adopted by others.
Altogether income adds a resource-based antecedent on top the competence-related antecedents studied so far. We expect that income will be positively related with household sector innovation and diffusion, also if education and technical work experience are controlled for.
Motives associated with household sector innovation
Until recently, national surveys of household sector innovation only considered innovations that consumers had developed for personal need. This was an unintended consequence -not noticed at the time -of terming non-producer innovators "users" (e.g., von Hippel, 2005). In the past few years, however, it has become evident that household sector innovators often are motivated by a range of incentives in addition to personal use (Raasch and von Hippel, 2013). These additional motives include: fun/enjoyment of innovation development work (Hienerth, 2006); learning and skill improvement (Bin, 2013;Hienerth, 2006;Lakhani and Wolf, 2005); helping others (Kogut and Metiu, 2001;Lakhani and von Hippel, 2003); and making money by selling the innovation (Meyer, 2005). In a survey of household sector innovators in Finland, a secondary data analysis (reported in von Hippel, 2017) of 408 household sector innovators incorporated these motivations. The analysis identified four major motivational clusters. "Users" (37% of the sample) expected their largest fraction of benefit to come from personal use of the innovation they had developed. "Participators" (43%) expected the largest fraction of their innovation-related benefits to come from self-rewards related to enjoyment plus learning from participating in the innovation process itself. "Helpers" (11%) were those whose strongest motivation asked about was to innovate in order to help others-altruism. "Producers" (9 percent of the sample) were most strongly motivated by the prospect of sales.
In this research note we will analyze the impact on household innovation frequencies of including this broader range of motives, rather than focusing on innovations motivated by personal need only. As was noted earlier, for many purposes, researchers and analysts do not care why a household sector innovation was developed -only that it was.
Sample and survey methods
We surveyed 5000 consumers in China aged 18 and over. Variation in income and education levels in China is substantial, making the country a good context for our research on the impact of these variables on levels of household sector innovation. In some geographical regions (e.g., Beijing, Shanghai) levels of income and education match those of the most prosperous regions across the globe. In other areas of China many have very low levels of income and education, similar to those encountered in much less developed parts of the world.
To identify the sampling frame for our survey, and also to carry out data collection, we collaborated with Dataway, a marketing research company based in Beijing. Because an exhaustive sampling frame with details of all Chinese citizens was not available, the initial sample was obtained with a random number generator covering both cell phones and landlines. To minimize the probability that we would get in touch with businesses, generated phone numbers were first filtered with a public database of all companies and public organizations. Also, data collection was done in the evenings to diminish the probability of contacting businesses, and the introduction to the survey explicitly mentioned our interest in consumer behavior, not business-related innovation. Our research approach insures that each citizen has an equal chance of being sampled (Malhotra and Birks, 2007). (Phone ownership in China is nowadays close to 100%. Cell phone ownership is 95%, and together with landlines coverage is close to complete (Pew Research Center, 2014).
Expert Dataway interviewers were selected and trained by our team, and only these few individuals were allowed to work on data collection for our study. Over a period of three months 37,403 Chinese citizens were sampled by these Dataway interviewers. For numerous reasons 11,120 citizens were impossible to contact (e.g., answering machine, no reply, unobtainable after five attempts). Another 21,283 refused to participate in the survey after being successfully contacted. At the conclusion of the data collection phase, responses were obtained from 5000 citizens, age 18 and older. The overall response rate was 19.0% of the individuals actually contacted.
With respect to demographic characteristics, 61% of the survey respondents were male. For education, 20% percent had a degree (bachelor, master or PhD), 33% had completed a secondary or college vocational school, and 47% had completed only high school or less.
As in previous survey studies of household sector innovation, we corrected for potential response bias by applying weights -to give the best possible population estimates. Compared to population characteristics taken from the China Population and Employment Statistical Yearbook (NBS, 2016) there was overrepresentation of males (population share 51%), highly educated (population shares: 9% has a bachelor/master/PhD degree, and 15% a secondary/college vocational degree), and younger citizens (population share of citizens <45 years is 52%). In contrast the poorest consumers were underrepresented. To obtain population estimates we computed weights for all respondents. Dataway provided us with a table which broke down the population of Chinese citizens aged 18 and over, across various combinations of gender, education and age classes. A similar table was obtained from our data, and weights were specified to be the ratio of the percentages in corresponding table entries.
Identification of household sector innovators
To identify innovations we applied the procedure summarized by de Jong (2016). We first identified if people had created or modified any items in the past three years. At the start of our survey we stated: "My next questions are about what you do in your free time. I would like to offer you some everyday items that you might have created or modified in your free time." In line with previous surveys respondents' recall was assisted by offering a list of nine specific cues: Had they created any (1) computer software; (2) household items; (3) vehicle-related; (4) tools or equipment; (5) sports, hobby or entertainment; (6) child or educationrelated; (7) health, care or medical; (8) fashion or clothing-related; or (9) any other items. Out of 5000 respondents, 803 reported developing or modifying at least one product in the past three years.
We then applied screening questions, to see if reported cases were household sector innovations. Specifically, to be included in the sample as a household sector innovation: (1) the product developed or modified must have been developed during leisure time; (2) must have been developed up to the level of a prototype actually used or applied in everyday life, and not simply be a not-yet-implemented idea; (3) must embody some functional novelty not available from items available on the market. We also asked respondents open-ended questions to describe what they had developed, and why. This enabled us to "double check" claims of functional novelty, enabled us to exclude esthetic or design-related innovations, and also enabled us to exclude homebuilt versions of products that could be purchased on the market. (We allowed coders to apply their own knowledge of what already exists in the market to, in clear cases only, exclude a development as non-innovative.) Each description was independently coded by two members of the research team. Cohen's Kappa was 0.91 indicating almost perfect agreement (Landis and Koch, 1977). In case of deviant codes, the descriptions were discussed to reach full agreement.
Out of 5000 respondents, 803 initially reported to have developed at least one product or product modification in the past three years. Our cross-check to identify and exclude job-related innovations reduced this number by 166, leaving us with 637 individuals who had created or modified at least one product in their free time during the past three years. Next, after our check on functional novelty we were left with 185 individuals who fulfilled all of our criteria for household sector innovators.
Previous surveys (e.g., von Hippel et al., 2012) applied an extra screening question, asking respondents if they had developed the innovation for personal need, to identify user innovators. In this survey we were interested in household sector innovators independent of motivation, and so did not screen out innovators with other motivations. Instead, we coded the motives driving respondents to develop the innovation (see later) to learn about the relative frequencies of household sector innovation independent of motive for development, and innovations developed specifically for personal use.
Variables collected via survey
Having established if a respondent was a household sector innovator, we then asked questions identical to those used previous surveys, in order to achieve comparability across all national surveys of household innovation. In the case of innovators who reported developing multiple innovations, we asked them to answer with respect to their most recently finished development only (see Table 2). In line with previous studies we asked for gender (dummy for males), education level (ordinal variable with six categories), being technically educated, and having work experience in a technical job.
To explore the relationship between innovation likelihood and personal income, we included an ordinal variable with nine categories ranging from an annual income of less than 10,000 Chinese Yuan, (about $1600 US at time of writing), to 300,000 Yuan, (about $47,000 US at time of writing) or more. (The latter is comparable to median incomes levels in the US and many European nations.) We also added as a control variable whether the respondent lived in a rural area or village, a town, or a city/urban area. We did so because income may also indicate that individuals have better access to social capital for innovation. For example, in China currently, high-income people are more likely to live in densely populated areas and have access to supportive innovation infrastructure such as makerspaces. To ensure that income only reflects the theorized resource-related dimension and not social connectedness, we controlled for urbanity in our analysis.
We also asked questions regarding diffusion, including both commercial and peer-to-peer diffusion as alternative pathways (von Hippel, 2017), and respondents' time investment and if they had collaborated in order to innovate. Time investment and collaboration have been shown to influence diffusion (e.g., (Ogawa and Pongtanalert, 2013) and we wanted to control for these to better access the role of income. Finally, we asked for the innovator's most important motive for innovating (out of the five motives reported in Table 2). This question enabled us to analyze household sector innovation more broadly that previous surveys which, as noted earlier, had analyzed innovations motivated by personal use only.
Findings
In this section, we first present descriptive statistics with regard to the frequency of innovation, and the relationship of frequency to demographic variables including income. Next we analyze determinants of innovation and diffusion more deeply with regression models. Table 3 gives the percentages of innovators we observed in China across demographic variables. Notice that a weighting scheme has been applied to provide population estimates. As can be seen, and in line with previous studies, household sector innovators are more likely to be male, well-educated, technically trained, and have technical work experience. (We also measured age as a variable, but do not include it in Table 3 for space-saving reasons. No significant differences between age categories were found.)
Frequency of innovation and demographic variables
In a finding novel to the China survey, we can clearly see a strong relationship between income and household sector innovation. In the top categories (200,000-300,000 Yuan and >300,000 Yuan annual income) the observed frequency of innovation is around seven times the frequency in the lowest categories (<10,000 Yuan and 10,000-30,000 Yuan). Table 3 also shows that citizens living in urban areas are more likely to innovate than those in rural areas. However, this finding vanishes when income is controlled for -respondents living in rural areas in China tended to have much less income than those living in cities (see Section 4.2).
Determinants of innovation
Next, we estimated probit regression models to explore the relationship between income and household innovation (and also diffusion) while controlling for competence-related and other independent variables. Table A1 (see appendix) offers descriptive statistics and correlations, showing that multicollinearity is not a concern. Regression output shown in Table 4.
Model I shows that innovation frequency is significantly related to educational level and (at marginal significance) with male gender and technical work experience. The model also confirms that income is significantly related to innovation, even when gender, age, technical education, technical job experience and education are controlled for. This is in line with our supposition that income reflects a resource-related dimension of innovation ability, and differs from the competencerelated indicators studied previously.
To better interpret our findings Table 5 shows the predicted frequencies of innovation at various levels of income and education, the two most significant variables in our regression models. At the lowest level of education the predicted innovation frequency is only 0.7% when other variables are controlled for. For those with a master degree (the best educational attainment) it is 7.2%. Likewise, for the lowest income category (<10,000 Yuan) the predicted frequency is 1.4%. In the highest income category (300,001 or more Yuan) it is 5.9%.
In the bottom portion of Table 5, we report the predicted frequency of household innovation at a number of combinations of income and education. For example the predicted frequency for those without education and the lowest income is 0.4%, while for those with a master degree and the highest income it is 15.9%.
Determinants of diffusion
We next tested whether income also increases the likelihood of diffusion directly to peers, and diffusion via commercial pathways (i.e. transfer of the innovation to an existing producer, or commercialization via a startup venture). In model II of Table 4 we see that innovation collaboration and male gender are associated with peer-to-peer diffusion. In addition, income is positively related to diffusion to peers. We followed up estimating predicted frequencies of innovation at various income levels. Overall the predicted frequency of peer-to-peer diffusion is 33.2%. This implies that around one of three household sector innovations is adopted by peers. At the lowest income level (<10,000 Yuan) the frequency was 19.8%, while at the highest level (300,001 or more Yuan) it was 53.0%.
In model III of Table 4 we see that technical education and male gender are related to commercial diffusion. In addition, income is (again) significant. Following up with predicted frequencies, at the lowest incomes (<10,000 Yuan), only 0.6% of all innovations are predicted to diffuse commercially. At the highest level (300,001 or more Yuan) it was 23.2%.
These observations are in line with our presupposition that high income provides individuals with resources, which reasonably lowers their threshold to engage in diffusion. Specifically, income provides individuals with better access to diffusion resources (e.g., internet access, memberships) and probably also proxies the pursuit of higherorder life goals (e.g., self-actualization) which make diffusion more important to them personally.
Income-related robustness checks
We estimated various other regression models to check the robustness of our findings regarding income. These are available on request. First, our findings were maintained if we did not apply weights to our dataset.
Second, our descriptive statistics in Table 3 suggest that the relationship between income and innovation may be non-linear. We estimated probit regression models in which we included dummy variables for each income category, rather than income as an ordinal variable. For those with incomes of 150,001-200,000 Yuan household innovation frequency was not significantly higher than the baseline category of <10,000 Yuan. This is probably be due to chance: drawing a random sample from any population does not lead to identical estimates, and occasionally, estimates based on a subsample may be lower Number of people who provided help, assistance or advice to develop the innovation Peer diffusion Innovation has been adopted by peers (0=no, 1=yes) Commercial diffusion Innovation has been adopted by commercial firms and/or diffused in a venture (0=no, 1=yes) Key motive Respondent's motive to innovate was related to (multiple answers possible) (1) personal need, (2) to sell or make money, (3) to learn or develop skills, (4) to help other people, (5) fun/enjoyment (or higher) than one would usually expect. Apart from this issue, for models I and II our findings were confirmed: at higher innovation categories innovation and peer diffusion were significantly higher compared to the baseline category. (Model III did not converge, most likely as a result of entering too many independent variables in a model with few positive outcomes. Recall that only 5.2% of validated innovations diffused commercially.) Third, we checked the robustness of models II and III by estimating sequential logit regression models (we thank an anonymous reviewer for this suggestion). Sequential logistic regression analysis estimates the effect of a set of independent variables on the odds of passing a specified number of transitions (Buis, 2011). We applied this model to predict the effect of male gender, age, urban (vs rural), technical work experience, technical education, education attainment, and income, on the 'transition' to household innovation (step 1) and subsequent diffusion to peers (step 2). When estimated simultaneously the significant variables related with innovation and peer diffusion were the same as those reported in Table 4. Next, we estimated a sequential logit model with commercial diffusion in the second step. Again, our findings were maintained, including that income is related with commercial diffusion.
Fourth, we recognized that income may be endogenous to household innovation and diffusion. Thus, we estimated an instrumental variables (IV) probit regression of household innovation. We used as instruments: region and mode of transport. Respondents in our dataset were from China's 31 regions (e.g., Shanghai, Beijing, Tianjing, Tibet, Inner-Mongolia). They had also indicated their main mode of transport: foot, bicycle, motorbike/electric cart, bus, subway/rail, car, taxi, and other (includes plane, boat, etc.). The IV probit model confirmed our findings with regard to the significance of technical work experience, education and income. However, the Wald test on the exogeneity of instrumented variables was marginally significant at p < .10, suggesting that our instruments were not sufficient. Therefore we explored the viability of our instruments more deeply by estimating a 2SLS regression model in which we (again) instrumented income by region and transport mode. On the positive side, the F-value of the instrumental variables was >10, while partial R 2 (of income on region and transport mode) was 0.1797, showing that the instruments were sufficiently related with income. However, Wooldridge's robust test was significant at p < .01, suggesting that the instruments are related with the structural error term. We therefore cannot exclude that endogeneity is present in our data. In our discussion section we call for longitudinal and multiplesource data to address these concerns in future research.
Frequency of innovation as a function of innovator motivations
Recall from Section 2.3 that, for all previous national surveys of household sector innovation except Finland and UAE, data collection was restricted to innovators motivated by personal need. Recall also that our present survey, conducted in China, had collected data on several innovation motives in addition to personal need. Our validated innovators reported the following (multiple answers possible) as important motivations driving their innovation development work: Notes: Data were weighted to gender, age and education. Average adjusted predictions are shown, based on the output of model I in Table 4. All predicted frequencies differ significantly from zero at p < .01.
As the basis for the second major finding in this study, we next analyzed the effects on innovation frequency from adding the aforementioned types of motivation to personal need. As can be seen in Table 6, the consequence of including innovations motivated by four additional sources of motivation is that the estimated percentage of household sector innovators documented in China increases by a factor of 1.4 ( = 2.1/1.5). Personal use-motivated innovation in China is 1.5% of the population, or 16.5 million household sector individuals innovating in the previous three years. Personal use-motivated innovation plus innovations most importantly motivated by the additional motivation types listed above is 2.1% of the population, or 23.2 million individuals. This is, of course, a very substantial difference.
In an initial effort to determine whether this Chinese "correction factor" finding might be generally applicable in future studies of household sector innovation, we went back to the two earlier national household innovation surveys that had collected data on the same additional motivations, Finland and UAE. Reanalysis of data collected in those two studies show a very similar increase in innovations documented. That is, when one adds innovations motivated by these sources of motivation those motivated by user need, the percentage increase found is of significant size, and also very close to that found in China ( Table 6).
Note that the levels of household sector innovation in China are clearly smaller than those measured in most other nations to date (c.f. Table 1). However, this may be largely a function of lower incomes and education levels in present-day China. In general across the world, citizens' education and income are improving over time. In practice these variables are related: better-educated individuals usually have higher incomes and vice versa. Assuming that economic growth in China continues in future decades, we expect that, along with increasing education and income, higher levels of household sector innovation will eventuate.
Discussion
As explained in our introduction, the first-of-type findings derived from this China survey study of household sector innovation are two: (1) the relationship between income levels and the likelihood that an individual householder will innovate and diffuse; and (2) a factor 1.4 increase in household sector innovation frequencies found when innovators' motives in addition to personal use value are allowed.
With respect to the impacts of income we found that, in China, when personal incomes are higher, household sector innovation frequencies and diffusion likelihoods are also higher. (This connection to income will likely be generally found: it has also been observed in studies of individuals' likelihood of inventing (Bell et al., 2017)). As we saw in Table 4, for Chinese citizens with annual incomes of between 200,000 and 300,000 Yuan, the household sector innovation rate was 6.3%. In 2017 the average incomes in the UK and Finland were in this range (25,500 Euros and 38,400 Euros respectively (www.tradingeconomics. com)) as were their household sector innovation rates of 6.1% and 5.6% respectively (von Hippel et al., 2012;de Jong et al., 2015). We therefore anticipate that the average percentage of household sector innovators in China will rise over time, as China continues its rapid development, and citizens' average incomes continue to rise.
With income our study adds a resource-related dimension of innovation ability, which differs empirically from competence-related ability measured by education level, technical training and technical work experience. Of course, in practice income and education are correlated, and collectively reflect consumers' general level of development. At the extremes of the development distribution, we found very strong differences between individuals at low income and no education, versus higher income and high education. It implies that in general, policies to advance a nation's level of development will go together with enhanced levels of household sector innovation and diffusion. Our findings also suggest that policy makers may want to put different priority to innovation by consumers at high versus low levels of development. At low levels of development consumers face more limitations in terms of innovation competences and physical resources, and would benefit more from policy interventions such as innovation tools and makerspaces. For China, today's education levels are still significantly lower compared to the US and Europe (OECD, 2017). However, major efforts are being made in China to increase educational levels over time (Jacob et al., 2018). Success at this will likely further improve future levels of household sector innovation and diffusion in the country.
With respect to motivations of household sector innovators, we suggest that it is reasonable that future national surveys also include, as we did here, innovations motivated by factors in addition to innovators' benefits from personal use. Consider that, for purposes of measuring the economic effects of household sector innovation, the specific motivations of innovators are a secondary issue. What counts is the number and value of innovations developed independent of motive. It is interesting to note that our reanalysis of data collected in household innovation surveys conducted in Finland and Emirates to include motives in addition to personal use value also produced adjustment factors in the range of 1.4. Therefore, researchers intending to use data from national surveys already done might reasonably consider using this ratio as an adjustment factor to findings as published to approximate household sector innovation frequencies.
Study limitations
It is important to note that surveys of household sector (HHS) innovation to date, including this one, have been conducted by academics who do not have "insider" access to exhaustive sampling frames (de Jong, 2016). It would clearly be preferable to use a sampling frame in which demographic data are known at the level of any potential respondent, so that potential response bias can be controlled for more thoroughly. The optimal scenario would be that a formal statistical office includes household innovation questions in an official consumer survey. In our study, we sampled respondents by means of a random telephone number generator. Given that the phone penetration rate for Chinese adults is well over 95% this is a viable method. Still, in company with all other national household innovation surveys conducted to date, it means that we could only control for response bias by weighting our data.
Second, although we do not believe that endogeneity has seriously affected our findings, the possibility certainly merits awareness. With regard to potential reverse causality, it is unlikely that household innovation and diffusion will have enhanced the incomes we observed. (2015);. 3 Data source: von Hippel et al. (2017).
Only 5% of the innovators had commercial motives, and also only 5% of the validated innovations had diffused commercially, so it is unlikely that innovation and diffusion 'caused' reported incomes. Also, with regard to common method bias our survey carefully followed Podsakoff et al. (2003) recommendations to minimize this potential problem, including upfront guarantees of anonymity, and carefully developed and tested questions. We avoided common answer formats and anchors, and our questions were mostly about 'facts' about very specific cues (e.g., 'Computer software by programming original code. In the past three years, did you ever create or modify this in your free time?'). Our survey did not include the multiple-item measures used in psychological research which are more sensitive to common method bias. Finally, absolute values of the correlation coefficients between variables (see Table A1) were mostly minor (r < 0.10) and less than the correlations expected in the presence of common method bias when bivariate relationships are lacking (Podsakoff et al., 2003). Nevertheless, it would clearly be valuable for future research to investigate household innovation from a longitudinal perspective to deeply explore causal relationships. Alternatively, endogeneity threats can be minimized by collecting data from multiple sources.
Conclusion
Evidence for the importance of the phenomenon of household sector innovation is now strong. Ten national surveys, including this one, have all documented its importance. We are pleased we can add two novel findings to the growing research in this field via the study presented here. We very much look forward to further explorations and developments by fellow researchers. | 2020-02-27T09:13:29.115Z | 2020-05-01T00:00:00.000 | {
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28806935 | pes2o/s2orc | v3-fos-license | fastBMA: scalable network inference and transitive reduction
Abstract Inferring genetic networks from genome-wide expression data is extremely demanding computationally. We have developed fastBMA, a distributed, parallel, and scalable implementation of Bayesian model averaging (BMA) for this purpose. fastBMA also includes a computationally efficient module for eliminating redundant indirect edges in the network by mapping the transitive reduction to an easily solved shortest-path problem. We evaluated the performance of fastBMA on synthetic data and experimental genome-wide time series yeast and human datasets. When using a single CPU core, fastBMA is up to 100 times faster than the next fastest method, LASSO, with increased accuracy. It is a memory-efficient, parallel, and distributed application that scales to human genome-wide expression data. A 10 000-gene regulation network can be obtained in a matter of hours using a 32-core cloud cluster (2 nodes of 16 cores). fastBMA is a significant improvement over its predecessor ScanBMA. It is more accurate and orders of magnitude faster than other fast network inference methods such as the 1 based on LASSO. The improved scalability allows it to calculate networks from genome scale data in a reasonable time frame. The transitive reduction method can improve accuracy in denser networks. fastBMA is available as code (M.I.T. license) from GitHub (https://github.com/lhhunghimself/fastBMA), as part of the updated networkBMA Bioconductor package (https://www.bioconductor.org/packages/release/bioc/html/networkBMA.html) and as ready-to-deploy Docker images (https://hub.docker.com/r/biodepot/fastbma/).
Our Contributions
We have previously described ScanBMA [14], an implementation of Bayesian model averaging (BMA) [20] for inferring regulatory networks. ScanBMA is available from the "networkBMA" Bioconductor package [21], written in R and C++. It has been shown that ScanBMA generates compact accurate networks that can incorporate prior knowledge.
In this paper, we present fastBMA, which is completely written in C++, and uses more efficient and scalable regression and hashing methods. The algorithmic improvements increase the speed by a factor of 30 on smaller sets, with greater increases observed on larger sets due to improved scalability.
fastBMA is parallelized using both OpenMP and MPI allowing for further increases in speed when using multiple cores and processors. Although fastBMA uses the same core methodology as ScanBMA, the increased scalability allows for more thorough sampling of the search space to increase accuracy. The new probabilistic hashing procedure used by fastBMA is faster and utilizes 100,000 times less memory when analyzing large numbers of variables. This allows fastBMA to operate on genome scale datasets without limiting the possible regulators of a given gene to a smaller subset.
A new feature of fastBMA is the implementation of a novel method for eliminating redundant indirect edges in the network. The post-processing method can also be used separately to eliminate redundant edges from networks inferred by other methods. The code is open-source (M.I.T. license). fastBMA is available from GitHub (https://github.com/lhhunghimself/fastBMA), in R as part of the networkBMA package ((https://www.bioconductor.org/packages/release/bioc/html/networkBMA.html)) and as Docker images (https://hub.docker.com/r/biodepot/fastbma/). The Docker containers include all the supporting dependencies necessary for MPI and make it much easier to run fastBMA on a local or cloud cluster.
Bayesian model averaging (BMA)
We can formulate gene network inference as a variable selection problem where the dependent variable (target gene expression) is modeled as a function of a set of predictor variables (regulatory gene expression Time series data can also be modeled by using the expression at the previous time point to predict the next time point. (2) Different candidate models can be constructed from different sets of regulator genes. Models can be evaluated based upon a measure of their goodness of fit, such as the sum of residuals. However, in ge-netic analyses, the number of genes often exceeds the number of samples, and many different models can fit the data reasonably well. The core idea behind the BMA methods is to find the posterior probability for each model and make a consensus prediction giving proportionately more weight to the higher confidence models. In terms of gene regulation, the posterior probability that gene j is a regulator of gene i is the sum of the posterior probabilities of all candidate models that include gene j in the set of regulators of i. This posterior probability becomes the weight of the edge drawn from gene j to gene i in the gene network.
Estimating model posterior probabilities
Estimation of the posterior probabilities of the models can be accomplished by a variety of methods, some of which are very computationally intensive [12]. The original BMA [20] and iterative BMA (iBMA) methods [22] use the Bayesian Information Criterion (BIC) [23] which is simple to calculate and penalizes larger models which are easier to fit. However, BIC is an asymptotic approximation that is most accurate for large sample sizes. As an alternative, ScanBMA provided the option of using Zellner's g prior [24] to compute the posterior probabilities. The optimal g prior parameter is obtained by finding the value that maximizes the total posterior probability of the models. Adjusting the range of possible values for the g prior allows us to tune the method for smaller sample sizes and produce better networks.
fastBMA exclusively uses the g prior to estimate the posterior probabilities and uses faster C++ code for the expectation maximization (EM) optimization of the g parameter.
Sampling candidate models
The number of possible candidate models grows exponentially with the number of possible regulators, necessitating an efficient methodology to find a subset of reasonable models. In the original implementation of BMA, the leaps and bounds algorithm [25] is used to identify the n best models for a given number of variables. Occam's window [26] is then used to discard models with much lower posterior proba-bilities than the best model. The leaps and bounds algorithm scales poorly and is limited in practice to fewer than 50 variables. Iterative BMA (iBMA) uses a pre-processing step to rank all variables (genes), iteratively applies the original BMA to the top w variables (w=30 by default), and discards predictor variables with low posterior inclusion probabilities [13]. In the iterative step, new variables from the ranked list are added to replace the discarded variables. This procedure of repeatedly applying BMA and variable swaps is continued until the w top ranked variables have been processed. In contrast to iBMA, ScanBMA removes the restriction of the search space to an initial list of variables [14]. ScanBMA keeps a list of the best current linear regression models found so far and adds or removes a variable from these models to search for better models. The process is repeated until no new models are added or removed from the best set of models. ScanBMA's greedy approach and the implementation of its core routines in C++ enable it to typically run faster than iBMA. In this paper, we present fastBMA that uses the ScanBMA approach but exploits the fact that new models are based upon existing models. In particular, new models are fitted using the results from the existing models which increases the speed and scalability of the search.
Post-processing graphs by transitive reduction
BMA and other methods for reconstructing biological networks can generate edges between genes that are the result of indirect regulation through one or more intermediate genes. While having edges that represent either direct or indirect interactions is a perfectly acceptable graph, biological networks are usually represented by edges that represent direct interactions. Such networks allow for more straightforward identification of potential driver genes. For genetic networks, it is therefore desirable to remove edges between nodes where the regulation is indirect (transitive reduction). This can be done through post-processing of the inferred network. One intuitive approach is based on eliminating direct edges between two nodes when there is a better indirect path [27]. For example, Bosnacki recently proposed comparing p-values of the best edge in an indirect path with that of the direct path [28]. fastBMA introduces a similar approach that reduces transitive reduction to a shortest path problem which can be solved more efficiently for the sparse graphs typically found in gene regulatory networks . Table 1 summarizes the key differences between the different BMA implementation. Figure 1 shows an outline of fastBMA. In this section, we report our algorithmic and implementation contributions in fastBMA and our evaluation procedure. Pseudocode for the entire implementation is provided in supplemental materials.
Algorithmic outline of fastBMA
The core approach for fastBMA is similar to that used by ScanBMA. The best models are found using ScanBMA's search strategy with a starting value of g in the interval [1… NumberOfSamples]. Brent minimization [29] is then used to find the value g in the interval that gives rise to the set of models with the highest marginal probabilities. A graph is constructed by drawing edges between genes with an edge weight equal to the average posterior probability of the regulator over the set of reasonable models.
Transitive reduction is applied to this graph to remove edges that can be adequately explained by a better indirect path. A final graph is constructed by retaining edges with weights greater than a given cutoff.
There are 4 major algorithmic improvements that increase the speed, scalability and accuracy of fastBMA: 1. Parallel and distributed implementation 2.
Faster regression by updating previous solutions 3. Probabilistic hashing
Parallel and distributed implementation
Parallelization can be accomplished by using a shared memory system, such as OpenMP (http://openmp.org/wp/), which is designed for assigning work to different threads in a single CPU with multiple cores. In contrast, MPI (Message Passing Interface) (https://www.mpich.org/ ) launches multiple processes on one or more CPUs and passes messages between processes to coordinate the distribu-tion of work. Both of these approaches have their respective advantages and disadvantages. OpenMP is applicable only to CPU's on a single machine and is a bit slower for fastBMA. MPI is usable on a single machine or a cluster but requires some work to set up. fastBMA implements both approaches allowing the user to choose the preferred methodology based on their requirements.
Inferring the entire regulatory network involves finding the regulators for every gene in the set. Since each of these determinations is carried out separately, each thread or process can be assigned the task of finding the regulator for a subset of genes in the set. When OpenMP is used, it provides a scheduler that dynamically assigns the regression calculations for a given gene to each thread. Threads work simultaneously on their tasks and receive a new task when they finish the previous task. All threads share access to memory and the same input data for the regression is available to all the threads. The parallel code only extends to the regression loop -the final transitive reduction post-processing and output is done by a single thread.
When MPI is used, we initially split the tasks evenly among the available CPUs. In the case of MPI processes, memory is not shared. Instead the input data is read by a master process and distributed to all the participating processes using MPI's broadcast command. All processes then work on their tasks simultaneously in parallel and send messages to all the other processes so that all processes know which tasks are being worked upon. The length of time required for each calculation varies considerably and as a result, some processes will finish before others. A process that finishes early then works on tasks initially assigned to other processes that have not yet been started. When all the regulators for all the genes have been found, a master process gathers the predictions, performs transitive reduction post-processing and outputs the final complete network. OpenMP can also be used in conjunction with MPI to further subdivide the tasks among threads available to a CPU.
Faster regression by updating previous solutions
Even with the above parallel implementation, each individual calculation of regulators is still accomplished by a single process. If the regression procedure is too slow, this step can be rate- It is vital that the ScanBMA algorithm does not sample a model more than once to ensure that the method will converge and terminate. However, the methodology is quite tolerant of falsely excluding models that have not been sampled. ScanBMA only explores a small sample of the possible models -the vast majority of models are normally excluded. Furthermore, in the BMA approach, many models are averaged to obtain the final edges. Variables that are important appear in many models. In the rare case where a good model is falsely excluded, the impact is minimized because the key regulators in the falsely excluded model will be found in other models. When such false negatives are tolerated, an alternative to using a hash table is to ignore the collisions. This saves both time and space by removing the dependence on m for both time and space complexity. An example of a noisy or probabilistic hashing approach is the Bloom filter [30], which has been used for bioinformatics applications [31] due to fast computation and low memory requirements.
fastBMA includes an optimized implementation of a probabilistic hash (see Figure 2) selective criterion of comparing best edge in the path. The search is also bounded: once a path's distance exceeds the direct distance, there is no need to further explore that path. In addition, fastBMA produces graphs with few high weight edges and in practice, the algorithm is much faster than the worst case as most searches are quickly terminated.
Datasets used for testing
We have previously benchmarked ScanBMA [14] against other network inference methods (MRNET [5], CLR [32] , ARACNE [4], DBN [8], and LASSO [11,33]) on smaller test sets. In this study we compare fastBMA only to ScanBMA and LASSO which were the two most accurate methods in these benchmarks and are the only two methods that could infer networks from the larger datasets in a reasonable time.
We used the following 3 datasets for testing.
1. Simulated 10-gene and 100-gene time-series data (5 sets of each) and the corresponding reference networks from DREAM4 [34][35][36][37][38]. As these datasets are simulated, the true regulatory relationships are known and are used to evaluate the accuracy of the predicted networks.
2. Yeast time-series expression data (ArrayExpress E-MTAB-412) consisting of 3556 genes over 6 time points [39]. Being actual data, there is no absolute ground truth. Instead, we compared the regulatory predictions with the literature-curated regulatory relationships from the YEASTRACT database [40].
3. Human single-cell time-series RNA-Seq data GSE52529 (9776 genes) from GEO [41]. As no satisfactory gold standard was available, we only used this to demonstrate that fastBMA could scale to noisy human genome-wide expression data.
Assessment metrics
We define a true positive (TP) as an edge in the inferred network that is also present in the ground truth We primarily use AUPR and AUROC for the assessment as these metrics measure the overall performance of the methods. In practice, however, predicting some edges accurately, even if only for the most confident predictions is still valuable for narrowing down a set of potential interactions to be further explored. Hence, we also plot the precision-recall graph to assess where the differences in accuracy are occurring.
RESULTS
We applied our fastBMA algorithm to both simulated and real time series gene expression data. We had previously tested several methods on these datasets [14] and found that ScanBMA and LASSO were the fastest and most accurate methods. Therefore we compared the fastBMA results to ScanBMA and LASSO [33,42]. LASSO is a non-Bayesian linear regression method that uses a penalty term to prevent overfitting to models with many variables. It is written in Fortran and is one of the fastest network infer- ScanBMA. The x-axis is logarithmic, indicating fastBMA is orders of magnitude faster than ScanBMA when using the same parameters. Alternatively, one can use a larger odds ratio with fastBMA and obtain a more accurate result in same time it would take to run ScanBMA with a smaller odds ratio. On the same datasets, fastBMA is also more accurate and faster than LASSO, the degree and nature of improvement dependent on whether the user chooses to emphasize speed or accuracy through the choice of the odds ratio parameter.
One of the main advantages of the BMA methods is that they are able to incorporate prior information to improve inference. This was not possible for the DREAM4 dataset as it is a synthetic dataset. In this case, an uninformative uniform prior probability is used. However, for the yeast dataset we had access to priors from external data sources [12]. This also allowed us to triage the variables to be explored to the 100 variables with the highest prior probabilities, saving considerable computational resources. As expected, using informative priors increases the accuracy and decreases the running time relative to LASSO. In addition, we ran fastBMA, without informative priors and without restricting the number of variables (i.e. using all 3556). This is beyond the capabilities of ScanBMA when using wider search windows. Even on this computationally demanding task, inferring the yeast network without informative priors, fastBMA is faster than LASSO with increased accuracy as assessed by AUROC and AUPR.
A common use for computational network inference is to identify a small set of potential regulators that could be verified with further experiments. For this use case, an improvement in the precision of the most confident predictions is more important than a small improvement in the overall performance of the method. As some of the differences in AUC for the yeast dataset are relatively small, we plotted the precision recall curves in figure 5. We see that the precision of the most confident predictions (i.e. lowest recall) is increased. The advantage of using informative priors when available is very clear. However, even when prior knowledge is not available, the fastBMA algorithm is superior which is especially evident in the case of the Dream4 dataset.
The effect of post-processing is more limited. In figure 5, the precision-recall curves for the Dream4 dataset are almost identical for fastBMA and LASSO with and without post-processing. The same result was observed for fastBMA on the yeast dataset and for clarity, we did not plot the overlapping precision-recall curves for the post-processed networks for fastBMA. However, we do see that postprocessing has an effect on LASSO for the yeast dataset.
We also tested fastBMA on a human single cell RNA-Seq dataset with 9776 variables. Using a 32 core cluster on Microsoft Azure, fastBMA was able to obtain a network in 13 hours without using informative priors. Neither ScanBMA, nor LASSO is able to return results for this dataset. We do not have a gold standard for this test -the purpose was to demonstrate that fastBMA could handle a very large and noisy genomic sized dataset and return a network within a reasonable time even in the worst case scenario where the data is noisy and there is no prior information.
DISCUSSION AND CONCLUSIONS
We have described fastBMA, a parallel, scalable and accurate method for inferring networks from genome wide data. We have shown that fastBMA can produce networks of increased accuracy orders of magnitude faster than other fast methods even when using a single thread. Further speed increases are possible by using more threads or processes. fastBMA is scalable and we have shown that it can be used analyze human genomic expression data even in the most computationally demanding situation of noisy data, no informative priors and considering all genes a possible regulators. prove useful as an adjunct to methods and datasets that give rise to denser networks and are more prone to over-predicting edges.
Although we have focused on biological time series data, fastBMA can be applied to rapidly infer relationships from other high dimensional analytics data. Also the fastBMA methodology can be extended for even more demanding applications. For example, multiple bit filters could be used to (i.e. a Bloom filter) to hash larger search spaces. We anticipate that the efficiency of fastBMA will be especially use- We take the negative log of the probabilities (middle panel) to transform the multiplication into distances. The indirect path A→B→C is shorter than the direct path A→C which is equivalent to the probability of A regulating C through B being greater than the probability of A directly regulating C. As a result, the edge between A and C is removed. | 2018-04-03T01:17:56.494Z | 2017-01-06T00:00:00.000 | {
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208622577 | pes2o/s2orc | v3-fos-license | Comparative analysis of squamate brains unveils multi-level variation in cerebellar architecture associated with locomotor specialization
Ecomorphological studies evaluating the impact of environmental and biological factors on the brain have so far focused on morphology or size measurements, and the ecological relevance of potential multi-level variations in brain architecture remains unclear in vertebrates. Here, we exploit the extraordinary ecomorphological diversity of squamates to assess brain phenotypic diversification with respect to locomotor specialization, by integrating single-cell distribution and transcriptomic data along with geometric morphometric, phylogenetic, and volumetric analysis of high-definition 3D models. We reveal significant changes in cerebellar shape and size as well as alternative spatial layouts of cortical neurons and dynamic gene expression that all correlate with locomotor behaviours. These findings show that locomotor mode is a strong predictor of cerebellar structure and pattern, suggesting that major behavioural transitions in squamates are evolutionarily correlated with mosaic brain changes. Furthermore, our study amplifies the concept of ‘cerebrotype’, initially proposed for vertebrate brain proportions, towards additional shape characters.
U nderstanding the processes underlying the origin and diversification of morphology is a central goal in evolutionary biology and requires the integration of form, function and ecology. Particularly, there is compelling evidence across diverse vertebrate species that behavioural and ecological factors such as diet, habitat, locomotion, cognitive abilities and lifespan play an important role in driving brain evolution 1 . Previous studies evaluating the impact of environmental and biological factors on morphological brain characteristics have so far largely focused on neuroanatomical descriptions or volumetric, linear, and/or stereological measurements of whole-brains, major brain subdivisions, or brain endocasts. However, very few investigations have integrated multiple approaches to study brain evolution at various levels of biological organisation, ranging from molecules to cells and organ shape, within specific clades of vertebrates.
The cerebellum-major hindbrain feature-plays a critical role in sensory-motor control and higher cognitive functions such as memory and language 1 . Furthermore, this brain subdivision shows an extensive diversity in terms of morphology and neuroanatomy across vertebrates [1][2][3] . This suggests that cerebellar organisation is likely tightly linked to functional demands associated with ecological behaviours. Comparisons of vertebrate cerebella using complementary approaches could thus offer an excellent framework to address the complex evolutionary relationships between organ specialization and behavioural capabilities. Consistent with this, the size and/or neuroanatomical features of the cerebellum have been frequently linked with various behavioural or ecological strategies in mammals [4][5][6] , birds 4,7-11 , fish 12,13 , amphibians 14,15 and squamate reptiles 3,16 , but the poor shape correspondences of endocasts in the hindbrain region [17][18][19][20] as well as the taxon-specific or limited sampling in brain studies have typically hampered precise assessment and correlation analysis [21][22][23][24][25][26][27][28][29] . Furthermore, comparative studies have typically focused on identifying the influence of cerebellum volume on various ecological characters such as nest complexity, locomotor ecology, and/or prey capture behaviour, but additional volume-independent morphological parameters such as cerebellar foliation pattern and size could also be correlated 30,31 . As a result, despite the long history of volumetric assessment of individual brain subdivisions and stereological evaluation of neuroanatomical structures in vertebrates, quantitative evidence of cerebellar ecomorphology have so far relied on weight, linear dimension, or foliar measurements of freshly dissected or sectioned brains [30][31][32][33][34] . Hence, the extent to which interspecific cerebellar variations are reflected in terms of morphology, gene expression pattern, neuronal parameters, such as size, number, and spatial organisation and ecological behaviour is unclear. The cerebellum contributes to coordination, precision, and accurate timing of movement by receiving input from sensory systems of the spinal cord and from other parts of the brain, including the cerebral cortex. This structure is thus of key importance in the control of locomotion, a fundamental attribute to several ecologically crucial functions, such as feeding, predator avoidance and territorial defense. Thus far, comparative studies investigating locomotor adaptations within specific clades of vertebrates have largely focused on other functional and anatomical aspects such as limb morphology and performance, muscle type and physiology, axial skeleton morphology and proportion, as well as organisation of major descending motor pathways 35 .
Here, we perform a comparative, integrative characterisation of cerebellum evolution in vertebrates, using a quantitative approach combining morphological, phylogenetic, ecological, volumetric, cellular, and transcriptomic data. We hypothesise that locomotor behaviour could be a strong predictor of cerebellar complexity at different levels of organisation, including volume, shape, neuron spatial arrangement, and gene expression pattern. To test this hypothesis, we use squamate reptiles-lizards and snakes-as the main model system because of their high levels of morphological diversity, including in the size as well as general anatomical and cellular organisation of the cerebellum 15,[36][37][38][39] . Moreover, squamates exhibit unique ecological and behavioural features 35,40,41 , and both the locomotor pattern and locomotor performance of snakes and lizards are surprisingly well-characterised and known to be strongly influenced by limb and body shape and/or length 35,42 , as well as by several habitat attributes, such as temperature, substrate and inclination [43][44][45] . We assess cerebellar phenotypic diversification with respect to locomotor specialization in a representative dataset of squamate species, by integrating geometric morphometric and volumetric analysis of highdefinition 3D brain models along with investigation and quantification of neuron distribution based on 3D imaging of cleared whole-cerebella and immunohistochemistry. Furthermore, to provide molecular insights into developmental processes and/or mechanisms underlying cerebellar diversification, we use a transcriptomic approach to test whether differences in the species expression profiles are shaped more by their phylogenetic relationships or by differences in locomotor behaviours. Altogether, our characterisation of the squamate brain uncovers multi-level variations in cerebellar structure. Along with changes in cerebellar shape and size, we show heterogeneity in Purkinje cell spatial distribution and dynamic gene expression pattern, which are all associated with locomotor specialization. These data indicate that locomotor pattern is a strong predictor of cerebellar architecture, and highlight the importance of mosaic brain evolution in generating brain patterns shared by groups of squamate species with behavioural similarities.
Results and discussion
Overall morphology of squamate brain. To explore potential relationships between brain morphology and locomotor specializations, we used high-definition 3D reconstructions of wholebrains and isolated cerebella based on contrast-enhanced computed tomography (CT) and manual segmentation, using a representative panel of 40 lizard and snake species with different locomotor modes (Fig. 1 and Supplementary Table 1). Tissue fixation and staining procedures were performed using low concentrations of fixatives and contrast agents based on previous reports [46][47][48][49][50][51] and optimised protocols [52][53][54][55] , thus avoiding or limiting soft tissue artifacts such as shrinkage (see Methods and Fig. 2). Furthermore, as observed in our study (Fig. 2a), brain structures have been shown to preserve their shape and cytoarchitecture following contrast enhancer staining 47,49-51 , likely as a result to the small cell size and/or high neuron density. Major locomotor patterns were defined based on cranial and post-cranial anatomical features, habitat use, and movement type, which are all intimately associated with locomotor performance 35,40,42 and well-documented in quadrupedal, limbreduced, and limbless squamates (see all descriptions of categorisation in Methods and Supplementary Table 1). For example, the limbless burrower group contains both lizard and snake species showing elongated bodies with an increased number (>26) of presacral vertebrae, reduced limb skeletal elements, and cylindrical skulls to reduce energy expenditure during burrowing 41,56 , and at least four discrete types of locomotion have been recognised in snakes 35 . In addition to covering all major squamate groups, our dataset includes nine different species of scincid lizards, a geographically widespread family that displays a large array of post-cranial and limb morphologies, habitats, and locomotor behaviours ( Fig. 1a and Supplementary Table 1). The wide heterogeneity in both morphology and spatial configuration of squamate brain subdivisions is already apparent from a global inspection of our 3D models (Fig. 1b-h). Especially, the shape, size and degree of development of the cerebellum diverge extensively among species, ranging from a trapezoidal structure in snakes (Fig. 1i, j) to a more hexagonal conformation shared by quadrupedal lizards (Fig. 1k-m). Furthermore, snake and lizard cerebella generally display an opposite tilting relative to brain anatomical axes, caused by a flexure occurring, with varying extents, either on pial (for snakes; Fig. 1i, j) or ventricular (for lizards; Fig. 1k-o) surface. As a result, whereas the snake cerebellum is caudally tilted (Fig. 1b, c, i, j), the lizard counterpart extends dorsally (Fig. 1k-o) Brain shape diversity in squamates. We next quantified the shape of the squamate brain, based on organ outline, as a whole and as a set of main subdivisions, including hindbrain subregions such as the cerebellum barely accessible on brain endocast 18,27 , using 3D reconstructions and geometric morphometric approaches. Our landmark-based principal component analysis (PCA) performed on Procrustes coordinates ( Supplementary Fig. 1) generated a morphospace defined by three first principal components-PC1 to PC3-which together account for more than 60% of the total shape variation both for the whole-brain (Fig. 3) and each individual subdivision tested (Fig. 4). Remarkably, whole-brain shape variations along the PC1 axis, from negative to positive values, reflect the morphological transition from snakes to quadrupedal lizards (Fig. 3). Indeed, PC1 negative values contain all snake species, which are characterised by laterally compressed optic tectum and compact forebrain showing stout olfactory bulbs and tracts as well as ventro-laterally expanded cerebral hemispheres (see, e.g., Python regius; Fig. 3). In contrast, apart from two chameleon species with stunted olfactory tracts that positioned at negative PC1 values ( Fig. 3 and Supplementary Fig. 2b), all quadrupedal lizards are scattered along the positive PC1 axis, featuring antero-posteriorly compressed and laterallyprotruding cerebral and tectal hemispheres as well as thin and elongated olfactory lobes and tracts (see, e.g., Trioceros jacksonii; Fig. 3). Interestingly, limbless and limb-reduced lizards populate the central part of the PC1 axis and display features ranging from a snake-like organisation in burrower lizards (see, e.g., Melanoseps loveridgei) to morphologies resembling that of quadrupedal lizards in facultative burrowers (see, e.g., Chalcides chalcides; Fig. 3). Lizards also display appreciable whole-brain morphological variations along the PC2 axis, which is associated with changes in the length and curvature of both olfactory tracts and medulla oblongata as well as in the reciprocal arrangement and dorso-ventral expansion of cerebral hemispheres and optic tectum (compare, e.g., Bradypodion pumilum versus Xerotyphlops vermicularis; Fig. 3).
Importantly, snake and lizard species tend to segregate in all our analyses of whole-brain and individual subdivisions, with snakes clustering at extreme positions along PC1 and/or PC2 axes, except for the cerebellum that displays an increased overlap based on two landmark configurations (Figs. 4 and 5a and Supplementary Fig. 1). Consistent with this pattern, and although a significant phylogenetic signal was identified for all subdivisions using a multivariate K-statistic 57 (K-values ranging from 0.5684 to 0.7118; p-values < 0.001; Supplementary Fig. 2a Table 3). Notably, post hoc pairwise comparisons corrected for phylogenetic information indicate significant morphological differences between limbless burrowers and all other locomotor modes (phylogenetic ANOVA, p-values ranging from 0.0004 to 0.0216), as well as between any limbless or limbreduced and quadrupedal locomotion (p-values ranging from 0.0001 to 0.0147; Supplementary Table 4). The segregation of limbless burrower species with other locomotor groups is conspicuous within cerebellum morphospace at negative PC1 values, which reflect a thin, triangular-shaped cerebellum with a barely detectable curvature (see, e.g., Xenotyphlops vermicularis; Fig. 5a). Along PC1 axis, cerebella progressively increase their dorso-ventral extension, expand laterally, and show a gradual intensification of the ventricular surface flexure (see, e.g., Bachia flavescens and Phelsuma grandis; Fig. 5a). At extreme PC1 positive values, they are remarkably developed along the dorsoventral axis, with a considerable elongation of their medial regions that bend rostrally to overstep the tectal hemispheres (see, e.g., Trioceros jacksonii; Fig. 5a). Importantly, the PC2 axis distinguishes other limbless or limb-reduced locomotor modalities from quadrupedal locomotion, with cerebellar shape ranging from a dorso-ventrally stunted and laterally compressed structure typical of limbless or limb-reduced multi-habitat species and partially shared by some facultative burrowers at negative values (see, e.g., Chrysopelea ornata and Chalcides chalcides; Fig. 5a), to the dorsally and laterally developed and anteriorlyprojecting cerebellum of quadrupedal lizards at positive values (see, e.g., Phelsuma grandis; Fig. 5a). Together with these changes, PC2 further marks the inversion of the cerebellar flexure from the pial surface at very negative values (see, e.g., Python regius; Fig. 5a) to the ventricular surface (compare, e.g., Chrysopelea ornata and Chalcides chalcides; Fig. 5a). Interestingly, the distribution of scincid lizards, which belong to four different locomotor categories, illustrates well the locomotor behaviour trends by their scattering throughout most of the cerebellum morphospace (see exact positions in Supplementary Fig. 2a). Furthermore, distance-based convergence measures from Stayton 59 support the significant cerebellar convergence of lizard and snake species within most of limbless or limb-reduced locomotor groups (p-values ranging from 0.0020 to 0.0360 in limbless burrowers and limbless or limb-reduced facultative burrowers; Supplementary Table 5).
The lack of significant locomotor signal on the whole-brain with phylogenetic ANOVA (p-value = 0.8076; Supplementary Table 3) might derive from the complex relationship between neuroanatomical structures and their function, with morphological changes likely reflecting several differences in behaviour and ecology. Importantly, similar cerebellar distribution patterns and shape changes were obtained using alternative landmark-free methods ( Supplementary Fig. 2c, d), thus underscoring the robustness of our data. To better visualize and topographically map cerebellar shape Fig. 1 Phylogeny and cerebellar diversity of squamates. a Phylogenetic tree of all snake and lizard species used in morphological and volumetric analyses, adapted from the most inclusive phylogenetic study available for extant squamates 85 . The seven major locomotor modes for snakes (coloured squares) and/or lizards (coloured circles), as defined based on anatomical features, habitat use, and movement type, are indicated by the same colour code throughout the entire manuscript (see bottom left corner): limbless or limb-reduced burrower (red squares and circles); limbless or limb-reduced facultative burrower (purple squares and circles); limbless or limb-reduced multi-habitat using lateral undulation (orange squares and circles); limbless or limb-reduced multi-habitat using other movements (yellow squares); quadrupedal arboreal (dark blue circles); quadrupedal terrestrial (light blue circles); quadrupedal facultative bipedal/aerial (green circles). b-o 3D-volume rendering and high-resolution whole-brain segmentation of iodine-stained adult heads (b-h) highlighting the cerebellum structure (b-o, red colour) of selected representative squamates at indicated position in the phylogenetic tree: Pantherophis guttatus (b, i), Epicrates cenchria (c, j), Pogona vitticeps (d, k), Draco volans (e, l), Bradypodion pumilum (f, m), Anguis fragilis (g, n), Melanoseps loveridgei (h, o). High magnifications of 3D-rendered cerebella (i-o) are shown in pial surface (left panels) and lateral (right panels) views for each selected species. Scale bars: 1 mm (b-h), 500 μm (i-o). ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13405-w changes typical of each locomotor mode, we next reconstructed the mean cerebellar shape configuration for each locomotor group (Fig. 5b). Consistent with a significant correlation between locomotion and cerebellar morphology, considerable deviations from the overall mean shape for the entire dataset are apparent in specific areas for most locomotor groups. Especially, a large heterogeneity exists on both ventricular and pial surfaces, where bending and thickening diverge among locomotor groups, as well as in medial and lateral peduncular areas (Fig. 5b). For example, the pial surface curvature exhibits the highest degree of variation in limbless or limb-reduced multi-habitat species, whereas limbless or limb-reduced burrowers, and to a lesser extent facultative burrowers, rather show major changes on the ventricular side. Similarly to other vertebrates, the reptile cerebellum is well-known to be divided into an elaborate array of regions that form an exquisitely organised topographic map regulating sensory-motor behaviour 2,3 . Especially, the medial and lateral parts of the main cerebellar body in snakes and lizards, two regions that show major changes in limbless or limb-reduced species from our dataset, have already been proposed to control axial musculature and limb
Fig. 3
Whole-brain shape variation in squamates with different locomotor behaviours. 3D plot of principal component (PC) scores showing the whole-brain shape distribution of snakes (coloured cubes) and lizards (coloured spheres) with different locomotor modes (see colour code and symbols in bottom left corner). Numbers in brackets indicate the percentage of variance explained by each of the PC axes, and 68% confidence ellipses are shown for each locomotor mode. The 3D-rendered whole-brains corresponding to extreme (indicated by coloured solid arrows, with species names) or representative (coloured dashed arrows, with species names) lizard and snake species in both positive and negative directions along the first two PCs are shown in dorsal (top) and lateral (bottom) views. Source data are provided as a Source Data file. movement 2,3 , respectively. Furthermore, the presence/absence and degree of development of motor control brainstem nuclei and spinal pathways, including the red nucleus, vestibular nuclei, certain reticular nuclei, as well as the rubro-spinal, vestibulospinal, vestibulo-reticulo-spinal, and reticulo-spinal tracts, have been associated with the different body plans and locomotor behaviours of reptiles 60-64 , but also with their level of cerebellar complexity 60,61,64 . Our observed cerebellar shape changes are thus definitely expected to functionally correlate with both locomotor system anatomy and locomotor behaviour.
Cerebellum size variability in squamates. Because of the welldocumented impact of ecological parameters on the relative size of we next compared the volume of our 3D cerebellar models among locomotor groups. In parallel with the significant effect of evolutionary allometry on cerebellar shape (multivariate regression analysis of Procrustes distance on centroid size, percentage predicted = 12.6%, p-value < 0.0001), our volumetric data highlight substantial divergence in cerebellar size both in absolute and relative terms (Fig. 6a). As the homogeneity of regression slope assumption . Numbers in brackets indicate the percentage of variance explained by each of the PC axes, and 68% confidence ellipses are shown for each locomotor mode. The 3D-rendered cerebella corresponding to extreme (indicated by coloured solid arrows, with species names) or representative (coloured dashed arrows, with species names) lizard and snake species in both positive and negative directions along the first two PCs are shown in pial (left) and lateral (right) views. Source data are provided as a Source Data file. b Warped surfaces of cerebellum representing the reconstructed mean shape configuration for each indicated limbless or limb-reduced (left panels) or quadrupedal (right) locomotion mode are shown in pial surface (top row) and lateral views (bottom). Colour gradient, ranging from blue to yellow, reflects the relative Procrustes distance (in μm) of shape changes from the overall mean shape for the entire dataset. required for conventional phylogenetic analysis of covariance (ANCOVA) was violated in our dataset (Supplementary Table 6), we used the alternative Johnson-Neyman procedure 65 to compare the locomotion-specific regression lines by establishing region of significance among volume covariates. Although there is not yet a phylogenetically informed implementation of the Johnson-Neyman method, the major assumptions of this technique are similar to ANCOVA, and the negligible phylogenetic signal (equivalent to zero) observed in our phylogenetic ANCOVA analysis allowed us to confidently use this method. Unexpectedly, this approach revealed few significant differences in the relative size of cerebella between locomotor groups. Indeed, only limbless burrowers showed a significantly reduced cerebellum within a restricted range of low whole-brain values (log volume < 2.42 mm 3 ; Supplementary Table 7). Importantly, similar significant differences of limbless burrowers with all other locomotion groups were further confirmed using phylogenetic ANOVA analysis (p-values < 0.05) on the cerebellum-to-whole-brain volume ratio (Fig. 6b). Altogether, these results highlight the great phenotypic diversity of the cerebellum across squamates, and unambiguously indicate that both the cerebellar shape and size reflect locomotor behaviours. Brain size has been traditionally the preferred measured trait in past evolutionary studies, but we show here that size is only one metric, and other parameters such as shape can change independently of brain region volume. Particularly, our data clearly indicate that the cerebellar shape is likely a more relevant feature of vertebrate brain evolution than its relative size, as also suggested by recent adaptive radiation studies in several vertebrate lineages 66,67 .
Purkinje cell layout in the cerebellar cortex. Histological and morphological evidence suggest that species-specific characteristics of the cerebellar architecture could also be linked to behaviour and/or sensory ecology in vertebrates 3,10,11,30,31,68 , although this relationship is yet not well understood. To investigate this aspect in the squamate cerebellum, we first compared the complexity and general organisation of the principal layers and cell types of the cerebellar cortex, using histology and immunohistochemistry (IHC) methods. We particularly focused on comparing the spatial organisation of Purkinje cells (PCs), which is known to be either arranged in a monolayer or scattered in a few lizard and snake species, respectively 3,36-39 . Our comparative examination of the two predominant cerebellar neuron types-PCs and granule cells (GCs)-with specific markers such as CALB1 and ZIC1/2/3, respectively, corroborates previous observations and indicates heterogeneity in PC spatial arrangement among squamate species (Figs. 2b, c and 7). Particularly, 3D light-sheet fluorescence microscopy imaging of cleared wholecerebella indicates a scattered organisation of PCs throughout the molecular layer (ML) in Boaedon fuliginosus snakes, in contrast to the orderly monolayer of Pogona vitticeps lizards (Fig. 7a, b), thus confirming the atypical PC pattern of snakes 3,36,37 . To investigate further this phenotype in a larger squamate dataset, we explored the potential relationships between cell arrangement and squamate locomotor specialization, by quantifying the scattering of individual PCs with a numerical approach integrating IHC, image processing, and statistical analysis. We particularly focused on comparing groups of individuals belonging to different selected squamate species with specific locomotor modes. Post hoc pairwise comparisons following highly significant Kruskal-Wallis test (p-value < 0.0001) revealed four major groups reflecting significantly different PC distribution patterns within the ML (Fig. 7c): ordered monolayer (group I), ordered multilayer (II), scattered multilayer (III) and totally scattered (IV). The lack of significant segregation between some locomotor modes, including between burrower and facultative burrower species that all cluster in group II, might be due to species sampling limitations, especially the difficulty to get fully burrower species. However, the low number of species for some locomotor modes does not affect the overall significance of our data, as significantly different PC organisation was further confirmed by directly comparing the obtained four groups I-IV with similar Kruskal-Wallis statistical test (p-values < 0.0001). Interestingly, and consistent with our cerebellar shape and volumetric data, all quadrupedal species cluster in a single group with similar ordered monolayer of PCs (group I). In contrast, significant differences were observed among limbless and limb-reduced locomotion modes. The most striking example include the significantly different PC distribution pattern between groups that include both lizard and snake species, including burrower or facultative burrowers (group II) and multi-habitat species using lateral undulation (group III; Fig. 7c), suggesting that different degrees of PC scattering present both in snakes and lizards parallel locomotor specialization independently of species relationship. Altogether, our data indicate that the evolution of different locomotor strategies might require different kinds of cerebellar mediated coordination, resulting in a particular topological organisation of PCs likely different from the ectopic PC pattern previously reported in mutant mice or human patients with abnormal cerebellar development and neurological disorders [69][70][71][72] should reveal whether additional levels of cerebellar complexity are also ecologically relevant.
Patterns of cerebellar gene expression. Differences in gene regulation and expression pattern have long been recognised as crucial contributors to phenotypic diversity of the nervous system at both cellular and morphological levels 73 . Particularly, the accurate characterisation and quantification of orthologous transcripts across species are critical for understanding gene expression patterns and transcriptome-phenotype relationships, as evolutionary changes in gene expression contribute to behavioural phenotype and play a key role in phenotypic changes between species. Here, we employed comparative transcriptomics to determine the similarity of cerebellar gene expression across representative squamate species with similar or different locomotor behaviours. We restricted our analyses to a set of 630 protein-coding genes that could be identified as one-to-one orthologs across all 10 species RNA sequencing data (Fig. 8a). The difficulty to recover shared orthologs in our lizard and snake dataset could be justified by the lack of genome data information in most analysed models as well as by the abundance of rapidlyevolving genes showing accumulation of amino acid changes in squamates 74,75 , two factors that are known to affect the sensitivity of ortholog identification. Heat map analysis and hierarchical clustering based on normalised expression values of orthologous genes revealed three clusters with characteristic gene expression patterns, including similar (up-or downregulated, clusters 1 and 2) or differential (cluster 3) expression among squamate species (Fig. 8a). We further investigated the biological function of sets of genes characterising each identified cluster, using a functional enrichment analysis of Gene Ontology (GO) categories. Interestingly, whereas the GO distribution was very similar in the three different clusters for the first three high-level biological process terms, including cellular process, biological regulation, and developmental process, significant gene category enrichment was observed for more specific terms. Especially, cluster 3, representing differentially expressed genes, is highly enriched with genes associated to development (three enriched categories, corresponding to 105 genes) but also to locomotory behaviour (29 genes; Fig. 8b), indicating that functionally relevant genes can exhibit dynamic expression ranges across squamates. Importantly, most of the identified enriched genes linked to locomotory behaviour are already known to be predominantly expressed in the brain and to play a key role in motor coordination, balance, and/or locomotor activity in multiple vertebrate models such as zebrafish (e.g., Spatacsin), mice (e.g., Contactin-2, Astrotactin-1, Ephrin type-A receptor 4, Striatin, Alsin, Metabotropic glutamate receptors 1 and 5, Nuclear receptor coactivator 2, Hamartin), rats (e.g., Unconventional myosin-Va), and humans (e.g., Pumilio homolog 1). Among examples, knock-out mice for Contactin-2, a cell adhesion molecule expressed in the Purkinje fiber network and critical for neuronal patterning and ion channel clustering, exhibit severe ataxic phenotype consistent with defects in the cerebellum 76 , and mice that lack Astrotactin-1 have abnormal development of Purkinje cells associated with defects in balance, coordination, and walking behaviour 77 . Our data thus suggest that the evolution of different locomotor strategies might require expression level modulation of the identified cerebellar genes, indicating that differential gene expression might reflect the alternative locomotor patterns in squamates. By contrast, housekeeping and structural genes such as regulators of cytoskeletal actin, as well as GO categories not directly or exclusively linked to the specific movement from place to place of an organism (as defined for the locomotory behaviour category), are significantly over-represented in clusters 1 and 2 (Fig. 8b), thus also confirming the validity of our data and experimental design.
To test whether differences in the species expression profiles are linked to phylogeny or locomotor behaviour, we next performed hierarchical clustering on pairwise correlation to build a tree-like structure (dendrogram) where species are clustered into hierarchies according to their degree of expression pattern similarity. Strikingly, our expression phylogeny from the set of orthologous genes identified in cluster 3 (but also of all orthologous genes) clearly differs from the molecular phylogeny expected for squamates, and rather reveals a locomotion-dominated clustering (Fig. 8c). Furthermore, the majority of the groupings have relatively high bootstrap support, thus strongly indicating that lizard and snake species clustered according to locomotor modes rather than phylogenetic relationships (Fig. 8c). Notably, the variable expression patterns between locomotor groups precisely correlate with the phenotypic changes observed at both cellular and morphological levels, supporting the existence of tight functional links between gene expression, cerebellar architecture, and locomotor behaviours.
Concluding remarks. Altogether, our characterisation of the squamate brain uncovers multi-level variations in cerebellar architecture as well as unique relationships between a major ecological behaviour and organ specialization in vertebrates. Along with significant changes in cerebellar shape and size across squamate species, our study indicates a peculiar heterogeneity in cortical neuron spatial distribution and gene expression pattern, which are all intimately associated with locomotor specialization. These data unveil the existence of several relationships between brain complexity and locomotion in vertebrates, indicating that locomotion mode is a strong predictor of cerebellar size, shape, PC spatial organisation, and gene expression levels. Furthermore, this also suggests that major behavioural transitions in vertebrate locomotion patterns are evolutionary correlated with key changes in cerebellar structure, in addition to morphological changes expected in structures such as limb and post-cranial skeleton. In this context, it would be very interesting to assess additional levels of cerebellar complexity across vertebrate species, including neuron number and size as well as neuronal morphology, physiology, and circuit complexity. Importantly, the observed correlation between cerebellar shape and ecological behaviour in our geometric morphometric analysis across multiple brain regions demonstrates the existence of brain patterns shared by groups of species with lifestyle similarities, in a similar way to 'cerebrotypes' initially described for mammals based on size/volume data 78 . Furthermore, these findings on morphological features indicate that the main brain subdivisions in squamates are not uniform structures but rather evolved independently, thus supporting a mosaic model of brain evolution, at least with respect to locomotor capacities, as previously reported in different vertebrate groups 28,[79][80][81][82][83] . Altogether, our work provides a framework for the evolution of cerebellar structure and locomotor behaviours in vertebrates, which can be reinforced by future experimental works and dissection of molecular and developmental mechanisms. In addition, our complementary analyses highlight how future comparative vertebrate studies of organ system-ecology relationships could benefit from adopting a multi-level integrative approach. Table 1). An average of 2.6 individuals per species was examined (range 1-8 depending on species sampling difficulties and number of analyses performed; Supplementary Table 1), and specimens were used for multiple observations and/or analyses whenever possible. Species were carefully selected to cover all major groups of squamates 85 , thus enabling a comprehensive representation of the diversity of locomotor behaviours (Supplementary Table 1). Categorisation of locomotor groups was performed by cross-correlating data from reptile databases, available literature, and personal observations on different criteria: cranial and post-cranial anatomical features as previously defined 41,86,87 , including overall skull shape, limb characteristics (intact: well-developed limbs; reduced: limbs with skeletal elements reduced in size, lost, or fused; vestigial: retention of rudimentary limb skeleton; limbless: absence of external limbs), and degree of body elongation (elongated body: increased number (>26) of presacral vertebrae); habitat modes (aquatic: adapted for aquatic life, including marine environment; terrestrial: adapted for surface locomotion and foraging, including saxicolous; burrower: adapted for digging, living primarily and foraging underground; facultative burrower: terrestrial with some leaf-litter or burrowing behaviour; arboreal: adapted for locomotion between tree branches or bushes; semiarboreal: often inhabiting and frequenting trees but not completely arboreal; aerial: arboreal with aerial behaviour, including gliding); movement types associated with locomotor performance and species-specific locomotor typologies based on previous descriptions 35 Fig. 8 Comparative transcriptomics of the squamate cerebellum. a Two-way hierarchical clustering heat map showing three clusters of genes (rows) that behave similarly (clusters 1 and 2) or differently (cluster 3) across ten selected squamate species (columns). Z-score colour intensities reflect scaled gene expression values, ranging from low (blue) to high (yellow), for 630 one-to-one orthologous genes identified in all species. Source data are provided as a Source Data file. b Pie charts showing the distribution of orthologous genes (in %) among all significantly enriched gene ontology terms for biological processes (hypergeometric test with a false discovery rate multiple-hypothesis correction, p-value < 0.01) in clusters identified in a. c Hierarchical clustering of pairwise Pearson's correlation coefficients for 630 orthologous genes identified across all squamate species. Colour intensities of individual tiles in the heat map depict pairwise correlation coefficient values, ranging from low (blue) to high (yellow), between selected species with indicated locomotor mode (see colour code and symbols on the right). Numbers at nodes in the cluster dendrogram represent approximately unbiased p-values (in percentage) obtained by multiscale bootstrap resampling.
Methods
consisting of gripping with portions of the body while pulling or pushing other sections in the direction of movement; modified concertina: concertina movement involving active pushing against the sides of the tunnel to provide a static anchor; quadrupedal arboreal: locomotion using four limbs to move through trees, including morphological specializations; quadrupedal terrestrial: surface locomotion using four limbs; facultative aerial: locomotion occurring in the air, including gliding; facultative bipedal: form of terrestrial locomotion using the two rear limbs, including running). Seven major locomotor groups were defined based on these criteria, including two major categories (limbless or limb-reduced and quadrupedal) further divided into four (burrower, facultative burrower, multi-habitat lateral undulation, multi-habitat other movements) or three (arboreal, terrestrial, facultative bipedal/aerial) subgroups (see Supplementary Table 1): limbless burrower (burrower species using modified concertina or rectilinear locomotion); limbless or limb-reduced facultative burrower (facultative burrower species using slow lateral undulation locomotion); limbless multi-habitat lateral undulation (terrestrial, aquatic, or aerial/arboreal species using lateral undulation locomotion); limbless multi-habitat other movements (terrestrial or arboreal species using rectilinear, arboreal concertina, or sidewinding locomotion); quadrupedal arboreal (arboreal species using quadrupedal arboreal locomotion); quadrupedal terrestrial (terrestrial species using quadrupedal terrestrial locomotion); quadrupedal facultative bipedal/aerial (semi-arboreal or aerial species using facultative bipedal or aerial locomotion).
Generation of 3D whole-brain models. High-resolution 3D CT scans of adult squamate heads were performed at the University of Helsinki or University of Kuopio imaging facilities (Finland) using Skyscan 1272 or 1172 (Brucker, Belgium). Samples were fixed and conserved in 10% formalin (museum specimens) or fixed in 4% paraformaldehyde (fresh specimens), two solutions that give almost identical final concentrations of formaldehyde (Supplementary Table 1). Previous quantitative analyses of brain tissues have demonstrated minor shape and cytoarchitecture artifacts introduced by such fixatives, including by comparing specimens with different preservation procedures 47,50,55 , and limited shrinkage of brain tissues is expected because of the relatively low concentrations used 46,[48][49][50] . For optimal brain tissue visualization, samples were next stained with 0.3% phosphotungstic acid (PTA) or 1% iodine solutions, two contrast enhancers with optimised protocol and known penetration power [52][53][54][55] . Based on previous reports [47][48][49]51 and own observations (see Fig. 2a), such low concentrations of contrast enhancers show either no or limited shrinkage of brain tissues, including in museum samples 51 . Scans were reconstructed using NRecon 1.7.0.4 software (Bruker) and 3D volume rendering as well as segmentation were done using the software Amira 5.5.0 (Thermo Fisher Scientific, U.S.A.). We adopted a manual segmentation approach to gain detailed brain reconstructions and to accurately reproduce the prominent traits of all encephalic subdivisions, including their spatial relationships (Fig. 2a). Most brain structures were discernible and could be included in the reconstructions, except for the pituitary and pineal glands. Both the whole-brain and isolated cerebellum were segmented, thus allowing assessment of volumetric and geometric morphometric measurements of these structures. Furthermore, geometric morphometrics was performed on each individual brain subdivisions using different specific subsets of landmarks on the whole-brain reconstructions (see below and Supplementary Fig. 1). A coronal plane intersecting the atlanto-occipital junction was set as the posterior limit of segmentation. The overall fixation/staining procedure and accuracy of all generated 3D models were carefully controlled by directly comparing our reconstructions with freshly dissected brains along the three anatomical planes (see Fig. 2a), and/or by incorporating more than one specimen as well as both museum and fresh specimens for each species whenever possible (n = 1-4 individuals per species depending on sample availability; Supplementary Table 1).
3D whole-surface analyses and geometric morphometrics. A total of 61 anatomical landmarks, clustered in groups delineating the 3D profile of the five major brain subdivisions (telencephalon, diencephalon, mesencephalon, cerebellum, medulla oblongata; Supplementary Fig. 1), were digitised on whole-brain surfaces using the Landmark Editor software (Institute for Data Analysis and Visualization, IDAV, U.S.A.). We further took advantage of our manual segmentation of the isolated cerebellum structure to improve its overall shape representation, by adding 5 extra-landmarks at the interface between mesencephalon and cerebellum, a region inaccessible on whole-brain models ( Supplementary Fig. 1b). Measurement errors deriving from digitisation were estimated by repeating landmarking in two independent sessions (Supplementary Table 8), and obtained coordinate values were then averaged and used for subsequent analyses 88 . The accuracy of our 3D reconstructions and landmarks was further validated by controlling for shape outliers in the R-package geomorph v3.0.7 89 (https://CRAN.R-project.org/ package=geomorph). A Generalized Procrustes Analysis (GPA) was used to simultaneously superimpose all configurations and extract shape data by removing the effects of scale, orientation, and position 45 . Centroid size was computed as the square root of the sum of the squared distances of all landmarks from their centroid, and a multivariate regression of shape onto centroid size was used to test for allometry 90 . A Principal Component Analysis (PCA) performed on the covariance matrix of shape variables was used to visualize the main patterns of morphological variation with 68.27% confidence ellipses for each group mean (5000 replicates), and the Rpackage pca3d v0.10 (https://CRAN.R-project.org/package=pca3d) was used to generate 3D PCA plots. Phylogenetic signal was calculated from shape data with a generalised K-statistic 47 in R-package geomorph v3.0.7 89 , using the most inclusive and recent phylogenetic studies available for extant squamate species 85 , and phylogenetic relatedness was corrected in subsequent statistical analyses to take into account independent observations across different species. To correct for allometry, all statistical analyses were also performed on residuals of the multivariate regression of shape onto size. The influence of locomotor modes on brain shape was tested with post hoc pairwise tests using phylogenetic ANOVA based on an advanced generalized least squares (GLS) approach 58 in geomorph package (function 'advanced.procD.lm'). Convergent evolution in the different locomotor groups was assessed using the distance-based convergence measures C1-C4 59 in the R-package convevol v1.3 (https://CRAN.R-project.org/package= convevol). To reconstruct the mean cerebellar shape configuration of each major locomotor mode, the closest specimen to the overall mean shape in multidimensional space was first identified via the function 'findMeanSpec' in geomorph package, and a 3D CT-scan of this specimen was warped to the overall mean shape (0.0) based on Thin Plate Spline (TPS) method 91 using the function 'tps3d' in R-package Morpho v2.6 92 (https://CRAN.R-project.org/package= Morpho). Patterns of shape changes were then visualized as deviations from this overall mean shape configuration to the averages of each locomotion group, using warping and heatmap functions from R-package Rvcg v0.18 (https://CRAN.Rproject.org/package=Rvcg) and colour palette from R-package viridis v0.5.1 (https://CRAN.R-project.org/package=viridis). Phylogenetic ANCOVA was carried out with function 'phylolm' from R-package phylolm v2.6 93 (https://CRAN. R-project.org/package=phylolm), using the most inclusive and recent squamate phylogeny 85 . The Johnson-Neyman procedure was used as an alternative to ANCOVA 65 when both heterogeneity of regression slopes and negligible phylogenetic signal (equivalent to zero) were observed, as this method currently lacks phylogenetically informed implementation. All analyses were performed in R with custom R scripts 94 .
Our whole-brain reconstruction strategy allowed us to employ alternative landmark-free methods, including statistical particle-based models of shape (ShapeWorks) 95 and generalized Procrustes surface analysis (GPSA) 96 , to corroborate the shape data derived from manual landmarking ( Supplementary Fig. 2c, d). Default ShapeWorks parameters were used for the grooming step, whereas the following parameters were used for the optimisation step: number of particles: 2000; relative weight: 0; starting regularisation: 1000; ending regularisation: 10; iterations per level: 3000; decay span: 1000. For GPSA, reconstructed whole-brain surfaces were simplified to 100,000 vertices and converted to Polygon File Format (PLY) in Amira 5.5.0, before loading into the GPSA software. 3D between-group PCAs 97 for ShapeWorks and GPSA were used to visualize the main patterns of morphological variation among groups via function 'groupPCA' from R-package Morpho.
Purkinje cell distribution quantification. To quantify and compare the distribution of PCs in the cerebellar cortex of squamate species with different locomotor modes, IHC on paraffin sections covering one cerebellar half along the sagittal plane were selected in homologous positions along the medio-lateral axis in each specimen, because of the species-dependent heterogeneity in antibody penetration with whole-mount methods. IHC on sections was conducted according to standard protocol 84 , using heat-induced epitope retrieval and overnight incubation at 4°C with primary anti-CALB1 antibodies (see above) for specifically identifying PCs. The position of individual labelled PCs relative to the ML was calculated by NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13405-w ARTICLE NATURE COMMUNICATIONS | (2019) 10:5560 | https://doi.org/10.1038/s41467-019-13405-w | www.nature.com/naturecommunications measuring the shortest distance between the center of PC somata and the inner border of the granule cell layer (GCL). Due to high intra-and interspecies heterogeneity in ML thickness that could affect the relative positioning of PCs, particularly in species with scattered PC organisation, all individual PC-GCL distances were then normalised to the entire ML thickness at particular PC location, by measuring the distance between the inner border of GCL and outer border of ML (pial surface). The particular anatomical localisation and organisation of the GCL, situated as a sharply edged and densely packed block of cells on the ventricular surface of the squamate cerebellum, allowed us to use 4′,6′-diamidino-2-phenylindole (DAPI) nuclear counterstaining (Sigma-Aldrich, U.S.A.) as a suitable proxy to outline the GCL. In addition, IHC with GCL-specific markers such as Zinc finger proteins 1/2/3 (ZIC1/2/3; 1:300, rabbit polyclonal, LifeSpan BioSciences, U.S.A., cat# LS-C118695) showed a consistent overlapping with DAPI labelling on the ventricular surface of the cerebellum in all tested species (Fig. 2b, c). Distance measurements were performed in the package Fiji 99 by running the 'measure distance to line' macro. As significant deviations from normality were obtained in the gathered data based on multiple methods (Shapiro-Wilk, Lillefors, Jarque-Bera, and Anderson Darling tests), thus precluding the use of parametric tests, the Kruskal-Wallis test followed by Dwass-Steel-Critchlow-Flinger post hoc comparative analysis was carried out to determine potential variations in PC distribution among locomotor groups (n = 250-750 observations (individual PCs) per species, using a range of 1-3 individuals from different species per locomotor mode depending on sample availability; Supplementary Table 1). Because of the low number of species for some locomotor groups, similar Kruskal-Wallis statistical analysis was also performed by combining some locomotor modes (n = 3-4 species per group in this case).Violin plots of PC distribution were obtained with function 'ggplot' from R-package ggplot2 v3.1.0 (https://CRAN.R-project.org/ package=ggplot2).
High-throughput RNA sequencing and transcriptome analyses. Total RNA was extracted from freshly dissected cerebellar tissues of 10 different squamate species with different locomotor behaviours (n = 1-3 species per locomotor mode depending on sample availability; Supplementary Table 1), using the RNeasy Plus Micro Kit (Qiagen, Germany), according to the manufacturer's instructions. RNA sample concentrations were detected by Qubit 3.0 (Thermo Fisher Scientific, U.S. A.), and RNA Integrity Number (RIN) values were determined using Agilent 2100 Bioanalyzer (Agilent Technologies, U.S.A.). Libraries were produced using the TruSeq Stranded Total RNA Kit with Ribo-Zero (Human/Mouse/Rat; Illumina, U.S.A.), and paired-end sequencing (88 + 74 bp) was done using duplicates on a NextSeq 500 platform (Illumina, U.S.A.). Quality trimming and adapter sequence removal of reads were done using the Trimmomatic tool 100 , and the SortMeRNA pipeline 101 was used for ribosomal RNA (rRNA) filtering. Quality trimmed and rRNA-filtered reads (total average of 44.2 million reads per species) were de novo assembled in contigs with the Trinity software v2.8.5 102 , using its default parameters. The coding (CDS) and protein sequence of unigenes were then predicted using the TransDecoder program implemented in Trinity 102 , and functional categories were determined using the Blast2GO program (BioBam, Spain) 103 . Finally, the OrthoFinder algorithm 104 was used for inferring orthologous and species-specific unigenes from protein sequences (total average of 8942 unigenes per species). The relative abundance of transcripts were evaluated for each species as FPKM (fragments per kilobase of transcript per million mapped reads) using the RNA-Seq by Expectation Maximisation (RSEM) software 105 , and only orthologs expressed above a fixed expression threshold of 0.5 FPKM in at least one sample were retained to get a more accurate characterisation and quantification by excluding possible noise at very low expression levels. 630 one-to-one orthologs shared across all ten species (Source Data) were subsequently used for hierarchical clustering, correlation coefficient, and functional enrichment approaches. Expression heat maps and hierarchical clustering were generated with the Heatmapper software 106 , using Euclidean distance or Pearson's and Spearman's rank correlation coefficients. The robustness of hierarchical clustering was further assessed using Rpackage pvclust v2.0-0 107 (https://CRAN.R-project.org/package=pvclust), with 1000 iterations of multiscale bootstrap resampling. Functional enrichment analysis was performed on several sets of orthologous genes based on hierarchical clustering, using the Gene Ontology (GO)-term enrichment tool available on GO Consortium website 108 (http://geneontology.org/) and R-package GOstats 109 (https://www.bioconductor.org/packages/release/bioc/html/GOstats.html). The significance of the enrichment was tested by the hypergeometric test with a false discovery rate multiple-hypothesis correction, and only significantly enriched GO terms (p-value < 0.01) comprising at least ten genes were considered.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. | 2019-12-05T15:18:56.327Z | 2019-12-01T00:00:00.000 | {
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5630262 | pes2o/s2orc | v3-fos-license | Kidney growth curves in healthy children from the third trimester of pregnancy until the age of two years. The Generation R Study
Information about growth of kidney structures in early life is limited. In a population-based prospective cohort study, from foetal life onwards, we constructed reference curves for kidney growth from the third trimester of pregnancy until early childhood, using data from 1,158 healthy children. Kidney size, defined as length, width, depth and volume, was measured in the third trimester of pregnancy and at the postnatal ages of 6 months and 24 months. Analyses were based on more than 2,500 kidney measurements. In the third trimester of pregnancy and at 6 months of age all kidney measurements were larger in boys than in girls. At 24 months of age, these gender differences were only significant for left kidney structures and right kidney length. Both groups showed trends towards smaller left kidney measurements than right kidney measurements at all ages. Gender-specific reference curves based on post-conceptional and postnatal ages were constructed for left and right kidney length, width, depth and volume. We concluded that kidney size is influenced by age and gender. Left kidney size tended to be smaller than right kidney size, except for kidney length. The reference curves can be used for assessing kidney structures by ultrasound in foetal life and early childhood. Electronic supplementary material The online version of this article (doi:10.1007/s00467-009-1335-2) contains supplementary material, which is available to authorised users.
Introduction
Assessment of kidney size in children is important for clinical and epidemiological studies. Abnormal early kidney development may have perinatal and neonatal consequences [1]. Smaller foetal kidney size has also been suggested to be related to hypertension and renal disease in adulthood [2,3]. Recently, we showed in the same cohort that small kidney size in foetal life tends to persist in early childhood. Furthermore, maternal anthropometric characteristics, foetal biometric data and blood flow patterns were associated with kidney size in childhood. Higher growth rates in early childhood were positively associated with combined kidney volume [4]. These results suggest that variations in foetal and early postnatal exposure and growth might have persistent consequences for kidney size. Kidney size can be measured non-invasively and efficiently with ultrasound. Few studies have published reference ranges for kidney size in healthy children during foetal and neonatal life [5,6]. One study showed reference data on postnatal kidney growth from birth to 18 months of age [7]. Previous studies were based on the characteristics of postnatal kidney growth and focused mostly on kidney volume in relation to weight, height or body surface area. Recently, new reference centiles were generated to assess kidney size of children with 'single kidneys' to identify those patients with unfavourable courses and relevant single kidney growth impairment [8]. Currently, there are no studies that have evaluated normal kidney growth from late foetal life to early childhood. This perinatal period may be of importance in the identification of abnormal kidney size and growth, with subsequent short-term and long-term clinical consequences [9,10].
Therefore, the aim of this study was to construct reference curves for kidney structures including kidney length, width, depth and volume in children from the third trimester of pregnancy until the postnatal age of 24 months in a population-based cohort.
Study design
This study was embedded in the Generation R Study, a population-based prospective cohort study from foetal life until young adulthood in Rotterdam, The Netherlands [11,12]. Detailed assessments of foetal and postnatal growth and development were conducted in a subgroup of 1,232 mothers and their children [11,12]. In this subgroup foetal kidney ultrasounds were performed in the third trimester of pregnancy (gestational age 30 weeks) and postnatal kidney ultrasounds were performed at the ages of 6 months and 24 months. The study was approved by the Medical Ethics Committee of the Erasmus MC, Rotterdam. Written informed consent was obtained from all participants.
Population for analysis
In total, 1,232 women were enrolled in the Focus cohort. The analysis was limited to singleton live-born infants (n= 1,215) whose mothers had participated in the third trimester measurements. Twin pregnancies (n=15) and pregnancies leading to perinatal death (n=2) were excluded from the analysis. Kidney ultrasounds were successfully performed in 95% (n=1,158) of these subjects. Of the initial 1,215 singleton live-born infants, 74% (n=901) and 70% (n=856) underwent postnatal assessment at the ages of 6 months and 24 months, respectively. Kidney ultrasounds were successfully performed in 83% (n=747) and 80% (n=683) of these infants, respectively. Missing values were mainly due to the infants crying or to the unavailability of equipment or radiographer. Infants who had undergone a postnatal kidney ultrasound at the ages of 6 months and 24 months did not differ from the postnatal non-responders in foetal and birth characteristics. There were no kidney or ureterovesical anomalies other then mild pyelectasis over 10 mm (n=3) in our study population. In total, analyses were based on more than 2,500 kidney measurements. The numbers of kidney growth measurements available for the analyses are shown in Table 1.
Ultrasound measurements
Gestational age was established by foetal ultrasound. Crownrump length was used for pregnancy dating until a gestational age of 12 weeks and 5 days (crown-rump length smaller than 65 mm), and biparietal diameter was used for pregnancy dating thereafter (gestational age from 12 weeks and 5 days onwards, biparietal diameter larger than 23 mm) [13].
Foetal left and right kidneys were measured in the third trimester of pregnancy with an ATL-Philips HDI 5000 instrument (Seattle, WA, USA) equipped with a 2.0-5.0 MHz curved array transducer. In a sagittal plane the maximum longitudinal kidney length was measured, with the calipers placed on the outer edges of the caudal and cranial sides. Antero-posterior (kidney width) and transverse (kidney depth) diameters were measured perpendicular to each other, outer to outer, in an axial plane [14]. Values of maximum bipolar kidney length, width and depth were obtained from both the left and right kidneys. Kidney width and depth were measured at the level of the kidney hilum [14,15]. The images were magnified to ensure optimal measurements [14]. Foetal growth characteristics (head circumference, abdomen circumference, femur length) were measures at the same visit, and foetal weight was estimated [13].
Postnatally, two-dimensional ultrasounds of the kidneys were performed in children at the ages of 6 months and 24 months. The examination was carried out in a quiet room with the child quietly awake in a supine position. This position was standardised to prevent differences resulting from position [14,15]. Mean length, width and depth were calculated as the average of three measurements and used for data analysis. Foetal and postnatal kidney volumes were both calculated in cubic centimetres using the equation of an ellipsoid: volume cm 3 ð Þ ¼ 0:523 Â mean length cm ð Þ Â mean width cm ð Þ Â mean depth cm ð Þ [15,16]. The infants' anthropometric parameters, including weight and length, were all measured at the ages of 1.5 months, 6 months and 24 months. Date of birth, birth weight and gender were obtained from midwife and hospital registers.
For the foetal ultrasound measurements, intra-and interobserver studies showed the intraclass correlation coefficient (ICC) to be higher than 0.98 and the corresponding coefficients of variation (CV) to be lower than 6%. Bland and Altman plots to test agreement of measurements demonstrated 95% limits of agreement to be within 10% difference from the mean of measurements, indicating good reproducibility [17]. For the postnatal ultrasound measurements, the intra-observer ICCs ranged from 0.93 (left and [18].
Data analysis
Differences in foetal and postnatal characteristics between boys and girls were assessed by t-tests and chi-square tests for independent samples. Differences between left and right kidney were tested with paired-samples t-tests. Data were analysed as recommended by Altman and Chitty [19] and Royston and Altman [20]. For the reference curves for kidney growth, post-conceptional age was plotted against kidney length, width, depth and volume. From the original data, measurements of more than two standard deviations (SDs) from the regression line, fitted on our data, were considered to be outliers (n=10) and were therefore removed. They were probably a result of measurement error or a data entry error. The best-fitting curves were determined by second-degree fractional polynomials [21]. We fitted the curves, using repeated measurement analyses and taking into account the dependency in the data by specifying a constant covariance between measurements of the same subject [20,22]. The best-fitting fractional polynomial curves were chosen by our comparing the deviances, by Akaike's information criterion, and by our visually checking the goodness of fit. Next, regression lines were fitted for the dependency of the residual SD on conceptional age [23]. Subsequently, we plotted the SD scores against conceptional age to assess the correctness of the model.
Finally, centiles were derived and the curves were plotted on the data. The median age of 2-year-old children visiting the research centre was 25 months (95% range 23.6-28.3 months). Since only 34 children had undergone measurement beyond the postnatal age of 28 months (160 weeks after conception), the results are presented up to the postnatal age of 28 months. Reference curves for kidney growth were constructed for a post-conceptional age from 30 weeks to 160 weeks, corresponding to a gestational age of 30 weeks and a postnatal age of 28 months.
All statistical analyses were performed with the Statistical Package for the Social Sciences, version 15.0, for Windows (SPSS Inc, Chicago, IL, USA) and the Statistical Analysis System (SAS) for Windows, version 9.1.3.
Results
The percentage of boys was 52% ( Table 2). The overall median age at the third trimester of pregnancy was 30 weeks of gestation (total range 27.1-35.1 weeks). The overall median age of the infants at their 6-month postnatal visit was 6.3 months (total range 5.1-11.0 months), and at their 24-month postnatal visit it was 25.1 months (total range 21.6-31.6 months). Head circumference at the third trimester and postnatal weight and length at the ages of 6 months and 24 months were larger in boys than in girls (all P values <0.001). No difference was found for gestational age at birth between boys and girls. In total, 15 children in our study group were born with a small size for gestational age [<−2 standard deviation scores (SDS)], 18 children had a low birth weight (<2,500 g) and 23 children were born before term (gestational age <37 weeks). Table 3 shows that in the third trimester of pregnancy and at the age of 6 months all kidney measurements were larger in boys than in girls. At the age of 24 months, these gender differences were only significant for left kidney structures and right kidney length. Both groups showed trends towards smaller left kidney measurements than right kidney measurements at all ages (Table 4).
Reference curves for individual measurements of kidney growth and fitted centiles are given in Fig. 1. Formulae for growth reference curves describing the mean with the corresponding standard deviation are given in Table 5. Standard deviation increased linearly with gestational age.
Reference values for kidney length, width, depth and volume are given in the Appendix (Tables 1S-4S).
Discussion
We constructed gender-specific reference growth curves for kidney length, width, depth and volume using measurements from a large population-based prospective cohort study of healthy children followed from foetal life until early childhood. We observed differences in kidney structures between left and right kidneys and boys and girls.
The major strength of our study is its prospective design from foetal life and the size of the population-based cohort. Our reference curves were based on more than 2,500 kidney measurements. To our knowledge, no previous studies that focused on kidney size in early life were based on such large numbers. All foetal ultrasounds were carried out by two sonographers, and 86% of all postnatal ultrasounds were performed by one trained sonographer [24]. A limitation could be that in all children participating in the Generation R measurements at the ages of 6 months and 24 months, kidney measurements were successfully performed in 83% and 80% of these infants, respectively. Missing values were mainly due to the infants crying or to the unavailability of equipment or radiographer. Our results would have been biased if the subjects' characteristics had differed between those included and those not included in the analyses. However, we observed no differences in growth and foetal kidney characteristics between the subjects that had undergone postnatal kidney measurement and those that had not. Another limitation could be that the study was performed in a healthy population-based cohort study. The selection towards a healthy population in our cohort might lead to a limited generalisability to preterm children or to children that were small for gestational age at birth. These numbers were too small to be assessed in further detail.
In the third trimester of pregnancy and at the age of 6 months all kidney measurements were larger in boys than in girls. At the age of 24 months, these gender differences were only significant for left kidney structures and right kidney length. Several studies of healthy neonates and adults have also shown that male subjects have larger kidneys than those of female subjects [25,26]. One explanation for this finding might be a growth-stimulating effect of androgens, or Y-chromosome-related genes. Another explanation could be that during foetal life testosterone levels are significantly higher in boys than in girls [27,28].
Previously published data have shown conflicting results concerning differences between left and right kidney size. Some studies found no difference [29,30], whereas others have suggested the left kidney to be larger [31,32]. Most consistent findings have been reported for kidney length, for which the left kidney seems to be longer than the right [7,25,26,31,33]. We found that the left kidney was longer in both boys and girls at the postnatal ages of 6 months and 24 months. In foetal life we found no significant difference between left and right kidney length.
Kidney growth is fastest during foetal life and early infancy, and the rate of increase gradually slows through the remainder of the first year of life and finally stabilises [34]. In our study numbers and curves for the 3rd centiles showed decreasing kidney volume at older ages, from 140 weeks and onwards. The decreasing numbers are due to wider ranges, because of the low number of children with visits around 140 weeks and onwards. To deal with non-linear kidney growth, some sonographic standards, including means and standard deviations, for kidney size in relation to age have been published [34][35][36]. A few other studies created linear or non-linear polynomial regression equations for kidney size during the first year of life [29,33]. One study created reference material for kidney size in healthy children beyond the neonatal period [7]. The authors only focused on kidney volume in relation to weight, height and body surface area and did not report data about prenatal kidney growth. To our knowledge, our study is the first to provide prospective longitudinal reference material for kidney size that covers the whole period from foetal life to infancy in a healthy population.
In conclusion, kidney size differed between boys and girls from the age of 30 weeks of pregnancy until 24 months of age. The left kidney tended to be smaller than the right kidney. At the age of 24 months, the differences in right kidney size between boys and girls were attenuated. Genderdifferentiated reference growth curves for both left and right kidneys were constructed for kidney length, width, depth and volume. These reference curves may be of importance in the identification of abnormal kidney size and growth, with possible subsequent clinical consequences. | 2017-06-19T19:08:12.704Z | 2010-02-01T00:00:00.000 | {
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118482906 | pes2o/s2orc | v3-fos-license | Search for z~6.96 Ly-alpha emitters with Magellan/IMACS in the COSMOS field
We report a search for z~6.96 Ly-alpha emitters (LAEs) using a Narrow-Band filter, centered at 9680 Angstroms, with the IMACS instrument on the Magellan telescope at Las Campanas Observatory. We obtain a sample of 6 Ly-alpha emitter candidates of luminosity ~10^42 erg/s in a total area of 465 square arcmin corresponding to a comoving volume of ~ 72000 Mpc^3. From this result, we derive a Ly-alpha luminosity function (LF) at z~6.96 and compare our sample with the only z~6.96 Ly-alpha emitter spectroscopically confirmed to date (Iye et al. 2006). We find no evolution between the z=5.7 and z~7 Ly-alpha luminosity functions, if a majority of our candidates are confirmed. Spectroscopic confirmation for this sample will enable more robust conclusions.
Introduction
Over the last decade, significant progress has been made in determining the processes of galaxy evolution using both ground-and space-based telescopes. Currently, the limits of the observable universe are at z ∼ 6, which corresponds to ∼ 90% the age of the universe. At z>6, we are approaching the NIR domain : the sky is brighter and it is more challenging to detect the faint high redshift sources. Beyond this boundary lie the first ultraviolet (UV)emitting sources, which ionized the majority of the hydrogen in the universe. Their detection will allow us to probe the era of reionization, after the "Dark Ages". Galaxies formed at high redshifts play a key role in understanding how and when the reionization of the universe took place. They also help constrain the physical mechanisms that drove the formation of the first stars and galaxies in the universe.
Starbursting galaxies can emit a large fraction of their ultraviolet luminosity in the Lyα line. Because Lyα photons are resonantly scattered in neutral hydrogen, even a small amount of dust can quench this emission. Hence, selecting objects with strong Lyα emission lines is expected to reveal a set of objects in the early phases of rapid star formation. These could either be young objects in their first burst of star formation or evolved galaxies undergoing a starburst due to a recent merger. Selecting galaxies with strong emission lines also allows us to probe the high-redshift Lyα luminosity function (LF). Once the Lyα LF is determined, it is then possible to infer the ionization fraction of the intergalactic medium (IGM) at different redshifts (Malhotra & Rhoads 2004;Stern et al. 2005;Furlanetto et al. 2006;Kashikawa et al. 2006;Ouchi et al. 2010). The presence of neutral hydrogen in the IGM can reduce the Lyα flux of galaxies, it is therefore clear that the Lyα LF is sensitive to the ionization fraction of the Universe. If we knew the intrinsic LF(z) of galaxies at each redshift, a deviation of the observed LF from this intrinsic distribution could be attributed to the attenuation by HI, and hence be used to infer the ionization fraction. In practice, the approach is to do proceed to a comparison of Lyα LF at different redshifts, since the LFs of Lyα emitters (LAEs) don't evolve much between z=3-5.7 (Cassata et al. 2010;Malhotra et al. 2011).
With ground-based telescopes, the detection of very distant objects requires observation of UV spectral signatures that have been redshifted into the visible spectrum. The longer the wavelength of the observed Lyα line, the earlier the epoch at which we observe the galaxy, and the closer to the "Dark Ages". Therefore, one way of searching for the most distant galaxies is to search for the redshifted Lyα emission at the longest possible wavelength. However, this search is complicated by the presence of OH emission lines within the terrestrial atmosphere, at an altitude of ≈ 80km. This strong line emission limits the sensitivity of ground-based telescopes at near-infrared wavelengths. Fortunately, there are spectral intervals with lower OH-background that allow for a fainter detection limit from the ground.
We use a custom-built filter, NB9680, centered at λ = 9680Å and with a width of 90Åto use one of the low-sky windows. Narrow-band imaging is the most successful method to detect strong Lyα emission lines of galaxies, since it relies on a specific redshift interval as well as a selected low-sky background window. Adapting the spectral width of this filter allows for maximum detection of light from the celestial objects at that spectral line, while minimizing the adverse influences of sky emission.
The first z > 6 LAEs was detected with the narrow-band (NB) technique at the 10m KeckII telescope (Hu et al. 2002). This galaxy was spectroscopically confirmed to be at z = 6.56. Over 1, 000 LAEs have been photometrically selected and spectroscopically identified in this way. Extensive observations have been done at redshifts 5.7 and 6.5, two spectral domains free of sky lines in the optical spectrum, and different conclusions on the Luminosity Function are discussed by several groups (Malhotra & Rhoads 2004Ouchi et al. 2008;Cassata et al. 2010;Ouchi et al. 2008;Ota et al. 2008;Iye et al. 2006;Kashikawa et al. 2006;Shimasaku et al. 2006). Several surveys have attempted to observe z∼7.7 Tilvi et al. 2010) and z∼8.8 (Cuby et al. 2007;Willis et al. 2008), with no spectroscopic confirmation yet. We present here a new NB imaging survey with the IMACS/Magellan telescope -targeting z = 6.96 LAEs. This paper first presents the data (Section 2.1) and the data reduction procedure (Section 2.2). We then describe the method of selection and contamination of low-redshift interlopers for high redshift LAEs in Section 3. We present the final sample of z = 6.96 LAEs and Lyα luminosity function at this redshift in Section 4.
Observations
The data were taken with the IMACS instrument (the Inamori-Magellan Area Camera & Spectrograph), installed at the 6.5m Magellan Baade telescope at Las Campanas Observatory. This instrument offers two cameras: f/2 and f/4, with different imaging scales and dispersions. We observed with the f/2 camera which delivers an image of 27.4' diameter field at a scale of 0.2 arcsec per pixel. To ensure background limited performance, each exposure lasted 15 min. After each exposure, the telescope was offset and a new exposure was taken. The offset values, for each exposure, was randomly chosen. We targeted one COSMOS field (RA=10:00:29 Dec=02:12:21) covered by the Canada France Hawaii Telescope Legacy Survey (CFHTLS) and the WIRCam Deep Survey (WIRDS : PIs Willott & Kneib) 3 . The total area of the survey is 572 square arcminutes. We observed using the NB filter centered at 9680Å (NB9680) on the 17th, 18th and 19th March 2009. During these observations, the conditions were good : we obtained 19, 23, and 19 15-minute exposures during these nights, with a seeing varying between 0.45" and 0.8".
We observed the field again on the 21st, 22nd and 23rd March 2010. Between the 2009 and 2010 observing season, the filter was replaced with a new one built to have identical bandpass specifications. There is no major difference between the two filters' realized bandpass, and we treat data from the two identically. The night conditions were not as good as those in 2009 : the seeing varied from 0.7" to 1.3" and we lost on average 2 hours per night due to instrument problems. We obtained 16, 19, and 18 15-minute exposures in three nights. The total exposure time for each epoch of data is given in Table 1.
Data reduction
To process this data, we use the package MSCRED/IRAF. At the beginning of each night, we take 10 bias frames and 10 dome flats, we process the bias frames by overscan subtraction, trimming, and stacking to produce a master bias frame for each night. The science frames are bias subtracted and flat field corrected in the standard way. But the main difficulty was the fringing correction. We see fringes produced by the interference of light reflected between parallel surfaces in an instrument. All object frames do not always share the same fringe pattern because flexure and variations in illumination geometry can change its amplitude or period even on short timescales. In order to correct each individual image for fringes, we produce a median image per night. An illumination frame is also generated for each science frame using a two-stage median smoothing algorithm, applied on each frame with a first stage filtering on a 16 pixel scale, the second on a 24 pixel scale. The net effect is comparable to a 384 pixel median filter corresponding to 76.8 arcsec.
We then produce a median of the illumination frame for each night and correct the science frames for the illumination pattern. We subtract this illumination frame from the median of the science frame to obtain a fringe pattern for each night. It is then essential the WIRDS (WIRcam Deep Survey) consortium, and the Canadian Astronomy Data Centre. This research was supported by a grant from the Agence Nationale de la Recherche ANR-07- to find the optimal multiplicative scaling factor to use in removing the fringe pattern from each science frame, as we have long exposure frames and the fringe pattern changes with time. Once the frames are corrected for the fringes, we perform a sky subtraction by subtracting a normalized median image of all science frames from each frame. Finally, once the sky-subtracted frames are reconstructed as single extension images, we stack them using the mscstack /IRAF task. We made stacks per night, per epoch and a combined epoch stack.
Astrometric calibration. We perform the astrometric calibration on the individual images before the final stacking. We set an initial WCS information in the header of the frames based on the COSMOS catalog 4 . We need then to adjust the WCS parameters to obtain a more precise alignment. For this purpose we use mscwcs to apply a first offset on the (RA,Dec) coordinates and we refine the calibration by checking the alignment with msccmatch task.We finally obtain an astrometry calibration for each individual images with a precision of rms∼ ±0.1 arcsec in both directions.
Photometric calibration. The photometric calibration of the CFHTLS data is based on the SDSS data for stars with 17<i'<21 and the Megacam-SDSS color transformation equations of Regnault et al. (2009).The precision obtained in u * , g ′ , r ′ , i ′ , z ′ is between 0.03 and 0.02 mag. As the NB9680 filter is included in the z ′ -band filter, we used the MAG AUTO magnitude from the z ′ band SExtractor catalog to calibrate our NB9680 filter. We select 1048 non-saturated stars in the magnitude range 16< z ′ <20 to perform the calibration. Considering the photometric error on the broad-band (BB) calibration, we obtain a photometric calibration precise to 0.1 magnitude in NB9680.
Completeness. We estimate the limiting magnitude for the different bands by adding 200 artificial star-like objects per bin of 0.1 magnitude in blank regions of the different stacked images. We then run SExtractor on the image with the same parameters as previously used for object detection. We repeat this procedure 40 times. The average count on 40 times of the number of artificial stars retrieved in each magnitude bin provides a direct measure of the completeness limit. We report the 50% completeness limit in Table 1. We use this result to determine the Luminosity Function presented in Section 4.
Broad Band Data
The CFHT-LS provides extremely deep optical imaging data for our observed field. For the purpose of this study, we made use of the T0006 release. The CFHT-LS data products are available from the CADC archive to CFHT users and take form of image stacks in the u * , g ′ , r ′ , i ′ , z ′ filters and of ancillary data such as weight maps, catalogs etc. The spectral curves of the filter u * , g ′ , r ′ , i ′ , z ′ are similar to the ones of SDSS filters. In addition to the optical data, we also have the WIRDS survey, providing J, H, Ks.These optical data have been calibrated photometrically using the SDSS photometry and the NIR data using 2MASS photometry . Considering internal and external photometric error sources, the uncertainty on the optical and the NIR data photometry is ∼0.05 mag and ∼0.02 mag, respectively. As the broad-band data and the narrow-band data do not have the same pixel scale, we resample the broad-band data using the software SWARP to obtain optical and NIR images with 0.2 arcsec/pixel. The alignment in pixels is then verified using IRAF geomap/geotran tasks. Our complete set of data is therefore scaled at 0.2 arcsec/pixel and covers an area of 27.4' diameter. A summary of the observational data used in this paper is provided in Table 1. Figure 1 shows the transmission curves of the filters corresponding to the multi-band data used in this study.
Catalog generation and Selection
We generate the catalogs using the software SExtractor (Bertin & Arnouts 1996). We use the dual-image mode : the first image, for the detection, is settled as the combined NB9680 image, the second image, for measurement, corresponds to the resampled images from the optical and NIR bands. We choose to detect objects in 7 pixels above a threshold of 1.5σ, corresponding to a NB9680 ∼ 25.8. The aperture used for the photometry is 1 arcsec diameter.
As the IMACS instrument is composed of eight chips, we see an increase of the noise in the inter-chip regions. The sky noise is higher by a factor 2.5 in the interchip regions. We choose therefore to eliminate these regions from the area of the survey. From the total area covered by our survey, 572 square arcminutes, we obtain an effective area to search for z∼6.96 LAE candidates of 465 square arcminutes. b The filter used during 2010 observations has a better transmitted wavefront than the filter used in 2009. This explains the similar limiting magnitude between the two epochs although the observing conditions were different.
c By combining the two epochs of data, we should have expected a sensitivity increase of 0.37 mag. But the limit has increased by only 0.2 mag. This could be due to a systematic noise component like the fringes. Criterion#1 : Since we have two epochs of data, we define NB9680 selection criteria on individual epochs and on the combined images. We select objects with a 3σ detection in both of the individual epoch images and a 5σ detection in the combined NB9680 image. 59% (=16150 objects) of the objects present in the initial catalog pass this criterion. This eliminates variable sources from the catalog.
Criterion#2 : Due to the nearly complete absorption of the flux shortward of Lyα by the intergalactic Hydrogen, we should observe flux discontinuity at rest wavelength of 1216Å, and observed wavelength of 9680Å.We are therefore searching for objects which are not detectable in optical (u * , g ′ , r ′ , i ′ ) bands. A possible method is therefore to select objects with less than a 3σ detection in filters blueward of the expected Lyα emission : u * , g ′ , r ′ , i ′ . We use a χ 2 image generated by combining g ′ , r ′ and i ′ CFHT data to obtain deep photometry of candidates in the combined optical bands. 3% of remaining candidates are accepted on this basis.
Criterion#3 : Following the successful method of Rhoads & Malhotra (2001) used for the search of z=5.7 LAEs in the LALA field, we require that more than 50% of the NB9680 flux comes from an emission line for object selection, which can be translated as mag N B9680 − mag z ′ < −0.75mag. This criterion is indicated in Figure 2, showing the z ′ −NB9680 color versus NB9680 magnitude diagram. 88% of the objects selected after the criterion#2, pass this color restriction.
Criterion#4 : To avoid selecting extremely red objects (Cimatti et al. 2002), we choose to set a different criterion : mag N B9680 − mag J < 0. 98% of the objects selected after the criterion#3 pass this color criterion.
Criterion#5 : After applying carefully all the criteria presented above, we inspect each candidate visually. 33% of the objects inspected carefully are selected as serious candidates. the rest are rejected because they are near chip boundaries, or they are defects, etc.
The equations summarizing this selection are presented in the Table 2.
Contaminants
COSMOS photometric redshift catalog gives us a first insight on the low redshift emitters contaminating our high redshift candidates sample.
Transient objects. Our strategy of observing the same field during two different years, and our requirement that the eligible candidates have to be detected in each epoch data within 3σ level, allow us to rule out the contamination of our sample by transient objects such as supernovae, which would have appear in only one epoch of data. This strategy likewise prevents contamination by slow-moving solar system objects.
L-T dwarfs stars. Following the method described in Hibon et al. (2010), we determine the expected number of L and T-dwarfs present in this survey. From the Figure 9 of Tinney et al. (2003), representing the relation between the absolute magnitude and the spectral types of late-type dwarf galaxies, we find that we could detect L-dwarfs up to a distance of 871 to 3630pc and T-dwarfs up to a distance of 400 to 1260pc, depending on spectral type, from the coolest to the warmest. This field is located at high galactic latitude. Our sensitivity to L-and T-dwarfs is then extended beyond the scale height of the Galactic disk. However, the scale height applicable to L-, T-dwarfs is truncated at 350pc (Ryan et al. 2005). We estimate therefore a sample volume of ∼ 692pc 3 . Considering a volume density of L-and T-dwarfs of a few 10 −3 pc −3 , we expect less than one L-, T-dwarf in our field. In addition, L-,T-dwarfs have NB9680-J>0 so they would have fail Criterion#4 of our selection.
Lower redshift galaxies with strong emission lines should be actively star forming galaxies with blue continuum emission. Looking for such a continuum allows us to identify these lower-redshift line emitters, unless their equivalent qidth is very large. Foreground emitters. We estimate the minimum observed equivalent width a foreground line emitter would require to be selected with our criteria using the formula from Rhoads & Malhotra (2001): with f N B and f BB the flux in NB9680 and g ′ band respectively, ∆λ N B the width of the NB9680 filter, σ N B and σ BB , the flux uncertainties in NB9680 and g ′ band respectively. We obtain therefore an EW min ∼ 1545Å in observer frame. 1-Hα at z∼0.47 We first estimate the fraction of Hα emitters with EW obs ≥ 1545Å from Figure 2 of Straughn et al. (2009). Fewer than 1.2% of Hα emitters at z∼0.27 from Straughn et al. (2009) sample would have such an equivalent width. We then evaluate the number of Hα emitters at z∼0.47 present in our survey using the luminosity function from Figure 14 of Tresse et al. (2002). We find that ∼ 12 Hα emitters can be present in our NB9680 combined image. An upper limit of the number of Hα emitters at z∼0.47 passing our criteria is then 0.15. Considering the Hα luminosity function of Geach et al. (2010), we find that ∼ 52 Hα emitters can be present in our survey. An upper limit of the number of Hα emitters based on Geach et al. (2010) is therefore 0.62. Thus Hα emitters are not serious contaminants.
2-[O iii] at z∼0.95
We apply the same method for the Hα emitters to estimate the number of [O iii] emitters in our survey and contaminating our selection. From Figure 2 of Straughn et al. (2009), we estimate the fraction of [O iii] emitters at EW min to be one out of 136. We obtain therefore an upper limit of 0.74%. Kakazu et al. (2007)[O iii] emitter sample is covering a wide range of rest-frame equivalent width up to EW rest ∼ 1000Å. As the rest-frame EW of the [O iii] emitters that could contaminate our high redshift sample is around 792.1Å we are therefore able to use their sample to estimate the possible number of [O iii] emitters passing through our selection criteria. From the luminosity function Figure 13 of Kakazu et al. (2007) Straughn et al. (2009), we obtain an upper limit of 3.3%. Using the luminosity function presented in Figure 5 of Rigopoulou et al. (2005), 45 [O ii] emitters at z∼1.6 can be detected in our survey. However a maximum of 1.5 of these objects can pass through our selection criteria.
False Detections
In order to estimate the number of false detections that could pass our selection criteria, we create an inverse NB9680 image by multiplying the combined NB9680 image by -1. We then apply the same selection method and criteria and we did not find any candidates.
Comparison with COSMOS redshifts
We verify our sample of candidates by cross-correlating this catalog with the photometric redshifts catalog from the COSMOS 6 field. This catalog covers a redshift range up to z∼5.2 and a magnitude range from z ′ ∼18 to z ′ ∼25. None of the catalog's objects matches our candidate sample. Since most of the case against foreground emitters is discussed in the previous sections and a consistency verification performed, we can therefore conclude that it is very unlikely that our z∼6.96 LAE candidate sample is contaminated by low-redshift interlopers. A more detailed study of the foreground emitters (Hα at ∼0.47, [O iii] at ∼ 0.95 and [O ii] at ∼ 1.6) will be presented in a forthcoming paper.
Final sample
Our final sample contains 6 z∼6.96 LAE candidates over the range of NB9680=24.1-24.4 and SNR (NB9680)=5.6-7.3, as described in Table 3. In order to derive a luminosity function independent of the photometry aperture, we compare the automatic aperture magnitude, the 1" aperture magnitude and the isophotal magnitude for unsaturated objects. We then correct the 1" aperture magnitude from the difference found between the different magnitude types. The aperture corrected magnitudes are presented in Table 3. Because our photometric calibration was based on matching SExtractor's automatic aperture magnitudes to the z ′ filter photometric catalog from the COSMOS porject, this procedure should provide an unbiased estimate of the total (aperture-corrected) AB narrowband magnitudes for our objects. This approach also provides some robustness to crowding, thanks to the relatively small (1") apertures used for color measurements described in Section 3.1. We show our final sample in the NB9680 − Ks vs z ′ − NB9680 diagram represented in Figure 3 with the different possible contaminants described above. The colors for L-and T-Dwarfs have been computed using the L-and T-Dwarfs library from Dahn et al. (2002). We used GALAXEV Bruzual & Charlot (2003) to model the color tracks of early and dusty galaxies using the Padova 1994 evolutionary tracks with a Salpeter IMF.
We also report in Table 3 the lower limits of the rest-frame equivalent widths (EW ) derived from the photometric data following Malhotra & Rhoads (2002), defined as: where f N B9680 is the observed flux in the narrow-band combined image, f Z is the observed flux in the z ′ broad-band image, ∆λ N B9680 and ∆λ Z are the width of the NB9680 filter (90Å) and the z ′ band filter (928Å) respectively. Our first object, LAE#1 in Table 3, present a detection in Ks band (see Figure 4). This could be due to a very red continuum slope. However, looking at the Spitzer/IRAC data in 3.5µm and 4.8µm, none of the LAE candidates are detected. Alternatively, it could be due to the presence of another line, such as MgII. Looking at the UKIRT/WFCAM2 6 data in J band (J 50% = 25.6, AB, 5σ) for all our high redshift candidates, LAE#1 is detected in this data with a magnitude of 24.8 (AB) and a SNR(J)∼ 10. From the HST/NICMOS 6 data available in H band (H 50% = 26.7, AB, 5σ), LAE#3 is detected with a magnitude of 26.5 and a SNR(H)∼4. These detections in different broad bands confirm the reliability on these candidates. Although the other candidates have a strong single band detection, the tests realized in Paragraph 3.2.1 confirm that they are emission line objects. The remaining candidates are based on a ≥ 5σ significant single-band detection in the narrow band filter. For Gaussian statistics, the false positive probability at ≥ 5σ is 3 × 10 −7 , while our survey area contains ∼ 0.5 × 10 7 independent resolution elements (based on 0.65 ′′ seeing). The number of noise spikes entering the sample should thus be ∼ 1.5, comparable to the expected number of foreground Dahn et al. (2002). The green track represents early-type galaxies and the blue track dusty galaxies generated with Bruzual & Charlot (2003).
emitters. Non-Gaussian noise could increase this number, but the absence of detections in the negative-image test (see Section 3.2.1) support the conclusion that 5σ noise spikes are not a major contaminant of our sample.
Three out of six of our LAE photometric candidates are not detected in the z ′ band and we therefore use the detection limit in this band, deriving in turn lower EW limits.
Discussion
Variance. Two sources of variance can be involved in a such high redshift study : the Poisson variance and the fluctuations in the large scale distribution of the galaxies, also called the cosmic variance. We used the on-line calculator 7 by Trenti & Stiavelli (2008) to estimate the cosmic variance. This calculator requires numerous parameters such as the area of the survey, the mean redshift, the reshift interval, but also the completeness value and several cosmological parameters. We obtained, for our sample, a value of 54% for the cosmic variance. The Poisson noise is estimated to 58%. The 54% uncertainty from the cosmic variance and the 58% from Poisson statistics are comparable and we therefore consider both in our error estimation.
Luminosity Function. Our NB9680 filter being quite narrow (FWHM∼90Å), we assume that the narrow-band flux is entirely coming from the Lyα line. We fit to the Lyα luminosity function of this z∼6.96 LAE sample, a Schechter function, Φ(L), given by in order to compare with previous high redshift works Hibon et al. 2010;Tilvi et al. 2010;Ouchi et al. 2008;Ota et al. 2008;Kashikawa et al. 2006;Malhotra & Rhoads 2004). Considering the small number of candidates in our sample, we choose to fit two out of three of the Schechter function parameters. We set the faint end slope of the luminosity, α, to α = −1.5, and determine Φ * and L * by χ 2 minimization. We decide to obtain a best-fit Schechter functions for the z∼7 cumulative luminosity function, which has been derived by considering only our photometric candidates.
In Figure 5, we present the Lyα Luminosity Function not corrected for detection incompleteness as a black solid line. We then use the completeness result of Section 2.2.1 to correct the luminosity of our objects, and find a new Schechter fit, presented as a red solid line in Figure 5. This is the z 7 Lyα Luminosity Function corrected for incompleteness. Ouchi et al. (2010) and the z=5.7 Lyα LF from Ouchi et al. (2008).
Sample Incompleteness. We create a grid pattern of 15000 objects on a mock image, andrun SExtractor for different photometric apertures in double image mode, using the g ′ -band image as the measurement image. We remark that by increasing the photometric aperture size, the number of objects matching the optical criterion (Criterion #2 in Table 2) decreases. For an aperture of 5 pixels, we recover 75%±0.65%, for 10 pixels 63%±0.6% and for 20 pixels 46%±0.5%. We choose a photometric aperture of 5 pixels for the objects catalogs we used for the high redshift LAEs selection. We know then that we could miss 25% of the objects due to the photometric aperture size we choose. This corresponds to the possibility that we missed ∼1.5 objects in our high redshift sample. Assuming one more object in our sample, our conclusion about the best-fit LF will not change.
Interpretation. The previous studies presenting z∼7 Lyα emitters (Ota et al. 2008 has lead to the first spectroscopic confirmed z∼7 LAE, called IOK1. Their survey covers an area of 876 square arcmin with the filter NB973 (∆λ = 200Å, λ c = 9755Å) and reaches a 50% completeness of NB973=25.6 (AB, 5σ) (equivalent to a flux of 1.36e −17 erg s −1 , cm −2 ) in the SXDS (Subaru/XMM-Newton Deep Survey) and NB973=25.3 (AB, 5σ) (equivalent to a flux of 1.8e −17 erg s −1 , cm −2 ) in the SDF (Subaru Deep Field). IOK-1 has a flux of 2 × 10 −17 erg s −1 cm −2 . From the Table 1 of Ota et al. (2008), we are able to obtain a lower limit for the rest-frame equivalent width of IOK1, EW rest ∼ 49Å, using Equation 2. This rest-frame equivalent width is in agreement with the rest-frame equivalent limit lower limit we found for our candidate sample and presented in Table 3. Ota et al. (2010) find four new photometric candidates in a survey covering the Subaru/XMM Newton Deep Survey Field with Suprime-Cam and reaching a depth limit of NB973=25.4, corresponding to 72% completeness. These candidates are represented by pentagons in the Figure 5.
We show in Figure 5 the cumulative z∼7 LAEs LF obtained after correcting our points for the aperture and the detection completeness. This completeness correction has been applied by number weighting according to the NB9860 magnitude. The best-fit parameters do not vary significantly before and after correcting from the completeness, as seen in Figure 5 between the black solid line and the red solid line (without and with the completeness correction, respectively). By considering only our photometric candidate sample, we do not observe any strong L * or Φ * evolution between z=5.7 and z∼7, and therefore contradict a possible L * evolution between z=6.5 and z∼7. If none of our candidates is a real z∼7 LAE, we can then put an upper limit on the z∼7 Lyα LF, which will help constrain the neutral fraction of the IGM.
Conclusions
We observed 465 arcmin 2 from the COSMOS field using the narrow-band imaging technique on Magellan/IMACS with the NB9680 filter, in order to target the Lyα line at z∼6.96. We obtained a comoving volume of ∼ 72000Mpc 3 . After applying our selection criteria and verifying that our selection was not contaminated by low-redshift emitters and transient objects, we obtain a sample of six z∼6.96 LAEs. From this photometric sample, we are able to infer a possible z∼6.96 Lyα Luminosity Function. We find no evolution in luminosity function from z=6.5 to z∼6.96, if a majority of our sources are confirmed. It is now crucial to obtain spectroscopic follow-up observations to reveal the real nature of these objects and establish a firm conclusion on the z∼6.96 Lyα Luminosity Function. | 2011-08-31T20:00:05.000Z | 2011-08-31T00:00:00.000 | {
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319067 | pes2o/s2orc | v3-fos-license | Cause-Specific Excess Mortality in Siblings of Patients Co-Infected with HIV and Hepatitis C Virus
Background Co-infection with hepatitis C in HIV-infected individuals is associated with 3- to 4-fold higher mortality among these patients' siblings, compared with siblings of mono-infected HIV-patients or population controls. This indicates that risk factors shared by family members partially account for the excess mortality of HIV/HCV-co-infected patients. We aimed to explore the causes of death contributing to the excess sibling mortality. Methodology and Principal Findings We retrieved causes of death from the Danish National Registry of Deaths and estimated cause-specific excess mortality rates (EMR) for siblings of HIV/HCV-co-infected individuals (n = 436) and siblings of HIV mono-infected individuals (n = 1837) compared with siblings of population controls (n = 281,221). Siblings of HIV/HCV-co-infected individuals had an all-cause EMR of 3.03 (95% CI, 1.56–4.50) per 1,000 person-years, compared with siblings of matched population controls. Substance abuse-related deaths contributed most to the elevated mortality among siblings [EMR = 2.25 (1.09–3.40)] followed by unnatural deaths [EMR = 0.67 (−0.05–1.39)]. No siblings of HIV/HCV co-infected patients had a liver-related diagnosis as underlying cause of death. Siblings of HIV-mono-infected individuals had an all-cause EMR of 0.60 (0.16–1.05) compared with siblings of controls. This modest excess mortality was due to deaths from an unknown cause [EMR = 0.28 (0.07–0.48)], deaths from substance abuse [EMR = 0.19 (−0.04–0.43)], and unnatural deaths [EMR = 0.18 (−0.06–0.42)]. Conclusions HCV co-infection among HIV-infected patients was a strong marker for family-related mortality due to substance abuse and other unnatural causes. To reduce morbidity and mortality in HIV/HCV-co-infected patients, the advances in antiviral treatment of HCV should be accompanied by continued focus on interventions targeted at substance abuse-related risk factors.
INTRODUCTION
Co-infection with HCV is a marker for poor prognosis in HIVinfected individuals. It is unclear whether the prognostic impact is caused directly by the viral disease, or whether it is an epiphenomenon of the infection [1][2][3]. It has been suggested that HCV exerts a direct pathogenic effect by compromising the immune restoration induced by highly active antiretroviral therapy (HAART) [1,4,5], but it has been difficult to separate the direct pathogenic effects of HCV from indirect effects of associated risk factors. In most settings, HCV infection is closely linked with intravenous drug use (IDU), which is associated with risk factors such as mental health illnesses, depression and alcohol abuse [6,7].
In a recent attempt to uncover the impact of the indirect effects, we examined the mortality in siblings of HIV/HCV-co-infected individuals. We found three-to four-fold higher mortality among siblings of HCV/HIV-co-infected patients, compared to siblings of HIV-mono-infected individuals and siblings of population controls [8]. This indicates that risk factors shared by family members partially account for the excess mortality of HIV/HCV-coinfected patients. Given the high parenteral transmissibility of HCV, we hypothesized that HCV infection is a marker of highrisk lifestyle associated with intravenous drug use (IDU). However, the excess mortality could also be mediated through shared susceptibility to some infectious pathogens or increased prevalence of HCV infection among siblings of HIV/HCV-co-infected individuals.
To explore the excess mortality shared among family members, we used a nationwide population-based Danish cohort to compare cause-specific mortality rates, including mortality associated with drug and alcohol abuse, among three groups: siblings of HIV/ HCV-co-infected individuals, siblings of HIV-mono-infected individuals, and siblings of general population controls.
METHODS
The study setting and methods used to identify siblings have been described in detail previously [8]. Briefly, we used the populationbased Danish HIV Cohort Study (DHCS) [9] to identify all Danish HIV mono-infected and HIV/HCV co-infected patients treated in Danish HIV Centres since 1995. HIV treatment in Denmark is restricted to eight specialised centres, and the Danish health care system provides free tax-supported medical care, including antiretroviral treatment for HIV.
We identified up to 99 population controls per patient, matched by gender, age and residency from the Danish Civil Registration System (CRS). CRS is an electronic database established in 1968, which tracks vital status, migration, residency and kinship for all Danish inhabitants [10]. CRS records contain a unique 10-digit civil registration number that allows accurate record linkage with other registries. For both HIV-infected patients and population controls, we identified all full siblings born after 1951 (as registration of kinship was incomplete before this date) and obtained their dates of death or emigration from CRS. Based on the HIV patients' HCV serostatus or tests for HCV-RNA, siblings of HIV-infected individuals were classified as siblings of HIVmono-infected individuals or siblings of HIV/HCV-co-infected individuals [8]. A flowchart of eligible individuals is displayed in Figure 1.
We used the National Registry of Deaths (NRD) [11] We followed the siblings from age 20 until death, emigration, or their 50 th birthday, whichever came first. All subjects were censored after 31 December 2001, the latest date when electronically coded causes of death were available in the NRD. In the current study, the number of siblings, person years of follow-up, and number of deaths were slightly smaller than in our earlier study of mortality among siblings of HCV/HIV-co-infected patients, in which follow up was censored on 1 May 2005 [8]. Figure 1 displays a summary of the study design for selection of siblings.
We classified causes of death into five groups, based on death certificate information: 1. Alcohol or drug abuse-related; 2. HIV (excluding those related to substance abuse); 3. Natural causes (excluding those related to HIV or substance abuse); 4. Unnatural causes (including deaths due to accidents, suicide, or homicide and excluding deaths related to substance abuse); 5. Unknown causes.
To capture deaths related to abuse of alcohol and drugs (including some prescription drugs), we used diagnoses for both the underlying (main) and contributory causes of death. The causes of death defined as substance-abuse related were: a) mental disorders associated with alcohol or drug dependence (ICD-8 [accidental, suicidal or undermined whether accidentally or purposely inflicted] (ICD-8: E8530, E8540, or, E950 or E980 in combination with N9650, N9780, or N9790. ICD-10: X41, X42, or X60-X64 in combination with T40, Y11, Y12) [12]. For all other causes (group 2-5), only the underlying cause of death was used. In the Danish versions of ICD-8 and ICD-10, deaths with HIV as the underlying cause are coded as 0797 and B20-B24, respectively.
Among individuals with HIV as the underlying cause of death, the route of infection was identified from DHCS records. Deaths in those who were intravenous drug users were reclassified as a IDU-related death in a supplementary analysis, as proposed by Schulz-Schaeffer et al. [13]. We also performed a supplementary analysis of deaths with HCV infection or other liver diseases as the underlying cause of death.
For the three groups of siblings, we first computed total and cause-specific mortality rates (MR) by person-year analyses and used exact poisson confidence intervals. We then calculated total and cause-specific absolute excess mortality rates (EMR), comparing siblings of HIV-mono-infected and HIV/HCV-co-infected patients with siblings of population controls (the reference group). We calculated absolute EMR as the risk difference between two groups as described by Rothman [14] i.e. by subtracting the MR of siblings of population controls from the MR for siblings of each of the other two groups. We calculated 95% confidence intervals for the EMRs by using the standard error of the rate difference as described by Rothman [14,15]. We used Stata software, version 9.2 (StataCorp, College Station, TX, USA) for statistical analyses. The study was approved by the Danish Data Protection Agency.
Excess mortality rates of siblings
Total and cause-specific MR and EMR with 95% confidence intervals for all groups of siblings are displayed in Table 1. The allcause EMR for siblings of HIV/HCV-co-infected individuals was 3.03 per 1,000 PYR, compared with siblings of population controls. This excess mortality was mainly caused by substance abuse-related deaths (EMR = 2.25 per 1000 PYR) and to a lesser extent by unnatural deaths (EMR = 0.67 per 1,000 PYR) and by deaths with HIV as the underlying cause (EMR = 0.39 per 1,000 PYR).
Siblings of HIV-mono-infected individuals had an all-cause EMR of 0.60 per 1,000 PYR compared with siblings of population controls. This small excess mortality was due to deaths from an unknown cause (EMR = 0.28 per 1,000 PYR), deaths from substance abuse (EMR = 0.19 per 1,000 PYR), and unnatural deaths (EMR = 0.18 per 1,000 PYR).
Substance abuse in HIV-related deaths
Three siblings of HIV/HCV-co-infected individuals and 137 control siblings had HIV as their underlying cause of death. Of these, two siblings of HIV/HCV-co-infected individuals and four control siblings were registered in DHCS with IDU as the route of HIV transmission. When these deaths were counted as substance abuse-related deaths, the cause-specific mortality rate for siblings of HIV/HCV-co-infected individuals increased from 2.50 to 2.78 per 1,000 PYR, and the EMR increased from 2.25 to 2.52 per 1,000 PYR. The substance abuse-related mortality rate for controls changed slightly from 0.25 to 0.26 per 1,000 PYR.
HCV-related deaths
No siblings of HIV/HCV-co-infected patients had acute or chronic HCV infection or any other liver-related diagnosis as the underlying cause of death.
DISCUSSION
Our nationwide study showed that the increased mortality in siblings of HIV/HCV-co-infected patients is mainly caused by alcohol and drug abuse-related deaths. The study did not provide evidence that hepatitis C infection or increased inherited susceptibility to other infectious diseases contribute to the excess mortality in siblings of HIV/HCV co-infected patients.
These results lend credence to the hypothesis that excess mortality in HIV/HCV-co-infected patients, compared to HIV mono-infected patients, partly stems from factors other than the HCV disease itself, and a large part of the increased mortality in HIV/HCV co-infected patients -compared to HIV mono-infected patients -may be caused by similar family-associated and lifestylerelated factors. In the co-infected patients, substance abuse and associated risk behaviors may directly affect mortality. Indirectly, mortality in HIV/HCV-co-infected patients may be elevated by suboptimal health-seeking behavior, poor adherence to medical treatment, drug-or alcohol-related comorbidity, or health care providers' inattention to these patients. In addition, clinicians may choose to postpone antiretroviral therapy in HIV/HCV-coinfected patients due to concerns about behavioral factors or liver toxicity, despite evidence that early treatment initiation is beneficial for these patients [16].
The mechanisms underlying clustering of substance abuserelated deaths in families of HIV/HCV-co-infected individuals require better understanding. A likely explanation is that the common childhood and environment among siblings lead to a similar high risk of substance abuse. However, adoption and twin studies have shown that in addition to family environment, there is a strong genetic contribution to a familial predisposition for substance abuse [17,18]. In any case, familial clustering of the detrimental effects of substance abuse underscores the need for adequate control groups, and/or extensive control of confounding when effects of HCV are investigated in epidemiological settings.
Further evaluation is also needed to determine whether these family-related risk factors have the same influence on prognosis in HCV-mono-infected patients, because HIV/HCV-co-infected individuals may represent a more vulnerable subgroup than HCV-mono-infected individuals. In line with our results, a large population-based study has recently demonstrated that young people with HCV infection have higher mortality from continued drug uses than from the HCV infection [19].
Our study had a number of strengths and limitations. Use of longitudinal population-based Danish registries permitted complete long-term follow-up with minimal selection bias, while the matched selection of siblings assured comparability of groups in terms of birth year and residency [8]. Because it has been shown that relying exclusively on underlying cause of death leads to underestimation of alcohol-and drug-related mortality, we used both underlying and contributory causes [12,13] to specify these causes of death. Still, death certificate data are not entirely accurate [20], and potential differential misclassification could lead to underestimation of the contribution of substance abuse to the category of unnatural deaths [13,21]. We could not assess mode of HIV infection in all HIV-related deaths among siblings of population controls. However, the resulting bias is probably negligible, since both the MR of HIV-related deaths and the proportion of HIV infections acquired by IDU among those with HIV-related deaths were small in this group. Finally, it is important to note that the results of this study may not be generalizable to settings where HIV/HCV-co-infected patients do not have IDU as the main route of transmission, for example Africa [22].
In conclusion, HCV co-infection among HIV-infected patients seemed to be a strong marker for family-related mortality due to substance abuse. In order to reduce morbidity and mortality in HIV/HCV co-infected patients, advances in antiviral treatment of HCV should be accompanied by interventions targeted at substance abuse and associated risk factors.
Author Contributions
Conceived and designed the experiments: HS AH NL JG NO. Analyzed the data: AH NL. Wrote the paper: AH. Other: Interpretation of data: NO | 2014-10-01T00:00:00.000Z | 2007-08-15T00:00:00.000 | {
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14920018 | pes2o/s2orc | v3-fos-license | Aberrant Expression of TIMP-2 and PBEF Genes in the Placentae of Cloned Mice Due to Epigenetic Reprogramming Error
Cloned mice derived from somatic or ES cells show placental overgrowth (placentomegaly) at term. We had previously analyzed cloned and normal mouse placentae by using two-dimensional gel electrophoresis and mass spectrometry to identify differential protein expression patterns. Cloned placentae showed upregulation of tissue inhibitor of metalloproteinase-2 (TIMP-2), which is involved in extracellular matrix degradation and tissue remodeling, and downregulation of pre-B cell colony enhancing factor 1 (PBEF), which inhibits apoptosis and induces spontaneous labor. Here, we used Western blotting to further analyze the protein expression levels of TIMP-2 and PBEF in cloned placentae derived from cumulus cells, TSA-treated cumulus cells, intracytoplasmic sperm injection (ICSI), and natural mating (NM control). Cloned and TSA-treated cloned placentae had higher expression levels of TIMP-2 compared with NM control and ICSI-derived placentae, and there was a positive association between TIMP-2 expression and the placental weight of cloned mouse concepti. Conversely, PBEF protein expression was significantly lower in cloned and ICSI placentae compared to NM controls. To examine whether the observed differences were due to abnormal gene expression caused by faulty epigenetic reprogramming in clones, we investigated DNA methylation and histone modification in the promoter regions of the genes encoding TIMP-2 and PBEF. Sodium bisulfite sequencing did not reveal any difference in DNA methylation between cloned and NM control placentae. However, ChIP assays revealed that the level of H3-K9/K14 acetylation at the TIMP-2 locus was higher in cloned placentae than in NM controls, whereas acetylation of the PBEF promoter was lower in cloned and ICSI placenta versus NM controls. These results suggest that cloned placentae appear to suffer from failure of histone modification-based reprogramming in these (and potentially other) developmentally important genes, leading to aberrant expression of their protein products. These changes are likely to be involved in generating the abnormalities seen in cloned mouse placentae, including enlargement and/or a lack of proper placental function.
Introduction
The placenta connects the developing fetus to the mother's body, and its growth is an essential characteristic of mammalian pregnancy. Successful placental development is important for the survival of the growing conceptus, as the placenta specifically nurtures the fetus, protects it from harmful waste, executes the exchange of respiratory gases and a multitude of nutrients, and acts as an immunological barrier [1]. Therefore, the study of placentation is considered key to understanding the pregnancy failures of cloned animals, the pathogenesis of some congenital diseases, and the transplacental transmission of teratogenic microbial agents [2].
Many of the fetal losses and phenotypic abnormalities observed among cloned mammals are associated with placental abnormalities [2,3,4]. For example, cloned mouse fetuses have been shown to develop to term and yield placental overgrowth (placentomegaly) regardless of the sex or source of the donor nuclei [5,6,7,8]. This placentomegaly has been associated with expanded spongiotrophoblast layers, increased glycogen cell numbers, and enlarged trophoblastic cells [9]. Trophoblast cells develop into some extra-embryonic membranes and much of the placenta [10], so perfect reprogramming of transferred somatic nuclei (which is needed to properly establish the trophoblast cell lineage) is required for the correct development of reconstructed embryos. It has been speculated that placentomegaly in cloned mice could reflect the acquisition of epigenetic abnormalities, at least partly via inadequate nuclear reprogramming. Consistent with this hypothesis, errors in the epigenetic reprogramming of the somatic cell genome have been associated with the expressional dysregulation of developmentally imprinted genes in cloned embryos, fetuses and placentae, and abnormalities in the resulting cloned animals [11,12,13,14,15]. In this context, numerous researchers have observed abnormal gene expression patterns following somatic cell nuclear transfer [16,17,18,19]. However, the existence, importance, and regulation of epigenetic modification-related abnormalities in gene expression have not yet been fully elucidated in the context of placentation.
Chromatin-modification-related changes in gene expression [20], called "epigenetic changes," support: various processes that require accurate gene activation/inactivation during development; the assembly of histones and histone variants into nucleosomes; and the remodeling of chromatin-associated proteins (e.g., linker histones, polycomb proteins, nuclear scaffold proteins, and transcription factors) [21]. Changes in chromatin configuration are critical to normal development, and are primarily determined by genomic DNA methylation and the histone acetylation/methylation status. Successful placental development depends on precisely regulated gene expression and may be negatively influenced by the abnormal expression of developmentally significant genes [22]: aberrant gene expression in the placenta (i.e., that arising via epigenetic error) may alter the placenta and perhaps even the conceptus.
We had previously used two-dimensional gel electrophoresis and mass spectrometry to conduct a global proteomic analysis of cloned and normal mouse placentae, in an effort to identify differential protein expression patterns [17,18,19]. TIMP-2 and PBEF which was related with pregnancy and placenta development, are abnormally expressed in cloned bovine and mouse placentae [17,18,19] and differentially expressed during pregnancy [23,24]. TIMP-2 has been associated with trophoblastic invasion and extracellular matrix remodeling during pregnancy [25], while PBEF appears to function at the proximal end of the labor initiation pathway [26,27,28]. The development of mouse cloned embryos could be improved by trichostatin A (TSA, histone deacetylase inhibitor) treatment which could adjust pre-existing epigenetic state of donor cells or reconstructed embryos [29]. To examine the potential contribution of epigenetic modification to this gene expression process, we herein analyzed the protein and mRNA levels of TIMP-2 and PBEF in cloned, TSA-treated cloned, intracytoplasmic sperm injection (ICSI)-derived and normal mating-derived (NM control) placentae.
Following their confirmation as differentially expressed proteins, we examined the DNA methylation and histone acetylation patterns in their gene promoter regions to determine whether their expression changes were associated with differences in the epigenetic programming of cloned versus normal mouse placentae. The novel identification of these genes as being differentially expressed in the placentae of cloned mice and our assessment of their epigenetic regulation provide important new insights into the molecular mechanisms underlying placental development. In the future, this may help researchers improve the efficiency of cloning.
Placental samples
All animal procedures were approved by the Institutional Animal Care and Use Committee of Chungnam National University. Cloned, TSA-treated, normal mating-derived (NM controls), and ICSI-derived mouse placentae were generated as previously described [27]. The mice were maintained on a 14L:10D schedule and allowed free access to food and water. To generate NM controls, 6-to 8-wk-old B6D2F1 females were housed with adult B6D2F1 male mice and examined daily for vaginal plugs. Noon of the day on which a vaginal plug was found was designated as 0.5 days postcoitum (dpc). Cesarean sections were performed at 18.5 dpc, and the wet weights of fetuses and placentae were recorded separately. To generate ICSI-derived placentae, B6D2F1 mice were used as the sources of oocytes and spermatozoa, and embryos were generated and transferred to recipient surrogate mothers (ICR mice) as previously described [28]. To generate cloned placentae, the nuclei of B6D129F1 embryonic stem (ES) cells were injected into enucleated B6D2F1 oocytes; the reconstituted embryos were incubated for 72 h; and those that developed to the morula or blastocyst stages were transferred to the uteri of 2.5 dpc pseudopregnant ICR females, as previously described [5]. TSA-treated cloned placentae were generated using standard mouse cloning procedures [29]. Briefly, donor nuclei from somatic cells were injected into enucleated oocytes, and the reconstructed oocytes were activated by culture in Ca 2+ -free CZB medium containing 5 mM Sr 2+ . After 6 h, the activation medium was changed to KSOM. For TSA treatments, the activated oocytes were cultured in KSOM including TSA for 14 h, and then transferred to KSOM without TSA. The resulting cloned embryos were subjected to embryo transfer into surrogate mothers on the second day. Concepti were collected by Cesarean section at 18.5 dpc, and four placental samples per ICSI, cloned, TSA treatment and natural mating separated and frozen until use. Anesthesia was induced with ketamine (100 mg/kg) and xylazine (16 mg/kg), and 100% CO 2 gas was used for sacrifice in this experiment.
RT-PCR
Total RNA was extracted from placentae using the AccuZol™ reagent (Bioneer). Quantitative real-time RT-PCR using fluorogenic 5'nuclease assay technology with TaqMan 1 probes and primers (Applied Biosystems) were employed for RNA expression analysis. Amplification of individual genes was performed on the Applied Biosystems 7300 Real-Time PCR System using TaqMan 1 Universal PCR Master Mix and a standard thermal cycler protocol. TaqMan 1 Gene Expression Assay Reagents for mouse TIMP-2, mouse PBEF, and mouse GAPDH were used for specific probes and primers of PCR amplifications. The threshold cycle (C T ) was determined and relative quantification was calculated by the comparative C T method as described previously. [30] The experiments were conducted in triplicate, and the results were normalized with respect to the mRNA expression level of GAPDH.
Statistical analysis
Significant differences among samples were determined by ANOVA followed by Duncan's multiple range tests, using the GLM found in the SAS package (SAS Institute). P-values < 0.05 were considered statistically significant.
Overgrowth of cloned placentae
The weights and sizes of mouse cloned and TSA-treated cloned placentae were compared with those of ICSI-derived and NM control placentae. As shown in Fig 1, the cloned and TSAtreated cloned placentae tended to have larger diameters than the NM control placentae (Fig 1), and weighed (on average) 310% (cloned) and 282% (TSA-treated cloned) of the mean weights of the NM control (0.092 g). There was no significant difference in weight between the cloned and TSA-treated cloned placentae (average 0.286 and 0.260 g, respectively). In addition, ICSI-derived placentae (0.140 g) tended to have higher weights (152%) than the NM control (0.092 g).
Abnormal protein expression of TIMP-2 and PBEF in cloned placentae
We previously reported two proteins that appear to be differentially expressed during pregnancy and placental development: TIMP-2 and PBEF. The former contributes to extracellular matrix degradation and remodeling and is upregulated in cloned placentae [17,18,19], while the latter contributes to inhibiting apoptosis and inducing spontaneous labor [26] and is down-regulated in cloned placentae [19]. Here, we used Western blotting to analyze the protein expression patterns of TIMP-2 and PBEF in cloned, TSA-treated cloned, ICSI-derived and NM control placentae. A TIMP-2-immunoreactive band of the appropriate size (24 kDa) was found in all tested samples (Fig 2A). Consistent with our previous results, cloned and TSA-treated cloned placentae expressed markedly higher levels of TIMP-2 protein than ICSIderived and NM control placentae (Fig 2A and 2C). PBEF, which was successfully detected as a~56-kDa band in all tested samples (Fig 2B), was observed at significantly lower levels in cloned, TSA-treated cloned and ICSI-derived placentae compared to NM controls (Fig 2B and 2D). Notably, ICSI-derived and TSA-treated cloned mouse placentae had comparable expression levels of PBEF (Fig 2D).
Abnormal mRNA expression of TIMP-2 and PBEF in cloned placentae
Real time-PCR (RT-PCR) was used to examine the mRNA expression patterns of TIMP-2 and PBEF in all tested samples. The cloned and TSA-treated cloned placentae showed higher TIMP-2 expression levels compared to the NM control and ICSI-derived placentae (Fig 3A). The mRNA expression levels of PBEF were significantly lower in cloned, TSA-treated cloned, and ICSI-derived placentae compared with NM control placentae (Fig 3B). These findings are consistent with the observed protein expression pattern of PBEF (Fig 2B and 2D).
Abnormal epigenetic modification of the TIMP-2 and PBEF promoter regions
To evaluate whether the observed expression changes in TIMP-2 and/or PBEF were associated with epigenetic changes, we examined the DNA methylation and acetylation levels of their gene promoter regions. Bisulfite sequencing was used to monitor the DNA methylation levels in the TIMP-2 promoter region, which encompassed 641 bp and included 47 CpG sites ( Fig 4A). As shown in Fig 4B, most of the CpG sites in the TIMP-2 promoter were unmethylated, and there was no significant difference in methylation status between normal and cloned placentae. Similar results were obtained with the PBEF promoter, which did not demonstrate any difference in methylation status (Fig 4C and 4D). Next, ChIP assays were used to examine the acetylation levels of the TIMP-2 and PBEF genes in cloned, TSA-treated cloned, ICSI, and NM control placentae (Fig 5). Chromatin fragments were precipitated using antibodies against acetylated histone H3 (acetyl-lysines 9 and 14) and H4 (acetyl-lysines 5), and real-time PCR was used to amplify fragments encoding the promoter regions of TIMP-2 and PBEF. Our results revealed that the H3K9 and -K14 acetylation levels of the TIMP-2 gene in ICSI and NM control mouse placentae were significantly lower than those in cloned and TSA-treated cloned placentae (Fig 5B). This pattern was similar to that observed for the protein expression of TIMP-2 (Fig 2A and 2C). With respect to the PBEF gene promoter, the H3K9 and -K14 acetylation levels in cloned, TSA-treated, and ICSI-derived placentae were considerably lower than those in NM control placentae and those of the ICSI mouse placentae were comparable to those of TSA-treated cloned placenta (Fig 5E). This was consistent with the patterns observed for PBEF protein and mRNA expression (Figs 2B, 2D, 3B, 3D and 3F). In contrast, no significant difference in H4K5 acetylation was observed for the TIMP-2 or PBEF promoter regions in any tested sample (Fig 5C and 5F).
Discussion
Fetal growth and development during pregnancy is supported by the placenta, which is a highly specialized organ that precisely ensures the efficient exchange of nutrients and waste products between the maternal and fetal circulatory systems. During the gestation of cloned mice and bovines, researchers have observed a high frequency of hypertrophic placentae [3,11]. In mice, a comparative study of placental weight at term showed that placentae derived from somatic cell nuclear transfer tended to show placental overgrowth [11]. Placentomegaly in cloned mouse concepti, which is caused by expansion of the spongiotrophoblast layer, has been speculated to reflect abnormal gene expression [11]. We previously identified several placental proteins that were up-and downregulated in cloned animal placentae and could be used to distinguish between cloned and normal placentae [19]. Here, we quantitatively determined the mRNA and protein expression patterns for two of them, TIMP-2 and PBEF, in the placentae of cloned, TSA-treated cloned, ICSI-derived and NM control mice.
TIMP-2 has been associated with extracellular matrix remodeling [32,33], and we previously reported that it is overexpressed in cloned placentae, particularly at the end of gestation [17,18,19]. High-level TIMP-2 secretion from binucleate giant cells in bovine placentae reportedly inhibits the proteolytic activity of MMP-2 (matrix metalloproteinase-2), leading to decreased extracellular matrix degradation during the prepartum period [34]. At the end of pregnancy, the number of binucleate giant cells decreases, as does TIMP-2 protein production [34]. In addition, enzymatic extracellular matrix degradation increases, the placenta detaches, and the fetal membranes are released [33]. Thus, proper control of TIMP-2 secretion may be important for successful placentation and parturition. In the present study, markedly higher levels of TIMP-2 expression were observed in cloned and TSA-treated cloned placentae compared with ICSI-derived and NM control placentae. Together with the previous findings in mouse and bovine cloned placentae [17,18,19], our results suggest that cloned and TSA-treated cloned placentae, which maintain strong TIMP-2 expression at the end of gestation, are likely to exhibit decreased proteolytic activity and MMP-induced ECM degradation, potentially leading to placental dysfunction and enlargement.
The PBEF protein, in contrast, was downregulated in cloned, TSA-treated cloned and ICSIderived placentae. PBEF appears to function at the proximal pathway for labor initiation [35], and is reportedly upregulated following distension of human amniotic epithelial cells [36]. We previous reported that the protein and mRNA expression levels of PBEF in normal mouse placentae gradually decreased from 11.5 to 18.5 dpc, but remained detectable throughout this period [24]. Similarly, a previous study found that PBEF is constitutively expressed in placentae and may play a role during normal pregnancy [37]. PBEF reportedly increases when fetal membranes are transiently distended [38], and it has been suggested to act as a growth regulator, facilitating the accommodation of placental tissue and preventing it from rupturing prematurely when advancing gestation increasingly distends the membranes [35]. In addition, PBEF was recently reported to have a local protective antiapoptotic effect in fetal membranes during placentation [38,39]. Since distension of fetal membranes in vivo may increase apoptosis in the placenta [35], our present results and the previous reports collectively suggest a model wherein PBEF expression in the developing mouse placenta could protect against the fetal membrane distension-induced apoptosis of placental cells. Interestingly, PBEF expression was lower in cloned and ICSI-derived placentae than in NM control placentae. Placenta of survived ICSI fetuses have a normal level of TIMP-2 but a different level of PBEF compared with NM, which may cause a relatively mild placentomegaly in ICSI samples. These results indicate that insufficient levels of PBEF, which appears to be important for proper placental function, may be associated with the abnormal placental development and/or unsuccessful parturition of cloned and ICSI-derived animals [17,40].
After cloning, the transferred nucleus must undergo epigenetic reprogramming in order to support successful development. Previous studies have shown that epigenetic reprogramming defects often occur in cloned embryos, as reflected by aberrant gene expression [15,39,40] and abnormal DNA methylation patterns [14,41,42]. The sall3 locus, for example, has been shown to be an epigenetic hotspot for the aberrant DNA methylation associated with placentomegaly of cloned mice [43]. Thus, altered gene expression in the placenta appears to be at least partly due to abnormalities in DNA methylation. To examine whether the protein expression changes identified in the present work might reflect epigenetic programming defects, we investigated DNA methylation and histone acetylation in the promoter regions of TIMP-2 and PBEF genes. No significant difference was observed in the methylation status of TIMP-2 or PBEF among NM control, ICSI-derived, cloned and TSA-treated cloned placentae, indicating that the TIMP-2 and PBEF genes are correctly methylated in the tested system and the methylation status may not appear to affect the mRNA expression of TIMP-2 and PBEF in the placenta. In contrast, H3-K9 and -K14 acetylations of TIMP-2 and PBEF analyzed by ChIP were up-and downregulated, respectively, in cloned placentae, in accordance with their observed mRNA and protein expression levels. Thus, cloned placentae must have suffered from aberrant reprogramming of histone changes in these (and potentially other) developmentally important genes, causing unusual expression of their proteins. These changes could give rise to the placental abnormalities seen in cloned mouse placentae, including enlargement and/or lack of proper placental function. Effect of TSA treatment in reconstructed embryos could be transient during embryo development. During early development, nuclei of reconstructed embryos treated with TSA could change acetylation status on H3K9 or globally on histone residues, and later, the modified epigenetic status could be erased or changed. Finally fetuses that have less severe modifications or proper changes in epigenetics could survive at term.
In sum, our findings indicate that TIMP-2 and PBEF are involved in abnormal hypertrophic placental development in the mouse. In cloned placentae, the failure of histone modification reprogramming in these (and potentially other) developmentally important genes appears to trigger the abnormal expression of their protein products. This is the first report to demonstrate that aberrant TIMP-2 and PBEF protein expression in the placentae of cloned mice is closely related to altered epigenetic modification of placental development-related genes. | 2018-04-03T03:55:47.675Z | 2016-11-17T00:00:00.000 | {
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244355618 | pes2o/s2orc | v3-fos-license | Uric Acid in Inflammation and the Pathogenesis of Atherosclerosis
Hyperuricemia is a common metabolic syndrome. Elevated uric acid levels are risk factors for gout, hypertension, and chronic kidney diseases. Furthermore, various epidemiological studies have also demonstrated an association between cardiovascular risks and hyperuricemia. In hyperuricemia, reactive oxygen species (ROS) are produced simultaneously with the formation of uric acid by xanthine oxidases. Intracellular uric acid has also been reported to promote the production of ROS. The ROS and the intracellular uric acid itself regulate several intracellular signaling pathways, and alterations in these pathways may result in the development of atherosclerotic lesions. In this review, we describe the effect of uric acid on various molecular signals and the potential mechanisms of atherosclerosis development in hyperuricemia. Furthermore, we discuss the efficacy of treatments for hyperuricemia to protect against the development of atherosclerosis.
Introduction
Recently, the prevalence of hyperuricemia has been increasing among developed or high-income developing countries [1]. According to the National Health and Nutrition Examination Survey (NHANES) 2007-2016, the prevalence of hyperuricemia in the U.S. was 19.1% in 1988-1994, but it increased to 21.5% in 2007-2008 [2]. Since then, the prevalence has remained at this relatively high level. According to the report for 2015-2016, the prevalence was 20.2% in men and 20.0% in women [3].
Since ancient times, gout has been known to be associated with rich foods and excessive alcohol consumption. For this reason, gout has been referred to as the "disease of kings". In some eras, gout was perceived as socially desirable because of its prevalence among the politically and socially powerful [4]. The prevalence differs between regions because of variations in lifestyle [2]. Gout is associated with Western diets or lifestyles, so the spread of Western diets in non-Western countries led to an increased prevalence of gout. A Portuguese missionary who visited Japan in the 16th century reported that the country had few gout patients, unlike in Europe. The first report of a patient with gout in Japan was in 1898 [5]. Patients with gout were rare until the end of World War II, but after the 1960s, its prevalence rapidly increased as people adopted the Western lifestyle. At present, serum uric acid levels remain low in regions without a predominantly Western lifestyle [2].
Hyperuricemia is closely associated with hypertension [6], chronic kidney diseases [7], type 2 diabetes mellitus [8], and metabolic syndrome [9], which are well known to be related to atherosclerosis. The Western diet is also closely related to the progression of atherosclerosis. For the last half-century, the association between hyperuricemia and coronary artery diseases has been discussed in many epidemiological studies. Several studies have reported a positive correlation between hyperuricemia and cardiovascular Int. J. Mol. Sci. 2021, 22, 12394 2 of 19 diseases [10,11], while others have found no association [12,13]. However, several metaanalyses of prospective studies indicating hyperuricemia's association with cardiovascular diseases have been reported recently (see below).
Recently, chronic inflammation and the inflammatory signaling pathway have been shown to play essential roles in the development of atherosclerosis. In this review, we focus on the effect of uric acid on the inflammatory pathway and show the role of uric acid in the pathogenesis of atherosclerosis.
Mechanisms of Hyperuricemia
Uric acid is an end product of purine metabolism in humans. Purine is metabolized to hypoxanthine, and then xanthine dehydrogenase (XDH) oxidizes hypoxanthine to xanthine and then xanthine to uric acid. Uric acid is metabolized to allantoin by uricase (uric acid oxidase), which is expressed in the liver in mammals, except for Hominidae primates, such as humans, chimpanzees, gorillas, and orangutans. Serum uric acid levels in most mammals are extremely low, around 0.5-1 mg/dL, because of the presence of uricase [14]. Primates such as humans that have lost uricase have several mechanisms to regulate serum uric acid levels. One is the increased excretion of uric acid by uric acid transporters [15], and the other is the increased activity of hypoxanthine-guanine phosphoribosyl transferase (HGPRT), which recycles purines [16]. Hence, these effects limit the elevation of serum uric acid levels to only about 1-2 mg/dL. Two-thirds of the excretion of uric acid is from the kidney, and one-third is from the intestinal tract. Several urate transporters have been identified. Among them, NPT1, NPT4, OAT1, OAT2, OAT3, OAT4, URAT1, GLUT9, and ABCG2 are closely related to serum uric acid levels. NPT1, NPT4, OAT1, OAT2, OAT3, and ABCG2 are related to urate secretion, and OAT4, URAT1, and GLUT9 are associated with urate reabsorption [17,18]. These transporters are expressed in the kidney, while ABCG2 is also expressed in the intestinal tract. One of the causes of hyperuricemia is the mutation of HGPRT, which converts hypoxanthine to inosine monophosphate (IMP) and guanosine to guanosine monophosphate (GMP). The decreased activity of HGPRT results in increased hypoxanthine levels and hyperuricemia. HGPRT deficiency is known to cause Lesh-Nyhan syndrome or Kelly-Seegmiller syndrome [19].
Other than genetic factors, the risk factors of hyperuricemia and gout are as follows: the intake of purine-rich foods such as meat or seafood; the intake of beverages and food that contain high amounts of sugar, especially fructose; and the intake of alcohol, especially beer. Consuming these items causes the intake of excessive amounts of purine. Fructose is phosphorylated to fructose-1-phosphate by using ATP in the glycolysis cascade. Excess intake of fructose results in the consumption of large amounts of ATP, the production of ADP and AMP, and the enhancement of AMP deaminase activity, which converts AMP to IMP. This accelerates the production of uric acid [19,20]. Hypertension, metabolic syndrome, chronic kidney disease, high body mass index (BMI), and obesity are also known risk factors of hyperuricemia. One of the mechanisms of hyperuricemia is insulin resistance, which affects the renal clearance of uric acid [21] (Figure 1).
Epidemiology of Association between Uric Acid and Atherosclerosis
The association between serum uric acid level and cardiovascular risk has been studied for several decades. Several studies have suggested that serum uric acid (SUA) is correlated with cardiovascular disease, but some studies have reported contradictory results. However, recent meta-analyses of prospective studies have supported that hyperuricemia is an independent risk factor of cardiovascular diseases.
Zuo et al. performed a meta-analysis of 14 prospective studies comprising 341,389 adult participants. They reported that the relative risk (RR) of mortality from coronary heart diseases (CHD) was increased in patients with hyperuricemia (RR: 1.14, 95% confidence interval [CI]: 1.06-1.23). Notably, the overall risks of CHD and all-cause mortality were increased by 20% and 9%, respectively, for every 1 mg/dL increase in serum uric acid. According to gender subgroup analyses, hyperuricemia increased the risk of CHD mortality to a greater extent in women compared with men [22]. In addition, Li et al. reported that a meta-analysis of 29 prospective cohort studies (N = 958,410) showed that hyperuricemia was associated with increased risk of CHD morbidity (adjusted RR 1.13; 95% CI 1.05-1.21) and mortality (adjusted RR 1.27; 95% CI 1. 16-1.39). In this report, the CHD risk was also greater in women; the RR of CHD mortality for every 1 mg/dL increase in uric acid was 1.02 in males and 2.44 in females [23]. Another metaanalysis that included 11 prospective studies with 172,123 patients suggested that elevated SUA increased the risk of all-cause mortality (RR 1.24; 95% CI 1.09-1.42) and cardiovascular mortality (RR 1.37; 95% CI 1.19-1.57). However, in this study, the risk of
Epidemiology of Association between Uric Acid and Atherosclerosis
The association between serum uric acid level and cardiovascular risk has been studied for several decades. Several studies have suggested that serum uric acid (SUA) is correlated with cardiovascular disease, but some studies have reported contradictory results. However, recent meta-analyses of prospective studies have supported that hyperuricemia is an independent risk factor of cardiovascular diseases.
Zuo et al. performed a meta-analysis of 14 prospective studies comprising 341,389 adult participants. They reported that the relative risk (RR) of mortality from coronary heart diseases (CHD) was increased in patients with hyperuricemia (RR: 1.14, 95% confidence interval [CI]: 1.06-1.23). Notably, the overall risks of CHD and all-cause mortality were increased by 20% and 9%, respectively, for every 1 mg/dL increase in serum uric acid. According to gender subgroup analyses, hyperuricemia increased the risk of CHD mortality to a greater extent in women compared with men [22]. In addition, Li et al. reported that a meta-analysis of 29 prospective cohort studies (N = 958,410) showed that hyperuricemia was associated with increased risk of CHD morbidity (adjusted RR 1.13; 95% CI 1.05-1.21) and mortality (adjusted RR 1.27; 95% CI 1. 16-1.39). In this report, the CHD risk was also greater in women; the RR of CHD mortality for every 1 mg/dL increase in uric acid was 1.02 in males and 2.44 in females [23]. Another meta-analysis that included 11 prospective studies with 172,123 patients suggested that elevated SUA increased the risk of all-cause mortality (RR 1.24; 95% CI 1.09-1.42) and cardiovascular mortality (RR 1.37; 95% 4 of 19 CI 1. 19-1.57). However, in this study, the risk of all-cause mortality was associated with SUA in males but not in females, unlike in the previously mentioned studies [24].
In Mendelian randomization, the measured variation in genes of a known function is used to examine the causal effect of modifiable exposure to a disease; this method is used in observational studies. Several studies have detected a causal relationship between SUA level and CHD by using Mendelian randomization. Chiang et al. reported that hyperuricemia was associated with an increased cumulative lifetime risk of cardiovascular disease [25]. In another study, every 1 mg/dL increase in genetically predicted uric acid concentration had a significant effect on cardiovascular death (Hazard Ratio [HR], 1.77; 95% CI. 1.12-2.81) and sudden cardiac death (HR, 2.41; 95% CI, 1.65-5.00) [26]. However, other studies reported no significant relationship between hyperuricemia and CHD [27].
A recent study indicated that in addition to high SUA levels, variable uric acid levels might be a risk factor for cardiovascular diseases. A gout attack is known to occur when the SUA level fluctuates. The risks of major adverse cardiovascular events (MACE), myocardial infarction (MI), cardiovascular death, heart failure-related hospitalization, and total cardiovascular events were significantly higher in patients with highly variable SUA levels compared with those with stable SUA levels [28]. Fluctuating uric acid levels, such as those in gout attacks, may induce inflammation in coronary arteries and lead to the development of atherosclerosis.
The Role of Hyperuricemia in the Pathogenesis of Atherosclerosis
As mentioned above, many epidemiological studies have shown an association between hyperuricemia and atherosclerosis-related events. In addition, basic studies have characterized the role of uric acid in the pathogenesis of atherosclerosis.
Atherosclerotic plaques develop as follows. First, vascular endothelial injury caused by mechanical stress induces the expression of adhesion factors, and chemokines and monocytes attach to vascular endothelial cells and infiltrate the subendothelial layer. In the subendothelial layer, monocytes differentiate into macrophages. A high level of hyperlow-density lipoprotein (LDL) cholesterolemia results in the accumulation of LDL in the subendothelial layer. Reactive oxygen species (ROS) oxidize LDL to oxidized LDL (oxLDL), which induces inflammation in vascular endothelial cells. The recruited monocytes/macrophages take in oxLDL and differentiate into foam cells. Foam cells secrete inflammatory cytokines and various chemokines, which attract leukocytes such as T cells, other lymphocytes, and dendritic cells. These leukocytes produce TGF-β, which attracts fibroblasts and promotes the formation of fibrous caps, leading to the development of the atheromatous plaque. Oxidative stress and various intracellular signaling cascades are involved in this process, and uric acid affects them in a pro-atherogenic manner.
Oxidative Stress
Oxidative stress is one of the most critical factors in the development of atherosclerosis. Oxidative stress contributes to the pathogenesis of atherosclerosis via induction of the dysfunction of endothelial cells and vasodilation, induction of inflammation in inflammatory cells such as macrophages, aggregation of platelets, and oxidation of LDL.
Reactive oxygen species (ROS) are derived from oxygen molecules (O 2 ) and are unstable and powerful oxidizing agents. In vivo, they are produced in the process of oxidative phosphorylation (OXPHOS) in mitochondria. In addition, NADPH oxidases, xanthine oxidases, and lipoxygenases are known to produce ROS. ROS have essential physiological roles in preventing infection and mediating signal transduction. ROS oxidize intracellular proteins, lipids, and DNA and lead to cellular damage. There are inherent systems for antioxidant defense in the body. However, if this balance is lost and oxidative stress becomes dominant, atherosclerotic lesions progress. The level of oxidative stress is correlated with age [29], blood pressure [30], smoking [29], LDL cholesterol [31], and blood sugar level [32]. Patients with CHD have high levels of oxidative stress.
Uric acid itself is chemically characterized as an antioxidant. In particular, uric acid is an important antioxidant in the extracellular space. Most mammals can synthesize ascorbic acid, a powerful antioxidant, but some primates, including humans, do not have the enzyme required for ascorbic acid synthesis. Thus, uric acid is thought to work as an antioxidant instead of ascorbic acid [33]. In a prior study, uric acid suppressed ROS accumulation and protected against ischemic neuronal injury [34]. It is also known that exercise-induced acute kidney injury can occur in patients with hereditary renal hypouricemia. In such cases, kidney injury results from the insufficient removal of exerciseinduced oxidative stress due to the deficiency of uric acid as an antioxidant [35].
However, it is also known that intracellular uric acid plays a role in inducing oxidative stress. In the pathogenesis of atherosclerosis, hyperuricemia acts as an inducer of oxidative stress. The mechanisms by which oxidative stress accumulates under hyperuricemic conditions are as follows: 1.
ROS are produced due to the increased activity of xanthine oxidase in the metabolic process of uric acid; 2.
The expression and activity of NADPH oxidase increase; 3.
Mitochondrial ROS (mtROS) are produced due to mitochondrial injury.
Xanthine Oxidoreductase
Xanthine oxidoreductase (XOR) exists in two forms, which are xanthine dehydrogenase (XDH) and xanthine oxidase (XO). XDH oxidizes substrates with NAD+ and produces NADH, while XO oxidizes substrates with O 2 and produces O 2 -or H 2 O 2 . Only mammals have the XO type [36]. XDH is expressed in the liver and small intestine and released to the plasma, where it is converted into XO by a protease [37]. Most XOR exists as the XO type in vascular endothelial cells. XO plays a role in preventing infections by producing ROS in vascular endothelial cells in physiological conditions.
The accumulation of XO in atherosclerotic plaque leads to the production of ROS. An elevated ratio of XO to XDH was observed in response to oscillatory shear stress in plaques [38]. Furthermore, the expression of XO itself was increased in plaques [39]. ROS derived from XO is involved in a vascular endothelial injury. It has been reported that XOR inhibitors improved endothelial dysfunction in patients with chronic heart disease, diabetes mellitus with mild hypertension, smoking, and sleep apnea syndrome (SAS) [40][41][42][43]. The activity of XO is elevated in pathological conditions such as MI and ischemia-reperfusion [44]. Increased XO activity results in a burst of ROS, the attraction of neutrophils, and the induction of tissue injury [45].
The production of ROS by XO induces the migration, proliferation, and production of monocyte chemotactic protein-1 (MCP-1) in arteriolar smooth muscle cells [46] and contributes to the development of atherosclerosis. XOR contributes to foam cell formation. Knock-down of XOR suppressed lipid intake in macrophages and their differentiation into foam cells [47]. XOR was also reported to regulate lipid accumulation and be involved in adipocyte differentiation via activation of the transcription factor PPARγ [48]. XOR regulates inflammatory cytokine secretion. Increased plasma XOR activity was correlated with plasma IL-6 level and NF-kB activity [49]. In mouse macrophages, XOR regulated IL-1β secretion via NLRP3 inflammasome activation [50].
NADPH Oxidase
NADPH oxidase (NOX) is a complex of membrane-bound enzymes. In phagocytes, ROS produced by NADPH oxidase eliminates microorganisms. Four members of the NOX family (NOX1, NOX2, NOX4, NOX5) are expressed in vascular smooth muscle cells, endothelial cells, fibroblasts, and perivascular adipocytes. NOX-dependent ROS formation in endothelial cells and vascular smooth muscle cells (VSMCs) potentially activates the expression of adhesion molecules and the subsequent monocyte/macrophage infiltration into the arterial wall. It also contributes to endothelium activation and stimulates VSMC proliferation. NOX1 also contributes to the accumulation of the extracellular matrix and leads to aortic media hypertrophy. Thus, activation of NOX is involved in the pro-atherogenic process [51].
It has been reported that uric acid activates NADPH oxidase and produces ROS. Uric acid-activated NADPH oxidase and increased oxidative stress lead to the activation of p38 MAPK and ERK1/2, causing a subsequent decrease in NO bioavailability and increase in protein nitrosylation and lipid oxidation in adipocytes [52]. In the human aorta, uric acid-treated smooth muscle cells increased the cell proliferation and expression of endothelin-1 (ET-1). These effects were suppressed by a NOX inhibitor and siRNA of the NOX subunit (p47phox). ET-1 is a pro-atherogenic molecule derived from vascular endothelium and promotes strong vasoconstriction, the proliferation of smooth vascular cells, and the proliferation of fibroblasts. The uric acid-induced expression of ET1 is caused by the activation of ERK-AP1 via the production of ROS [53]. In kidney tubular cells, uric acid increased oxidative stress and apoptosis via up-regulation of NOX4 expression and mitochondrial injury. Furthermore, NOX signaling was dependent on the intracellular uptake of uric acid via URAT1, a uric acid transporter [54]. This indicates that uric acid works intracellularly. Uric acid regulates not only the expression level but also the activity level of NOX. Uric acid was observed to promote the phosphorylation of p47phox and the interaction of p-p47phox with p22phox, leading to the assembly of subunits and activation of NOX. In a hepatocyte cell line, uric acid-induced NOX activation caused endoplasmic reticulum (ER) stress, followed by mitochondrial dysfunction and the accumulation of lipids [55]. These results suggest that uric acid is involved in the pathogenesis of the metabolic syndrome.
Mitochondrial ROS
Mitochondrial dysfunction induces cell senescence and apoptosis of vascular endothelial cells and influences the development of atherosclerosis. Decreased copy numbers of mitochondrial DNA (mtDNA), a reduced oxygen consumption rate, and mitochondrial dysfunction were observed in human plaque. Mitochondrial dysfunction was found in the plaque of ApoE-knockout mice fed a high-fat diet. This was attributed to the decreased expression of complexes I, III, IV, and V in OXPHOS, which leads to the disorder of VMSC proliferation and macrophage apoptosis, subsequently increasing the vulnerability to plaques [56,57]. Mitochondrial dysfunction leads to ROS production, thereby increasing the production of inflammatory cytokines and promoting the differentiation of M1 macrophages [58].
Several studies have reported that uric acid induces mitochondrial disorder. A high concentration of uric acid caused increased mitochondrial ROS and mitochondrial damage. In hepatocytes, decreased membrane potential, mitochondrial DNA damage, and suppression of OXPHOS due to decreased levels of cytochrome C and succinate dehydrogenase (SDH) were observed [59]. Soluble uric acid-induced endothelial dysfunction in human aortic endothelial cells was related to reduced mitochondria and ATP production. Mitochondrial DNA damage and accumulation of oxidative stress were increased in hyperuricemic rats [60].
The mechanism of mitochondrial injury induced by uric acid remains unclear, but it is assumed that it involves the production of ROS by NADPH oxidase [54], suppression of AMPK [61], or activation of Rho kinase [62].
Inflammatory Signaling Pathway
As mentioned above, uric acid induces ROS production. ROS are vital mediators that activate various signaling pathways. Furthermore, uric acid itself may activate several intracellular signaling pathways that result in the production of inflammatory cytokines, adhesion factors, and chemokines and regulate cell proliferation and apoptosis, consequently leading to atherosclerosis development ( Figure 2). Next, we discuss the role of the signaling pathways activated by uric acid in the pathogenesis of atherosclerosis. intracellular signaling pathways that result in the production of inflammatory cytokines, adhesion factors, and chemokines and regulate cell proliferation and apoptosis, consequently leading to atherosclerosis development ( Figure 2). Next, we discuss the role of the signaling pathways activated by uric acid in the pathogenesis of atherosclerosis.
ERK/p38 MAPK Cascade
The intracellular mitogen-activated protein kinase (MAPK) cascade is crucial for bridging extracellular stimuli to intracellular reactions. In atherosclerosis, MAPK transduces the stimulation of cytokines or growth factors to intracellular signals and regulates the growth and survival of cardiomyocytes, smooth muscle cells, or macrophages. The MAPK pathway is composed of three steps of kinase activation: MAPK kinase kinase, MAPK kinase, and terminal MAPK. The main terminal MAPKs are extracellular signal-regulated kinase (ERK) 1/2, Jun N-terminal kinase (JNK), p38 MAPK, and ERK5. ERK, JNK, and p38 have crucial roles in the pathogenesis of atherosclerosis [63].
Activation of p38 MAPK has been characterized as pro-atherogenic. The p38 MAPK promotes the expression of adhesion factors, such as E-selectin and vascular cell adhesion molecule-1 (VCAM-1), and chemokines, such as MCP-1. Furthermore, activation of p38 MAPK and ERK1/2 leads to the proliferation and hypertrophy of VSMCs and the induction of RUNX2, which result in the calcification of the aortic wall and aortic valves. p38 MAPK is associated with the migration and proliferation of VSMCs and is involved in the induction of angiogenesis and the formation of atheromatic plaque. Activation of p38 MAPK promotes the adhesion ability of immune cells via the expression of CXCR2 and the expression of inflammatory cytokines [64]. p38 MAPK is involved in the uptake
ERK/p38 MAPK Cascade
The intracellular mitogen-activated protein kinase (MAPK) cascade is crucial for bridging extracellular stimuli to intracellular reactions. In atherosclerosis, MAPK transduces the stimulation of cytokines or growth factors to intracellular signals and regulates the growth and survival of cardiomyocytes, smooth muscle cells, or macrophages. The MAPK pathway is composed of three steps of kinase activation: MAPK kinase kinase, MAPK kinase, and terminal MAPK. The main terminal MAPKs are extracellular signal-regulated kinase (ERK) 1/2, Jun N-terminal kinase (JNK), p38 MAPK, and ERK5. ERK, JNK, and p38 have crucial roles in the pathogenesis of atherosclerosis [63].
Activation of p38 MAPK has been characterized as pro-atherogenic. The p38 MAPK promotes the expression of adhesion factors, such as E-selectin and vascular cell adhesion molecule-1 (VCAM-1), and chemokines, such as MCP-1. Furthermore, activation of p38 MAPK and ERK1/2 leads to the proliferation and hypertrophy of VSMCs and the induction of RUNX2, which result in the calcification of the aortic wall and aortic valves. p38 MAPK is associated with the migration and proliferation of VSMCs and is involved in the induction of angiogenesis and the formation of atheromatic plaque. Activation of p38 MAPK promotes the adhesion ability of immune cells via the expression of CXCR2 and the expression of inflammatory cytokines [64]. p38 MAPK is involved in the uptake of LDL [64]. Activation of JNK or p38 MAPK is required for foam cell formation induced by oxLDL [63].
Several reports have shown that uric acid activates p38 MAPK and ERK. ROS were produced in cardiomyocytes exposed to HUA, and ERK and p38 MAPK were sequentially activated. As a result, the viability of the cardiomyocytes exposed to HUA decreased.
In vivo, ERK/p38 MAPK was activated in the heart of a high-uric-acid mouse model, indicating that uric acid induces myocardial damage [65,66]. Activation of ERK1/2 and p38 MAPK was also observed in VSMCs and promoted the expression of MCP-1. This activation of MAPK was also caused by the production of ROS by HUA [67]. In pancreatic β-cells, uric acid activated ERK, decreased cell viability, and induced apoptosis and ROS production. Zurampic, a URAT1 inhibitor, inhibited the ERK pathway and attenuated uric acid-induced cell damage [68]. This observation reflects the effect of intracellular uric acid on MAPK activity.
Uric acid also regulates MAPK via phosphatase activity that inhibits the MAPK pathway. In macrophages, febuxostat activated MAPK phosphatase-1 (MKP-1) and deactivated JNK, which led to the suppression of MCP-1 expression [69].
AMPK
AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates the intracellular energy state. AMPK is activated by decreased intracellular ATP concentrations and an increased ratio of ATP to ADP or AMP. The activation of AMPK promotes glycolysis and OXPHOS and leads to the production of ATP. Currently, research is focused on AMPK as a key molecule connecting metabolism with inflammation. The suppression of AMPK induced inflammatory responses, such as the production of inflammatory cytokines in macrophages and activation of the NLRP3 inflammasome [70,71]. In a study on the pathogenesis of atherosclerosis, activation of AMPK suppressed the development of atherosclerosis in ApoE-KO mice [72,73]. Furthermore, atherosclerotic lesions were increased in endothelial cell-specific AMPK-KO mice [74]. The activation of AMPK causes attenuation of the migration of monocytes [72] and anti-inflammatory effects via inhibition of STAT3 and suppression of differentiation of monocytes to macrophages [73].
Uric acid has been reported to suppress AMPK. In a fructose-treated hepatocyte cell line, uric acid suppressed AMPK activity and was involved in gluconeogenesis and insulin resistance [75,76]. This suggests that uric acid is involved in the pathogenesis of metabolic syndrome via the regulation of AMPK. However, several studies reported that AMPK was activated by ROS induced by uric acid [77,78].
In a study of atherosclerosis, it was reported that AMPK was activated in blood cells and plaques, and serum IL-1β or TNFα was decreased in a urate-lowering mouse model fed HFD in which uric acid levels were decreased by the administration of allopurinol or overexpression of uricase. In vitro, uric acid attenuated AMPK activity and led to the activation of the NLRP3 inflammasome and the production of IL-1β [61]. Another study also reported the effect of allopurinol on AMPK. AMPK activity was reduced in rats fed a high-fructose diet, but administration of allopurinol rescued the activation of AMPK [79].
PI3K-Akt Pathway
Phosphatidylinositol-3 kinase (PI3K) is a phosphatidylinositol kinase that catalyzes phosphorylation at the 3 position of the inositol ring of phosphatidylinositol, a component of the cell membrane, and also has protein serine/threonine kinase activity. Downstream of PI3K, the most important effector molecule is Akt. Akt is a serine/threonine kinase and a key molecule involved in various signaling pathways, including cell proliferation, differentiation, apoptosis, and migration. Additionally, Akt is involved in the cell cycle and glucose metabolism via GSK3b and cell growth and survival via mTORC1 [80]. With respect to atherosclerosis, the PI3K-Akt pathway regulates the migration of monocytes and macrophages, lipid accumulation, cell proliferation, and endothelial dysfunction, which lead to the development of atherosclerotic plaques [80]. In vitro studies suggested that Akt may play a pro-atherogenic role. However, the development of atherosclerosis was aggravated in Akt1 knockout mice [81]. The role of Akt in atherosclerosis is still debated.
In human monocytes, uric acid was observed to phosphorylate Akt, activate mTOR, and subsequently suppress autophagy. These events resulted in the suppressed expression of IL-1R antagonist and increased production of IL-1β [82]. However, uric acid was also reported to suppress Akt. Uric acid was suggested to be involved in the progression of atherosclerosis via insulin resistance induced by the suppression of Akt [83].
Inflammasome
The inflammasome is an innate immune sensor and regulates the activity of caspase-1. Activation of the inflammasome is induced after the recognition of pathogen-associated molecular patterns (PAMPs) derived from microorganisms and damage-associated molecular patterns (DAMPs) from dead or dying host cells. The nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) inflammasome is involved in various infections and inflammatory diseases. The expression of NLRP3 is induced by the activation of NF-kB. Next, it assembles and forms a complex with an adaptor protein, ASC, and procaspase-1. Subsequently, procaspase-1 undergoes autolysis and matures to caspase-1. Caspase-1 processes pro-IL-1β and pro-IL-18 to mature IL-1β and IL-18. At the same time, pyroptosis is induced, and IL-1β is released to the extracellular space [84,85].
Sterile crystals activate the NLRP3 inflammasome. In gout, monosodium urate (MSU) crystals activate the NLRP3 inflammasome, induce the release of IL-1β and promote the development of arthritis. The mechanism of NLRP3 inflammasome activation by MSU crystals involves phagocytes such as macrophages or neutrophils; these cells take up the crystals, and the crystals lead to lysosomal rupture and release of cathepsin B to the cytosol. K+ efflux or production of ROS is also induced and triggers the activation of the NLRP3 inflammasome [84,86]. Recently, MSU crystals were shown to induce the translocation of Nrf2 into nuclei and alter intracellular ROS levels, which promotes activation of the NLRP3 inflammasome [87].
Recently, it has become clear that the NLRP3 inflammasome plays an important role in the pathogenesis of atherosclerosis. Canakinumab, an IL-1β inhibitor, suppressed the development of atherosclerosis [88]. Furthermore, colchicine, which inhibits the formation of the NLRP3 inflammasome, also protects against the recurrence of cardiovascular diseases [89].
In atherosclerotic plaques with hyperuricemia, deposition of MSU crystals was reported [90,91]. MSU crystals can be distinguished from calcium crystals by using dualenergy CT, which is useful for the detection of MSU crystals in gout and urolithiasis. Dual-energy CT was performed on 59 patients with gout and 47 controls, and the frequency of cardiovascular MSU deposition was analyzed. The frequency of the deposition in cardiovascular systems was higher among patients with gout (51 [ [90]. The expression of XO was increased, and there were significantly higher concentrations of uric acid in atherosclerotic plaques [39], which may also affect the deposition of MSU crystals. However, the findings of dual-energy CT may include artifacts [92]. The association between the deposition of MSU crystals and cardiovascular events is not yet clear. Andres et al. reported that MSU deposition in the knee or first metatarsophalangeal joints was related to calcification of coronary arteries [93].
Soluble uric acid, as well as MSU crystals, has been reported to activate the NLRP3 inflammasome [94]. Soluble uric acid induces the production of mitochondrial ROS and leads to the activation of NLRP3 inflammasome complexes. In another report, soluble uric acid suppressed AMPK, led to the production of mitochondrial ROS, and finally activated the NLRP inflammasome [61].
Inflammasome activation in patients with gout or hyperuricemia has been observed in several studies. Serum IL-18, an inflammasome-related cytokine, was reported to be higher in gout patients, and the serum IL-18 level was correlated with the level of C-reactive protein (CRP) and the erythrocyte sedimentation rate (ESR) [95]. The expression of IL-1β and IL-18 was increased in the peripheral blood mononuclear cells of patients with active gout [96]. A correlation of the plasma uric acid level with the plasma IL-18 level was also reported [97]. Furthermore, decreasing plasma uric acid levels by administering benzbromarone resulted in decreased plasma IL-18 levels [61]. 4.2.5. NO, HMGB1, RAA, ER Stress, and Mechanism of Sensing UA NO regulates blood vessel tonus and acts as an internal anti-atherogenic factor. However, NO is an unstable molecule and easily oxidized by ROS, and subsequently, the physiological activity is lost. A decrease in anti-atherogenic factors is one of the proatherogenic effects of ROS. The reactivity of O 2 -itself is weak, but it reacts with NO to produce peroxynitrite (ONOO-), a very powerful radical. In fact, the vasodilation ability was decreased in patients with CHD in whom NADPH oxidase and XOR were activated. Vasodilation impairment in hyperuricemia was improved by the administration of allopurinol, a xanthine oxidase inhibitor.
As mentioned above, NO is consumed by excessive ROS production, but NO production is also decreased by uric acid. The dephosphorylation of eNOS (endothelial NO synthase) was induced via uric acid transporter, and the production of NO was decreased in human umbilical vein endothelial cells (HUVECs). The decreasing production of NO was attenuated by benzbromarone, a URAT1 inhibitor [98]. Production of eNOS is also regulated by the HMGB1-RAGE pathway. In HUVECs, uric acid promoted the production of HMGB1 and the expression of RAGE, a receptor of HMGB1, and suppressed the expression of eNOS. Furthermore, the autocrine effect of HMGB1 leads to further production of HMGB1. The blocking of RAGE prevented the up-regulation of HMGB1 and the decrease in eNOS expression [99]. In a study to explore the relation between uric acid and HMGB1, uric acid activated the MEK-ERK pathway and regulated intracellular calcium morbidity and HMGB1 acetylation and translocation. The subsequent release of HMGB1 to the extracellular space was promoted, and inflammation was aggravated [100].
The RAA (Renin-Angiotensin-Aldosterone) system may be involved in the effects of uric acid. In HUVECs, uric acid induced the inflammatory response via the up-regulation of renin receptors [101]. Uric acid also up-regulated the expression of angiotensinogen, angiotensin-converting enzyme, and angiotensin II receptors and increased angiotensin II levels. This led to the production of ROS, cell apoptosis, and cellular senescence. The effect was diminished by probenecid, a urate transporter inhibitor [102].
Uric acid was also shown to induce endoplasmic reticulum (ER) stress. In rats with hyperuricemia induced by a uricase inhibitor, oxonic acid, cellular apoptosis, intestinal fibrosis, and diastolic heart dysfunction were exacerbated. Increased expression of calpain-1, a kind of protease, and increased ER stress markers (CHOP, GRP78, p-PERK) were also observed in rat myocardium. In the H9c2 cardiomyocyte cell line, increased apoptosis and ER stress were dependent on calpain-1 [103].
As stated above, intracellular uric acid alters various signaling pathways, including the production of ROS. However, it remains unclear how intracellular uric acid is recognized and activates signaling pathways. Recently, NAIP1 was reported to directly recognize intracellular uric acid and induce the activation of NLRP3 in mice [104]. However, the binding of uric acid with human NAIP1 is weak [104], so the effect of uric acid may be caused by other molecules in humans.
In summary, uric acid plays a pro-atherogenic role in several steps in the progression of plaques as follows. Uric acid promotes oxidative stress and destabilization of NO, which leads to vasoconstriction and endothelial dysfunction. The expressions of chemokines, such as MCP-1, are increased, and monocytes are recruited into the subendothelial layer. Macrophages in subendothelial are differentiated into foam cells depending on oxidative stress by uric acid and the effect of xanthine oxidase. These foam cells or macrophages secrete inflammatory cytokines, and uric acid promotes the production of the cytokines. The inflammatory cytokines attract further inflammatory cells and bring the formation of the necrotic core. Uric acid promotes proliferation and migration of VSMCs via activation of MAPK and oxidative stress, which leads to the progression of atheromatous plaque. Oxidative stress derived from mitochondrial dysfunction by uric acid results in the destabilization of plaques. Inflammation augmented by uric acid via activation of inflammasomes or several inflammatory signaling pathways contributes to the development of atherosclerosis in each atherogenic step (Figure 3). cells and bring the formation of the necrotic core. Uric acid promotes proliferation and migration of VSMCs via activation of MAPK and oxidative stress, which leads to the progression of atheromatous plaque. Oxidative stress derived from mitochondrial dysfunction by uric acid results in the destabilization of plaques. Inflammation augmented by uric acid via activation of inflammasomes or several inflammatory signaling pathways contributes to the development of atherosclerosis in each atherogenic step (Figure 3).
The Effect of Therapeutic Agents for Gout on Atherosclerosis
The Guideline for Management of Gout by the American College of Rheumatology (ACR) in 2020 provides the following recommendations [105]. Initiating urate-lowering therapy (ULT) is strongly recommended for gout patients with any of the following: ≥1 subcutaneous tophus; evidence of radiographical damage attributable to gout; or frequent gout flares, with frequent being defined as ≥2 annually. Initiating ULT is not recommended in patients with asymptomatic hyperuricemia. In randomized control trials (RCTs), ULT significantly suppressed gout flares in patients with asymptomatic hyperuricemia. However, the incidence of gout was low (<5%), so the effect of preventing an attack was too costly even in patients with comorbid chronic kidney disease, cardiovascular disease, urolithiasis, and hypertension. In the guideline for gout in Japan, initiating ULT to treat asymptomatic hyperuricemia is recommended in patients with an SUA of more than 8 mg/dL and complications such as chronic kidney disease, urolithiasis, hypertension, cardiovascular disease, diabetes mellites, and metabolic syndrome or in
The Effect of Therapeutic Agents for Gout on Atherosclerosis
The Guideline for Management of Gout by the American College of Rheumatology (ACR) in 2020 provides the following recommendations [105]. Initiating urate-lowering therapy (ULT) is strongly recommended for gout patients with any of the following: ≥1 subcutaneous tophus; evidence of radiographical damage attributable to gout; or frequent gout flares, with frequent being defined as ≥2 annually. Initiating ULT is not recommended in patients with asymptomatic hyperuricemia. In randomized control trials (RCTs), ULT significantly suppressed gout flares in patients with asymptomatic hyperuricemia. However, the incidence of gout was low (<5%), so the effect of preventing an attack was too costly even in patients with comorbid chronic kidney disease, cardiovascular disease, urolithiasis, and hypertension. In the guideline for gout in Japan, initiating ULT to treat asymptomatic hyperuricemia is recommended in patients with an SUA of more than 8 mg/dL and complications such as chronic kidney disease, urolithiasis, hypertension, cardiovascular disease, diabetes mellites, and metabolic syndrome or in patients whose SUA is more than 9 mg/dL [106]. Thus, the management approach for asymptomatic hyperuricemia varies by region.
Colchicine, nonsteroidal anti-inflammatory drugs (NSAIDs), and steroids are used to treat gout attacks. If they are not effective, the use of an IL-1 inhibitor is considered. When initiating ULT, administrating prophylactic anti-inflammatory therapy such as colchicine and NSAIDs for 3-6 months is recommended [105].
The reported effects of urate-lowering drugs on cardiovascular disease are described below (Table 1). Several small-scale randomized studies have reported the effect of allopurinol. Allopurinol has been the most frequently used xanthine oxidase inhibitor. In a prospective randomized trial of patients with chronic kidney disease (CKD) (eGFR < 60 mL/min/m 2 ), administration of allopurinol for 24 months decreased CRP and slowed the progression of CKD. Furthermore, allopurinol reduced the risk of cardiovascular events in 71% of participants [107]. However, in a recent RCT of 369 patients with stage 3 or 4 CKD, allopurinol did not slow the decline in eGFR compared with the placebo [108]. In a meta-analysis of nine RCTs on the efficacy of allopurinol in patients undergoing coronary artery bypass graft (CABG) after ACS or heart failure, the pooled odds ratio of periprocedural ACS and cardiovascular mortality was significantly lower in patients administrated allopurinol during CABG. Nevertheless, the efficacy of allopurinol in the secondary prevention of ACS or mortality in long-term outcomes was not observed [109]. In another randomized controlled prospective trial in 100 patients with ACS, the allopurinol treatment was compared with the placebo. One month of treatment with allopurinol improved the indicators of oxidative stress and inflammatory response (malondialdehyde-modified LDL, oxLDL, high-sensitivity CRP, and TNFα) and significantly increased the level of NO. The symptoms and frequency of angina pectoris were also significantly improved by allopurinol. However, the rates of stent implantation and cardiovascular events during the 2-year follow-up were not significantly different between the allopurinol and control groups [110]. Several largescale case-control studies showed that allopurinol decreased the risk of cardiovascular diseases such as acute myocardial infarction [111,112]. Another frequently used xanthine oxidase inhibitor is febuxostat. Allopurinol is a purine base analog, while febuxostat is a non-purine analog inhibitor, for which adverse effects related to purine metabolism, such as cytopenia, are less frequent. The effect of febuxostat on renal and vascular function in patients with type 1 diabetes after 8-week administration was examined to explore its endothelial function. Febuxostat had a modest systolic blood pressure-lowering effect, but flow-mediated dilation (FMD) and production of NO were not altered [113]. In a smallscale study comparing febuxostat and benzbromarone, vascular endothelial function was not improved by febuxostat but was improved by benzbromarone [114]. In Japanese patients with asymptomatic hyperuricemia, the development of atherosclerosis was assessed by carotid intima-media thickness. In this RCT, 24 months of febuxostat treatment did not effectively delay the progression of carotid atherosclerosis [115]. In 2018, a large-scale randomized study to compare cardiovascular outcomes associated with allopurinol and febuxostat in patients with gout and cardiovascular disease was reported (CARES study). The study included 6190 patients. The rates of adverse cardiovascular events were not significantly different between febuxostat and allopurinol. However, all-cause mortality and cardiovascular mortality were higher with febuxostat than with allopurinol (HR for death from any cause, 1.22 [95% CI, 1.01-1.47]; HR for cardiovascular death, 1.34 [95% CI, 1.03-1.73]). However, a limitation of this study was its high drop-out rate. The trial regimen was discontinued in 56.6% of patients, and 45.0% discontinued follow-up [116]. After the CARES study, in 2020, a large-scale RCT was reported on the cardiovascular safety of febuxostat (FAST study). Patients who were 60 years or older, already receiving allopurinol, and had at least one additional cardiovascular risk factor were eligible, and 6128 patients were enrolled. Febuxostat was not associated with an increased risk of cardiovascular events, death, or serious adverse events compared with allopurinol [117].
As a uricosuric agent, probenecid was associated with a decreased risk of MI, stroke, and chronic heart failure exacerbation and mortality compared with allopurinol in patients with gout who were 65 years or older in a retrospective, propensity score-matching study [118].
In several studies described above, ULT was effective for the prevention of cardiovascular events. However, a recent meta-analysis of RCTs in which cardiovascular risks were compared between a group-administered ULT and a control group reported that ULT had little effect in preventing cardiovascular events. ULT significantly reduced the total incidence of cardiovascular events and hypertension, but the risks of MACE and mortality were not improved [119,120]. The effect of ULT on atherosclerosis has been contradictory among studies. A large-scale RCT to investigate the effect of allopurinol on cardiovascular outcomes in patients with ischemic heart diseases was performed in the UK (ALL-HEART study). The results are yet to be published [121].
In addition to these urate-lowering agents, the efficacy of colchicine and IL-1β antagonist for the prevention of CV events was proven by large-scale RCTs. Canakinumab, an IL-1β monoclonal antibody, led to a significantly lower rate of recurrent cardiovascular events in patients with previous myocardial infarction and hsCRP levels of 2 mg or more per liter [88]. The administration of colchicine, an inflammasome inhibitor, also improved the prevention of CV events in patients with acute and chronic coronary diseases [89,122]. However, these drugs are not typically used for the long-term treatment of gout. If deposition of MSU is related to cardiovascular events, long-term use of colchicine may be considered for the prevention of gout attacks and cardiovascular events. Allopurinol was associated with the reduction of odds of periprocedural ACS but not with that of long-term secondary prevention of ACS.
RCT [116] Allopurinol
Patients with gout and cardiovascular disease. 6190 All-cause and cardiovascular mortality were higher in the febuxostat group than in the allopurinol group (HR for all death, 1.22; HR for cardiovascular death, 1.34).
RCT [117] Allopurinol
Patients were ≥60 y.o., already receiving allopurinol, and had at least one additional cardiovascular risk factor.
6128
Febuxostat is non-inferior to allopurinol therapy as the primary cardiovascular endpoint and not associated with an increased risk of death.
Xanthine oxidase inhibitor (XOI)
Meta-analysis: 81 RCTs [119] Placebo or no treatment RCTs comparing purine-like or non-purine XOI with placebo or no treatment (control) for a period equal or superior to 28 days in adult patients.
10,684 (6434 pt·yr) XOI did not significantly reduce the risk of MACE and death but reduced the risk of TCE and hypertension.
7757
Any ULT did not demonstrate a significant difference in any cardiovascular death, non-fatal myocardial infarction or non-fatal stroke, or all-cause mortality.
RCT [89] Placebo
Patients had any evidence of coronary disease and have been in a clinically stable condition for at least 6 months.
5522
A primary endpoint event occurred in 6.8% in the colchicine group and 9.6% in the placebo group (HR 0.69, 95% CI 0.57-0.83). | 2021-11-19T16:18:07.603Z | 2021-11-01T00:00:00.000 | {
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19998667 | pes2o/s2orc | v3-fos-license | Naringin enhances osteogenic differentiation through the activation of ERK signaling in human bone marrow mesenchymal stem cells
Objective(s): Naringin has been reported to regulate bone metabolism. However, its effect on osteogenesis remains unclear. The aim was to investigate the effect of naringin on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) through the activation of the ERK signaling pathway in osteogenic differentiation. Materials and Methods: Annexin V-FITC assay and MTT assay were used to measure the effect of naringin on cytotoxicity and proliferation of hBMSCs, respectively. Alkaline phosphatase activity analysis, Alizarin Red S staining, Western blotting, and real-time PCR assay were used to evaluate both the potential effect of naringin on osteogenic differentiation and the role of ERK signaling pathway in osteogenic differentiation. Results: Our results showed that naringin had no obvious toxicity on hBMSCs, and could significantly promote the proliferation of hBMSCs. Naringin also enhanced the osteogenic differentiation of hBMSCs and increased the protein and mRNA expression levels of osteogenic markers such as Runx-2, OXS, OCN, and Col1 in a dose-dependent manner. In addition, we found that the enhancing effect of naringin on osteogenic differentiation was related to the activation of phosphor-ERK, with an increase in duration of activity from 30 min to 120 min. More importantly, both the enhancing effect of naringin on osteogenic differentiation and the activity effect of naringin on ERK signaling pathway were reversed by U0126 addition. Conclusion: Our findings demonstrated that naringin promoted proliferation and osteogenesis of hBMSCs by activating the ERK signaling pathway and it might be a potential therapeutic agent for treating or preventing osteoporosis.
Introduction
Osteoporosis, characterized by low bone mass density and deterioration of bone microarchitecture is regarded as one of the challenging clinical entities in the elderly population (1)(2)(3). Bone mass homeostasis depends on mainly the balance between bone formation and bone resorption, which is tightly regulated by bone-forming osteoblasts and boneresorbing osteoclasts (2). There is now a mounting body of scientific evidence to suggest that the impaired osteogenic differentiation of resident bone mesenchymal stem cell (BMSCs) may play a decisive role in mediating osteoporotic bone loss (4). In general，MSCs differentiate into osteoprogenitors, preosteoblasts, and subsequently mature osteoblasts, which are accompanied by the mineralization of the extracellular matrix (ECM) in the form of hydroxyapatite (5). Recently, several researchers have demonstrated that patients with bone fracture treated with MSCs present promising results. A study found that directing MSCs to the bone surface could augment bone formation and increase bone mass (6). Thus, enhancing osteogenesis of MSCs is considered as a potential therapeutic strategy for osteoporosis.
Osteogenesis is an intricate process, which is mediated by the action of several key transcriptional factors, including Runt-related transcription factor-2 (Runx-2) and osterix (OSX), and is usually accompanied by the increased expression of bone matrix proteins, such as alkaline phosphatase (ALP), type I collage (ColA1), and Osteocalcin (OCN) (4). ERK signaling pathway has been intensively investigated in regulating many cellular functions, such as meiosis, mitosis, apoptosis, and differentiation (7). Recently, it was reported that the activation or inhibition of ERK signaling pathway is involved in the commitment of MSCs into osteogenic lineages (8,9). Several studies have found that ERK1/2, activated by mitogen-activated protein kinase 1/2 (MEK1/2) through phosphorylation at a threonine and an adjacent tyrosine residue, could strongly increase the Runx-2 protein level though an increase in acetylation and a decrease in ubiquitination (8,10,11). In addition, many studies have shown that traditional medicinal herbs such as Salvianolic acid B (12) and Chlormadinone acetate (13) could enhance the osteogenic differentiation of MSCs through the activation of ERK signaling pathway.
Naringin, a major traditional Chinese medicine with a long medicinal history, is the main effective component of rhizome dryariae (14). Rhizome dryariae, specifically naringin, is commonly used to manage orthopedic disorders (15). In addition, numerous studies revealed that naringin could enhance the proliferation and osteogenic differentiation of the MC3T3-E1 cell line (16). A study by Zhang et al also demonstrated that naringnin had anti-osteoporotic and enhancing bone-unifying properties through the promotion of proliferation and osteogenic differentiation of human BMSCs (17). However, it remains unclear whether the effect of naringin for enhancing osteogenic differentiation is related to the activation of the ERK signaling pathway. Thus, the present study seeks to investigate the effects of narignin on the proliferation and osteogenic differentiation of hBMSCs by activating the ERK signaling pathway.
Cell Cultures and differentiation
Bone marrow aspirates were obtained from four healthy volunteers (A-D) (A, 27 years old; B, 29 years old; C, 34 years old; D, 47 years old) during the routine orthopedic surgical procedure in Shanghai 9th People's Hospital and hBMSCs were isolated and expanded using the previous method (18). All protocols of human tissue handling were approved by the Research Ethical Committee of our Hospital. Briefly, the aspirates were immediately re-suspended in DMEM supplemented with 10% fetal bovine serum (FBS, Thermo scientific), 1% penicillin-streptomycin (Thermo scientific) (growth medium, GM), and plated at 4×10 4 cells/cm 2 in 100 mm culture dishes (Falcon, USA) with the medium changed every three days at 37 °C in a humidified atmosphere containing 5% CO2. After the cells were cultured for 1 day, the growth medium was replaced with differentiation medium (DMEM with 10 -8 M dexamethasone, 50 ug/ml ascorbic acid, and 5 mM glycerol phosphate), which was changed every 2 days during osteogenic differentiation. To evaluate the effect of naringin on osteogenesis, the cells were treated with osteogenic differentiation medium supplemented with various concentrations of naringin (0, 10, 50, and 100 ug/ml). Three independent experiments were performed in quadruplicate.
Cell viability
The hBMSCs from passage 3-5 were seeded in 6well plates at a density of 1×10 4 cells/well and cultured in growth or osteogenic medium supplemented with various concentrations of naringin at 0, 10, 50, and 100 ug/ml. Then, cell viability analysis was performed at 1, 3, and 7 days based on a known method (19). The cells were washed twice in PBS and dissociated with 0.25% trypsin/EDTA, after which the cells were collected and stained using an Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) according to the manufacturer's protocol, and finally analyzed using a fluorescence-activated cell sorter (FACS) flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).
MTT assay
The MTT assay was used to test the effect of naringin on cell proliferation based on an existing method (19). Briefly, hBMSCs at a density of 5×10 3 cells/well were seeded in 96-well plates and cultured in growth or osteogenic medium supplemented with various concentrations of naringin (0, 10, 50, and 100 ug/ml). Following that, the cells were cultured for 0, 1, 3, 7, and 14 days, respectively. At each time point, the cells were washed twice with PBS, and then 200 μl DMEM supplemented with 20 μl 5 mg/mL MTT (Amresco, USA) solution was added and incubated at 37°C for 4 hr. Subsequently, the medium was replaced with 200 μl DMSO and vibrated for 10 min to dissolve the MTT formazan crystals. Finally, the absorbance was measured at 450 nm using an ELx Ultra Microplate Reader (BioTek, USA).
ALP staining and activity assay
hBMSCs were seeded in 6-well plates at a density of 1×10 4 cells/well and cultured in DMEM with various concentrations of naringin, as described above. After the cells were cultured for 14 days, the cells were washed with PBS twice and fixed with 4% paraformaldehyde for 10 min. Then, the cells were equilibrated by ALP buffer (0.15 M NaCl, 0.15 M Tris-HCl, 1 mM MgCl2, pH 9.0) twice, and incubated with ALP substrate solution (5 l BCIPl ml ALP buffer) at 37 ℃ in the dark for 60 min. Then the reaction was stopped by distilled water and the plate was visualized using a microscope. The ALP activity was determined using p-nitrophenylphosphate as the substrate. Absorbance at 405 nm was measured and the protein concentration of cell lysates was measured using the Bradford assay at 595 nm on a microplate spectrophotometer (Bio-Rad, USA). ALP activity was normalized according to the total protein concentration.
Alizarin Red S staining and quantification determination
To detect mineral accumulation in differentiation osteoblasts, the hBMSCs treated with osteogenic induction for 14 days were washed with PBS and fixed with 4% paraformaldehyde for 10 min. After washing with deionized water, the cells were stained with 0.1 % Alizarin Red solution (Sigma) at pH 4.2 for 30min at room temperature. Orange Red staining indicated the position and intensity of calcium deposits. For quantitation determination, 0.5 ml 100 mmol/l cetylpyridinium chlorides (CPC) solution (Sigma-Aldrich, MO, USA) was added to each well and shaken for 30 min. The mixed liquid was diluted with 100 mmol/l CPC solution to 1/20 of the original concentration and quantified by measuring the OD of the extract at 550 nm.
Inhibition of ERK
U0126, a highly specific and potent ERK inhibitor (20), was used to examine the role of ERK signaling pathway in hBMSCs osteogenesis stimulated by naringin. The experimental specimens were treated with 10 μM U0126. Then, 100 ug/ml naringin stimulation was performed. Control cells were cultured in the absence of U0126. The inhibitor was replaced every 3 days, along with the growth or osteogenic medium. At day 14, ALP staining and activity analysis were used to examine the osteogenic differentiation of the hBMSCs and Alizarin Red S staining was used to evaluate the formation of calcium deposits. Western blotting and real-time PCR were used to measure the proteins and genes expression of Runx-2 and OSX.
Statistical analysis
The results were presented as mean±standard deviation (SD) from three individuals, and each of them was the mean of triplicate experiments. Statistical analysis was performed using the SPSS software (version 13.0). One-way analysis of variance was used to compare the differences between the experimental group, where a value of P<0.05 was considered to be statistically significant.
Effect of naringin on viability and proliferation of hBMSCs
To explore whether the naringin treatment had toxicity on cells, the cell viability was measured. Our results showed that naringin exposure at various concentrations (10, 50, and 100 ug/ml) did not influence the viability of hBMSCs cultured for 1, 3, and 7 days as compared to control ( Figure 1A).
The MTT assay was used to compare the proliferation of hBMSCs, after being cultured in growth or osteogenic medium supplemented with different concentrations of naringin (0, 10, 50, and 100 ug/ml) for 1, 3, 7, and 14 days. Our findings showed that the cell numbers increased with increasing culture time, and naringin at various concentrations resulted in a high proliferation potential after 3 days of culture as compared to control ( Figure 1B). These results indicated naringin seemed to be safe and effective for cell growth.
Naringin promoted osteogenic differentiation and mineral accumulation of hBMSCs
To examine whether naringin stimulated osteogenesis, ALP staining and activity analysis were carried out in the presence and absence of naringin after the cells were cultured for 14 days. ALP, a significant marker for the differentiation of osteoblast, was expressed at an early phase of differentiation and involved in the initiation of mineralization (6). Our results showed that naringin increased the ALP activity of hBMSCs in a dose-dependent manner, and the increase in the activity peaked at the concentration of 100 ug/ml ( Figure 1C). Concurrently, naringin treatment increased the mineral accumulation and calcium deposition in a dose-dependent manner, which was confirmed by Alizarin red S staining ( Figure 1D). Taken together, these results suggested that naringin promoted osteoblast differentiation and mineralization of hBMSCs.
Naringin promoted osteogenic markers expression
To further investigate the effect of naringin on osteogenic differentiation in hBMSCs, Western blotting and real-time PCR were used to detect the expression of several osteogenic differentiation-related markers after the hBMSCs were treated with osteogenic medium supplemented with vehicle control or naringin for 14 days. Our results showed that naringin at various concentrations increased the expression level of Runx-2, OSX, and Col1 as compared to the vehicle control in osteogenesis induction condition (Figures 2A, B, and D). OCN, a major non-collagenous bone matrix synthesized protein, was secreted by normal maturing osteoblasts (6). Our findings showed that naringin at the concentration of 100 ug/ml increased the expression level of OCN, while there was no difference at the concentration of 10 or 50 ug/ml naringin as compared to control ( Figure 2C). Taken together, these results supported that naringin promoted osteogenic differentiation of hBMSCs.
Naringin activated the expression of the ERK signaling pathway components
To determine whether naringin-mediated osteogenesis was related to the ERK signaling pathway, We initially analyzed the effect of naringin (100 ug/ml) on the compounds of ERK signaling pathways by Western blotting. As shown in Figure 3, our results indicated that 100 ug/ml naringin induced the activation of phosphor-ERK1/2 from 30 min to 120 min and peaking at 60 min as compared to control. Therefore, these results suggested the hypothesis that naringin was sufficient to induce the activation of ERK1/2.
U0126 inhibited the effect of naringin for osteogenic differentiation
To demonstrate whether the activation of the ERK signaling pathway is required for the naring-mediated osteogenic activity, U0126, an inhibitor of MEK1/2, was used to block the activation of p-ERK.
As shown in Figure 4A, the higher protein expression levels of p-ERK activated by naringin treatment were decreased in the presence of U0126, suggesting that U0126 completely inhibited the naringin-induced activation of ERK1/2. Furthermore, our results indicated that pretreatment of U0126 effectively decreased the enhancing effect of naringin on ALP activity, which was shown by ALP staining. Concomitantly, Alizarin Red S staining revealed that U0126 treatment significantly inhibited the naringinpromoted calcium deposition ( Figure 4B).
Then, we examined the effect of U0126 on the expression level of naringin-promoted osteogenic marker genes such as Runx-2 and OSX. Our findings demonstrated that the protein expression levels of Runx-2 and OSX elevated by naringin treatment were reduced in the presence of U0126 ( Figure 3D). Moreover, the increased mRNA expression levels of Runx-2 and OSX due to naringin treatment were effectively attenuated by the addition of U0126 ( Figure 4D).
These results supported the hypothesis that naringin stimulated osteoblast differentiation and mineralization of hBMSCs through activating the ERK signaling pathway.
Discussion
Previous studies have demonstrated that the enhancing osteogenesis of MSCs to osteoblast is a potential therapeutic method for osteoporosis (4,6). Many natural products capable of regulating osteogenic differentiation have been explored to treat osteoporosis (8,11,12). In the present study, we demonstrated that naringin could enhance osteogenic differentiation by up-regulating the expression level of osteogenic transcription factors and activating the ERK signaling pathway.
Naringin has been reported to have antioxidant, anti-inflammatory, and anti-apoptosis effect, which could exert its potential therapeutic benefits for a wide gamut of human disorders such as atherosclerosis, cardiovascular disorders, diabetes, and cancer (14). A study demonstrated that naringin at a dose of 0.5-5 mmol/l significantly suppressed iron-induced lipid peroxidation, protein oxidation and DNA damage (21). Liu et al showed that naringin at a dose of 50-200 uM medicated its anti-inflammatory effect by inhibiting IL-8, MCP-1, and MIP-1 secretion and mRNA expression as well as by inhibiting the phosphorylation of ERK1/2, JUK, and p38 MAPK signaling pathway (22). Recently, a study revealed that naringin (10 nM to 1 µM) increased cell proliferation and ALP activity in rat osteoblast-like UMR-106 cells, which presented a potential therapeutic effect for osteoporosis and bone loss (23). A study indicated that naringin at a dose of 2 µg/ml could improve osteogenic proliferation and differentiation in MC3T3-E1 cells via up-regulation of Runx-2, Col1, and OCN protein expressions as well as modulation of BMP-2 (24). Recently, research showed that naringin at a dose of 1-100 µg/mL also enhanced the proliferation and osteogenic differentiation of hBMSC (17). According to these published studies, we estimated that a high concentration of naringin is required to exert its anti-apoptosis and anti-inflammatory effects. However, a low concentration of naringin is enough to promote proliferation and induce differentiation of cells. A study demonstrated that naringin could increase the proliferation and ALP activity of human adiposederived stem cells (hADSCs) in the range of 1-100 ug/mL, while a dose of 200 ug/ml naringin resulted in reduced cell numbers (25). Thus, in the present study, a naringin dose of 10-100 ug/ml was utilized to evaluate the enhancing effect of naringin on osteogenic differentiation of hBMSCs.
The development of osteogenesis involved in several proteins and transcription factors and their coordinated function has been envisaged. ALP, a cell membrane-associated enzyme increases if there is active bone formation occurring, which is used as an indicator of early osteogenic differentiation (6). ALP activity is associated with matrix formation in osteoblasts formation prior to the initiation of mineralization (6). In the present study, our outcomes indicated that naringin treatment enhanced the osteogenic differentiation, as measured by ALP staining and significantly increased the activity of ALP in hBMSCs. As expected, naringin enhanced calcium nodule formation, a functional marker of mineralization, as presented by Alizarin Red staining. Next, we evaluated the changes of osteogenesis-related genes in naringin-treated hBMSCs, such as Runx-2, OSX, OCN, and Col1. Runx-2，a pivotal osteogenic transcription regulator, plays a crucial role in the process of osteoblast maturation (26). OSX, as a downstream factor of Runx-2, is required for osteoblast differentiation in developing bones (27). OCN, characterized by mature cells of the osteoblastic lineage, is usually involved in controlling the mineralization process and appears at a late stage of osteogenic differentiation (28). Col1 is the most abundant protein in the bone matrix; it constitutes about 90% of the organic bone matrix and high levels of Col1 mRNA expression would be observed during proliferation (27). As expected, our findings indicated that naringin up-regulated the protein and mRNA expression levels of Runx-2, OSX, OCN, and Col1 in a concentration-dependent manner. These findings indicated that naringin could promote the osteogenic differentiation of hBMSCs by controlling these biosynthesis-related genes during osteogenic differentiation.
Previous studies have shown that ERK signaling pathway plays an important role in the regulation of osteogenesis by enhancing the osteogenic transcription regulators such as Runx-2 and OSX (28). Studies found that the activation of ERK signaling pathway involved in taurine (11), lactoferrin (8), and phosphatidylserine (29) promoted osteogenic differentiation. In the present study, we found that naringin significantly activated the phosphorylation of ERK1/2 in a dosedependent manner. To further confirm our findings, we also checked whether blocking ERK1/2 signaling pathway could impair the effect of naringin on osteogenesis of hBMSCs. Our outcomes indicated that a higher phosphorylation level of ERK1/2 activated by naringin treatment could be blocked completely by the addition of U0126. In addition, U0126 also inhibited the ALP activity and reduced the mineralized nodule formation that was enhanced by naringin in hBMSCs. Moreover, the relatively higher expression levels of Runx-2 and OSX due to naringin treatment were reversed by U0126; indicating Runx-2 was a target of the ERK1/2 pathway. These results demonstrated that ERK signaling pathway was related with naringinmediated regulation of osteogenic differentiation of hBMSCs.
Conclusion
According to our findings, naringin promoted osteogenesis of hBMSCs through activating the ERK signaling pathway. This might be one of the mechanisms by which naringin increased bone mass. More studies were needed to explore other potential mechanisms involved in the differentiation of naringintreated hBMSC, which might help the application of naringin in osteoporotic patients. | 2018-04-03T02:00:27.977Z | 2017-04-01T00:00:00.000 | {
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251660515 | pes2o/s2orc | v3-fos-license | Effects of Minority Stress on Group Identification and Collective Action among Sexual Minorities: A Longitudinal Test of the Rejection-Identification Model
Minority stress remains pervasive in various aspects of life among sexual minorities. Driven by the awareness of social injustice, some sexual minority individuals may undertake collective action to counteract discrimination, but this does not apply to all members of sexual minorities. The present study used a prospective, longitudinal research design to examine how different dimensions of minority stress (i.e., perceived discrimination and internalized stigma) interact to affect group identification and collective action. A total of 628 sexual minority individuals in Hong Kong were involved in the study. The results showed that prior discriminatory experiences were positively associated with collective action at follow-up through increased levels of group identification and commitment to social justice. The moderating effect of internalized stigma was found in which perceived discrimination was not significantly related to group identification and collective action among those with high levels of internalized stigma. The study extends the literature on the rejection-identification model by understanding collective action as a form of group-level coping in the face of discrimination. It highlights the importance of fostering group identification, strengthening collective action, and mitigating internalized stigma among sexual minorities in psychological practice.
Introduction
Collective action is the involvement in group-oriented actions by members of a group in the pursuit of common goals and interests, usually with the aim to improve the social conditions of the group (DeBlaere et al., 2014;van Zomeren et al., 2008). There are various forms of collective action, such as signing petitions, donating, engaging in community organizations, and participating in public campaigns and protests concerning the rights of a group. In the context of disadvantaged groups, the basis of collective action originates from critical consciousness (Freire, 1970). To become liberated from oppressive environments, members of disadvantaged groups have to recognize how social oppression affects their life and subsequently take action to tackle the oppressive forces (Freire, 1970). The present study examines how minority stress experiences contribute to the group identification process and commitment to social justice, and thereby drive collective action among sexual minorities.
Minority Stress and Collective Action
Minority stress theory (Meyer, 2003) proposes that the minority group members are exposed not only to the general stress that is experienced by dominant social group members (e.g., physical, financial, and social stress) but also to excessive minority stress resulting from their marginalized status. The theory conceptualizes minority stress on a distal-proximal continuum. Distal stressors refer to objective prejudicial events and conditions that affect minority group members, such as discrimination and victimization (Meyer, 2003). In contrast, proximal stressors are more subjective and dependent on perceptions and appraisals of minority group identity (Meyer, 2003). As a proximal stressor, internalized stigma refers to the personal acceptance of negative societal evaluation toward oneself as a member of a stigmatized group (Corrigan & Watson, 2002). Internalized stigma among sexual minorities reflects the internalization of heterosexist biases and prejudices from society as part of their value system and self-concept (Herek et al., 2009). As posited by minority stress theory (Meyer, 2003), experiences of discrimination are viewed as a distal stressor, whereas internalized stigma (which has also been named as internalized homophobia, internalized homonegativity, and internalized heterosexism) is a form of proximal stressors representing internal and subjective interpretations of one's sexual minority identity. Altogether, distal and proximal stressors subject sexual minority individuals to a hostile, stressful social environment, subsequently leading to adverse mental health outcomes (Meyer, 2003). The tenets of minority stress theory are well supported by the results of meta-analyses (Dürrbaum & Sattler, 2020;Newcomb & Mustanski, 2010), which have shown that discrimination and internalized stigma are related to depression, anxiety, substance use, and suicidal ideation among sexual minorities.
Although discrimination is detrimental to mental health, it may trigger group resilience and social change progress. Several studies have demonstrated that perceptions of discrimination are positively associated with minority group members' involvement in social change efforts on behalf of their group (Cronin et al., 2012;Simon et al., 1998). This has also been the case for sexual minority individuals who engage in collective action to challenge heterosexist oppression and strive for lesbian, gay, bisexual, and transgender (LGBT) rights (DeBlaere et al., 2014). Previous research showed that experiences of gendered heterosexist discrimination were positively associated with commitment to sexual minorityrelated collective action among sexual minority women (Friedman & Leaper, 2010). Similarly, Dunn and Szymanski (2018) found that heterosexist discrimination was positively related to political activism among sexual minorities.
Nevertheless, previous findings have indicated that the mechanisms underlying discrimination and collective action are complex. Although members of disadvantaged groups often experience stigma and discrimination, only a small proportion of them participate in collective action to improve their group status in society (Stürmer & Simon, 2004a). It is plausible that collective action engagement may vary among members of disadvantaged groups depending on ingroup identification and the perception of injustice (Duncan, 2018;Radke et al., 2016). Therefore, it is vital to explore the potential mediating and moderating mechanisms that may explain the relationship between discrimination and collective action.
Group Identification and Collective Action in the Face of Discrimination
The rejection-identification model (Branscombe et al., 1999) posits that perceived group-based discrimination and stable attribution to prejudice lead to an increase in group identification and subsequently affect well-being among members of disadvantaged groups. On the basis of social identity theory (Tajfel, 1978;Tajfel & Turner, 1986), the model assumes that people derive their sense of self from the social groups to which they belong. In the face of group-based discrimination and rejection, members of disadvantaged groups may develop low selfesteem because their group is excluded from valued positions in society (Schmitt & Branscombe, 2002). To cope with the devaluation, members of disadvantaged groups tend to identify themselves more with their ingroup. Group memberships may act as psychological resources from which individuals can draw strength when responding to discrimination (Jetten et al., 2018). The members can gain a sense of belonging by identifying with their ingroup when they are unable to gain acceptance from the mainstream society (Jetten et al., 2001). Such an increase in ingroup identification may compensate for the psychological harm caused by group-based discrimination. It is therefore postulated that discrimination from the outgroup can stimulate collective response and cultivate ingroup identification (Branscombe et al., 1999;Jetten et al., 2001).
The rejection-identification model has also been employed to understand collective action among members of disadvantaged groups (Branscombe et al., 1999;Molero et al., 2011). As postulated in the rejection-identification model, members may cope with discrimination by adopting group-based strategies that strengthen their identification with an ingroup and entail a distancing from the mainstream society (Schmitt & Branscombe, 2002). They may turn toward their ingroup when confronting discrimination and respond collectively with a view to challenging the status quo (Jetten et al., 2018). This proposition is consistent with Duncan's (2018) integrated model of personality and social psychological theories of collective action, which emphasizes that life experiences (e.g., personal experiences with discrimination) are linked to group consciousness and collective action. Empirical studies have found support for this claim and indicated that group identification explains the association between perceived discrimination and collective action (Cronin et al., 2012;Friedman & Leaper, 2010;Simon et al., 1998). People with higher ingroup identification may be more concerned with and commit to collective goals and interests, instead of individual prosperity. Specifically, Duncan and colleagues (2017) showed that queer consciousness (i.e., a politicized collective identity around sexual orientation) was related to LGBT-related political activism among sexual minorities. In a sample of people living with HIV/AIDS, Molero and colleagues (2011) found that perceived group-based discrimination was positively linked to ingroup identification, which predicted intentions to engage in collective action. Friedman and Leaper (2010) also showed that heterosexist discrimination was associated with greater identification with sexual minorities and higher commitment to collective action. Recent studies also revealed that perceived group discrimination was positively associated with group identification, which in turn was associated with higher levels of collective action intention among people with physical disabilities (Molero et al., 2019) and people with hearing and visual impairment (Pérez-Garín et al., 2021).
Furthermore, previous studies proposed that group identification may lead to a stronger commitment to social justice among disadvantaged groups and motivate participation in collective action. As outlined in the integrated model of personality and social psychological theories of collective action (Duncan, 2018), experiences of discrimination may foster group consciousness by strengthening group identification and facilitating members' appraisals of group disadvantage and injustice. This in turn may enhance their willingness to engage in group-based strategies that change the status quo (Duncan, 2018;van Zomeren et al., 2008). This notion is supported by previous work which indicates that members of disadvantaged groups must recognize their devalued group status as unfair before attempting to undertake social change efforts on behalf of their group (Cronin et al., 2012;Tyler et al., 1997). Klandermans (2002) also revealed that people would have higher levels of protest and union participation when they perceive the unjust treatment of their identified group. In addition, Dunn and Szymanski (2018) found that heterosexist discrimination was indirectly associated with collective action through heterosexism awareness, implying that discrimination may trigger one's awareness of group-based oppression and motivate collective action among sexual minorities.
Although previous studies have found associations between perceived discrimination, group identification, and collective action (Bourguignon et al., 2020;Simon et al., 1998), these studies have some important limitations. First, the majority of the studies have relied on cross-sectional data and, thus, were not able to draw conclusions about the direction of the relationships between discrimination, group identification, and collective action (Bourguignon et al., 2020;Dunn & Szymanski, 2018;Friedman & Leaper, 2010;Simon et al., 1998). Longitudinal research is needed to make inferences about the direction of the effects and to test the rejection-identification model accurately. Second, the mediating role of group identification on the association between discrimination and collective action has rarely been examined among sexual minorities in areas where social stigma against LGBT individuals remains highly prevalent. Most of the previous work has been conducted in the United States and other Western liberal democracies in which sexual minority rights and civil society participation are considered fundamental human rights (Bourguignon et al., 2020;Dunn & Szymanski, 2018;Friedman & Leaper, 2010). Given the widespread stigma against sexual minorities and the growing political solidarity for LGBT rights in less democratic societies (e.g., Hong Kong), it is important to disentangle the dynamics of discrimination and collective action among sexual minorities in these regions (Chan & Mak, 2021b). Third, although several studies found that rejection-identification is linked to collective action, the effect appears to be small (Cronin et al., 2012;Friedman & Leaper, 2010;Simon et al., 1998). These findings imply that only some members of disadvantaged groups are motivated to participate in collective action even though most of them have experiences of discrimination and sympathize with the goals of collective action (Stürmer & Simon, 2004a). As such, it is critical to identify the moderating factors that explain why some people who have similar experiences of group-based discrimination are more or less likely to undertake collective action on behalf of their group.
Considering the Moderating Role of Internalized Stigma
While group-based discrimination and attribution to group prejudice are positively associated with group identification and collective action (Branscombe et al., 1999;Molero et al., 2011), internalized stigma is considered to diminish group identification and undermine collective action (Herek et al., 2009). This is in accordance with the tenet of minority stress theory (Meyer, 2003) that distal and proximal stressors are two distinct yet related stress processes affecting members of disadvantaged groups. Although experiences of discrimination (distal stressors) may signify social exclusion and marginalization, it does not guarantee that all members of disadvantaged groups would necessarily internalize the societal devaluation of their identity (proximal stressors). In contrast to discrimination that is imposed externally, internalized stigma is a proximal stressor that involves negative subjective interpretations of one's stigmatized identity (Meyer, 2003). It may influence how members of disadvantaged groups view their groups and make sense of their experiences of groupbased discrimination (Corrigan & Watson, 2002). People with internalized stigma often perceive societal rejection as legitimate and inevitable, and this perception is associated with the emotions of fear and hopelessness, which further predict disengagement and avoidance from their ingroup and related collective action (Corrigan & Watson, 2002). In a sample of people with mental illness, Pérez-Garín and colleagues (2017) found that group discrimination was positively associated with collective action, whereas internalized stigma was negatively associated with collective action. Herek and colleagues (2009) also indicated that sexual minority individuals experienced more internalized stigma when they reported less positive affect toward their group membership. Internalized homophobia was related to lower levels of ingroup identification and LGBT collective action among sexual minorities (Górska et al., 2017).
Not only may internalized stigma undermine collective action, but it may also mitigate the association between discrimination and collective action. Particularly, when members of disadvantaged groups believe that discrimination is legitimate or they are blameworthy for the stigma, the protective effect of attributions on prejudice would diminish, and they are less likely to undertake collective action (Major & Crocker, 1993). This view is consistent with social identity theory which argues that group identification is attenuated and collective action is less likely when members perceive intergroup status differential as legitimate (Tajfel & Turner, 1986;van Zomeren et al., 2008). The theory suggests that the extent to which group members consider their low group status to be legitimate or illegitimate may determine whether they are motivated to identify with their ingroup and undertake attempts to improve their group's status position collectively (Bettencourt et al., 2001;Ellemers et al., 1993;Tajfel, 1978). Nevertheless, while social identity theory may shed some insights on how perceptions of the ingroup may influence one's identification and collective action, it is important to note that the perceived legitimacy of low ingroup status is not conceptually identical to internalized stigma (van Zomeren et al., 2008). Whereas the former is a more distal sociostructural characteristic of intergroup relations, the latter is a more proximal psychological process through which individuals accept society's devaluation of their groups and integrate the views into their value systems (Corrigan & Watson, 2002). The role of internalized stigma on group identification and collective action has rarely been examined in previous studies. As revealed by McNamara et al. (2013), having negative beliefs about one's stigmatized identity would lead to active suppression of stigmatizing experiences and consequently reduce future community action. People with concealable stigma may hide their stigmatized identity from outsiders and socially withdraw themselves from the stigmatized group in order to avoid discrimination, and this may undermine the process of group identification and thus hinder collective action. Given the lack of research in this area, the moderating effect of internalized stigma on the association between discrimination and collective action warrants further investigation.
Overview of the Present Study
Grounded in minority stress theory (Meyer, 2003), the rejection-identification model (Branscombe et al., 1999), and social identity theory (Tajfel, 1978;Tajfel & Turner, 1986), the present study used a prospective, longitudinal research design to (1) examine the mechanisms underlying minority stress processes (i.e., perceived discrimination and internalized stigma), group identification, and collective action among sexual minority individuals and (2) test the moderating effect of internalized stigma on the association between perceived discrimination and collective action. It was hypothesized that perceived discrimination would be positively associated with private and public collective action via increased levels of group identification and commitment to social justice (Hypothesis 1). Also, it was hypothesized that internalized stigma would be negatively associated with collective action via reduced levels of group identification and commitment to social justice (Hypothesis 2). In addition, it was hypothesized that the association between perceived discrimination and collective action would be moderated by internalized stigma (Hypothesis 3), such that the association between perceived discrimination and collective action would be weaker among sexual minority individuals with higher levels of internalized stigma.
Participants and Procedure
This study was part of a longitudinal study of the LGBT community in Hong Kong. As this study focused on the experiences of cisgender sexual minority individuals, those who identified as transgender and non-binary were not included in the analysis. The inclusion criteria of this study included: people who (1) were 16 years of age or above, (2) identified as lesbian, gay, bisexual, or otherwise not heterosexual, (3) had a gender identity that aligned with the sex assigned at birth, and (4) lived in Hong Kong. Participant recruitment messages were disseminated through LGBT social media, community organizations, and social venues. People who met the inclusion criteria were directed to an online survey hosted by Qualtrics. They were presented with the study information and were asked to provide informed consent before participating in the study. After completing the questionnaire at baseline (T1), they were asked to provide a personal email address for follow-up contact.
Participants who provided consent and personal email were followed up at one-year post-baseline (T2) and invited to complete an online questionnaire. Participants were followed up over a one-year time interval because one year has been considered an appropriate time frame for examining long-term effects in between-person studies (Masa et al., 2021;van Stekelenburg et al., 2013). A study of the German gay movement by Stürmer and Simon (2004b) also used a longitudinal panel design with a one-year interval between two points of measurement, showing the effect of collective identification on gay movement participation. In addition, large-scale collective actions such as pride parades and other LGBT community events are often organized once a year. By administering the questionnaire at the same time in the subsequent year, we had sufficient time to observe the effects of minority stress on collective action while minimizing seasonal effects that may potentially change the opportunities for collective action at different times of the year (van Stekelenburg et al., 2013).
Perceived Discrimination
Perceived discrimination was measured with 10 items from the Heterosexist Harassment, Rejection, and Discrimination Scale (Szymanski, 2006) at Time 1. The measure assesses experiences of rejection and discrimination in different areas, such as education (e.g., "How many times have you been treated unfairly by teachers or professors because of your sexual orientation?"), employment (e.g., "How many times have you been treated unfairly by your employer, boss, or supervisors because of your sexual orientation?"), and service provision (e.g., "How many times have you been treated unfairly by people in service jobs (by store clerks, waiters, bartenders, waitresses, bank tellers, mechanics, and others) because of your sexual orientation?") (Szymanski, 2006). The items were rated on a 6-point Likert scale from 1 (the event has never happened to you) to 6 (the event happened almost all the time; more than 70% of the time). Higher scores represent more frequent experiences of discrimination in the past year. The measure also included a response option of not applicable ("not applicable to your situation; there is no such person or group of people in your life"). Responses of not applicable were not included in the analysis. The internal consistency (Cronbach's alpha) of the scale in the present study was 0.92.
Internalized Stigma
Internalized stigma was measured by the nine-item Self-stigma Scale (Mak & Cheung, 2010) at Time 1. The scale assesses the extent to which participants endorse negative stereotypes of and attitudes toward their identity as a sexual minority person. A sample item includes "The identity of being a LGBT person taints my life." The items were rated on a 6-point Likert scale from 1 (strongly disagree) to 6 (strongly agree). Higher scores indicate higher levels of internalized stigma. The Cronbach's alpha for the scale in the present study was 0.90.
Group Identification
Group identification was assessed by three items based on the three-factor model of social identity (Cameron, 2004), i.e., in-group ties ("I feel strong ties to other sexual minority people"), in-group affect ("I am proud to be a sexual minority person"), and identity centrality ("Being a sexual minority person is a very important aspect of my life") (Chan & Mak, 2021a;Mohr & Kendra, 2011) at Time 2. The items were rated on a 6-point Likert scale from 1 (strongly disagree) to 6 (strongly agree). The items have been used to measure group identification among people living with HIV (Chan & Mak, 2021a). Higher scores represent higher levels of identification with sexual minority identity. The Cronbach's alpha for the scale in the present study was 0.74.
Commitment to Social Justice
Commitment to social justice was measured by the Social Justice subscale of Lesbian, Gay, and Bisexual Positive Identity Measure (Riggle et al., 2014) at Time 2. The subscale consists of five items assessing the extent to which one's LGB identity has increased one's concern with all forms of oppression and social justice. A sample item includes "My LGBT identity makes it important to me to actively educate others about LGBT issues." The items were rated on a 7-point Likert scale from 1 (strongly disagree) to 7 (strongly agree). Higher scores indicate a stronger commitment to social justice. The Cronbach's alpha for the scale in the present study was 0.90.
LGBT Collective Action
Collective action was measured by the 12-item LGBT Collective Action Scale (Chan & Mak, 2021b) at Time 2. The scale consists of two subscales, including private collective action (e.g., "Discuss LGBT issues with family and/ or friends to raise their awareness of LGBT rights") and public collective action (e.g., "Take part in demonstrations, protests, marches, and rallies for LGBT rights"). The items were rated on a 5-point Likert scale from 1 (never) to 5 (always). Higher scores indicate more frequent participation in collective action. The Cronbach's alpha for the private collective action and public collective action subscales was 0.63 and 0.89 respectively.
Data Analysis
Prior to the main analysis, all variables were screened for normality (i.e., skewness ≤ 3.0 and kurtosis ≤ 10.0) (Weston & Gore, 2006). Skewness and kurtosis for all variables were within an acceptable range. Less than 0.1% of baseline data were missing at the item level. Pearson's correlation coefficients were used to examine the relationships between the study variables.
A sequential mediation model (Fig. 1) was examined to test whether perceived discrimination (T1) and internalized stigma (T1) would be related to collective action (T2) through group identification (T2) and commitment to social justice (T2) (Hypotheses 1 and 2). A two-step structural equation modeling was used to test the hypothesized sequential mediation model. First, a measurement model was estimated using confirmatory factor analysis (CFA) to determine how well the items or parcels represented the latent constructs. The latent constructs of group identification, commitment to social justice, and private collective action were manifested by their corresponding items. For perceived discrimination, internalized stigma, and public collective action, three sets of item parcels were created to represent the latent constructs (Russell et al., 1998). Upon the confirmation of the latent factor structure, a structural model was estimated to examine the structural relationships between the latent constructs, controlling for demographic variables (i.e., gender, age, ethnicity, education level, employment status, and monthly income level). Mplus version 8.6 was used to examine the measurement and structural models using full information maximum likelihood estimation, which can accommodate missing data and use all available data to estimate the parameters of the model. The goodness-of-fit of the models was evaluated by using the χ2 statistic, comparative fit index (CFI), Tucker-Lewis index (TLI), root mean square error of approximation (RMSEA), and standardized root mean square residual (SRMR). Criteria for acceptable model fit were as follows: CFI and TLI values of 0.90 or above, RMSEA value of 0.08 or below, and SRMR value of 0.08 or below (Tabachnick & Fidell, 2007). A bootstrapping analysis was conducted to estimate the indirect effects of perceived discrimination (T1) and internalized stigma (T1) on collective action (T2). The bias-corrected 95% confidence intervals (CI) for the indirect effects were estimated using 1,000 bootstrap samples.
A latent moderated structural equation modeling approach (LMS) was used to examine the hypothesized moderated mediation model (Hypothesis 3), as depicted in Fig. 2. A latent moderated structural equation model was estimated by including the latent interaction terms (i.e., perceived discrimination × internalized stigma). The observed indicators of perceived discrimination and internalized stigma were standardized prior to model estimation. When the latent interaction terms were significant, we examined the indirect effects of perceived discrimination (T1) on collective action (T2) at low (1 SD below the mean), medium (the mean), and high (1 SD above the mean) levels of internalized stigma (T1).
A Sequential Mediation Model of Minority Stress and Collective Action
The measurement model showed a good fit to the data, χ 2 (155) = 511.25, p < .001, CFI = 0.94, TLI = 0.92, RMSEA = 0.06, SRMR = 0.06. All loadings of the measured variables on the latent constructs were statistically significant (p < .001). This suggested that all latent constructs were adequately operationalized by their respective items or parcels.
The results of the bootstrapping analysis indicated that there were significant indirect effects of perceived discrimination on private collective action (b = 0.03, 95% CI = 0.01, 0.08) and public collective action (b = 0.04, 95% CI = 0.01, 0.09) via group identification. It was also found that group identification significantly mediated the effects of internalized stigma on private collective action (b = -0.06, 95% CI = -0.12, -0.02) and public collective action (b = -0.08, 95% CI = -0.15, -0.03). As shown in Table 3, the sequential mediating hypothesis was supported. There were significant indirect effects of perceived discrimination on private collective action (b = 0.03, 95% CI = 0.01, 0.07) and public collective action (b = 0.02, 95% CI = 0.003, 0.05) via group identification and commitment to social justice, which provided support for Hypothesis 1. It was also observed that the indirect effects of internalized stigma on private collective action (b = -0.06, 95% CI = -0.12, -0.03) and public collective action (b = -0.04, 95% CI = -0.09, -0.01) via group identification and commitment to social justice were significant, which supported Hypothesis 2.
Moderating Effect of Internalized Stigma
LMS was conducted to examine whether internalized stigma would moderate the effects of perceived discrimination on group identification, commitment to social justice, and collective action (see Fig. 2). The results showed that the interaction effect of perceived discrimination and internalized stigma was significantly associated with group identification (b = -0.12, p = .04). Specifically, the positive association between perceived discrimination and group identification was stronger among sexual minority individuals who had low levels of internalized stigma (b = 0.16, 95% CI = 0.002, 0.32). On the other hand, the association between perceived discrimination and group identification was not significant among those who had high levels of internalized stigma (b = 0.05, 95% CI = -0.03, 0.12). Table 4 shows the conditional effects of perceived discrimination on group identification at low, medium, and high levels of internalized stigma. ( In addition, the indirect association between perceived discrimination and collective action varied by levels of internalized stigma. Specifically, the indirect association between perceived discrimination and public collective action via group identification was stronger among sexual minority individuals who had low levels of internalized stigma (b = 0.08, 95% CI = 0.01, 0.14), whereas the indirect association was not significant among those who had high levels of internalized stigma (b = 0.02, 95% CI = -0.004, 0.05). Furthermore, the indirect effect of perceived discrimination on private collective action via group identification and commitment to social justice was stronger among sexual minority individuals who had low levels of internalized stigma (b = 0.05, 95% CI = 0.01, 0.10), but the indirect effect of perceived discrimination on private collective action was not significant among those with high levels of internalized stigma (b = 0.02, 95% CI = -0.002, 0.04). The results provided support for Hypothesis 3.
Discussion
The present study is one of the first studies to examine how different minority stress processes (i.e., perceived discrimination and internalized stigma) interact to influence group identification and collective action among sexual Table 4 Conditional effects of perceived discrimination at low, medium, and high levels of internalized stigma A major strength of this study is the use of longitudinal data, which may serve to elucidate the directionality of the associations between the variables and overcome the limitations of previous cross-sectional studies (Bourguignon et al., 2020;Friedman & Leaper, 2010;Simon et al., 1998). The prospective research design and longitudinal analytic technique take into account the temporal ordering of the variables under naturalistic conditions and provide a more precise estimation of the hypothesized direction of effects.
The study design makes significant contributions to understanding the dynamic relationships between minority stress and collective action over time.
Effects of Minority Stress on Group Identification and Collective Action
Consistent with previous cross-sectional research (Dunn & Szymanski, 2018;Friedman & Leaper, 2010), the longitudinal findings in the present study demonstrated that, across the period of one year, prior exposure to discrimination was positively related to participation in private and public collective action among sexual minorities. The findings suggested that perceptions of discrimination may be a necessary impetus for efforts to change the group's status position through collective action (Cronin et al., 2012). Most importantly, the results of mediation analysis provided empirical support for the rejection-identification model (Branscombe et al., 1999) in which prior heterosexist experiences were associated with increased identification with sexual minorities and subsequently predicted participation in collective action for LGBT rights. Perceptions of pervasive discrimination represent marginalization and rejection from the broader society. In the face of stigma and discrimination, having a collective identity is a common response because it can provide a positive frame of reference and allow sexual minority individuals to seek inclusion and support from the ingroup. This in turn may propel private and public collective action for LGBT rights.
The hypothesized sequential mediation model of minority stress and collective action was supported, which provided evidence for Hypothesis 1. Sexual minority individuals who encountered discrimination and rejection at baseline were more likely to identify with sexual minority identities and show concern with oppression and social injustice toward sexual minorities, which led them to undertake collective action for LGBT rights (Duncan, 2018;Dunn & Szymanski, 2018). They were willing not only to participate in private collective action to foster awareness and support for LGBT rights in their immediate environment, but also to take public collective action to advocate for structural changes for sexual minorities. Taken together, the findings extended previous research on the rejection-identification model (Branscombe et al., 1999;Molero et al., 2011) and showed that group identification resulting from discriminatory experiences might increase one's awareness of oppression and mobilize collective action. Furthermore, it is important to note that the majority of participants were in early adulthood when identity was actively being formed. Group consciousness is most likely to be developed during this developmental stage in which individuals begin to explore different roles and identities (Duncan, 2018). There might be developmental and/or generational differences in life experiences and access to opportunity structures for collective action between sexual minority individuals in different stages of life. Building on the current work, future research could explore whether the developmental stage may moderate the relationships between minority stress, group identification, and collective action.
Although minority stress theory posits that both distal and proximal stressors contribute to negative health outcomes among sexual minorities (Meyer, 2003), the present study argues that distal and proximal stressors may play differential roles in predicting collective action. On the one hand, distal stressors such as discrimination and rejection were shown as a facilitator of collective action. On the other hand, it was found that proximal stressors such as internalized stigma were a hindrance to collective action (Herek et al., 2009). Consistent with Hypothesis 2, the negative relationship between internalized stigma and collective action was mediated by group identification and commitment to social justice. In accordance with previous research (Górska et al., 2017), sexual minority individuals who had higher levels of internalized stigma were less likely to identify with sexual minorities and feel connected to the LGBT community. They were also less concerned with prejudice and discrimination against LGBT individuals and other stigmatized groups, and thus were less likely to participate in collective action for LGBT rights.
Moderating Effect of Internalized Stigma
While the rejection-identification model asserts that discrimination as a form of rejection from the outgroup may lead minority group members to identify with their ingroup (Branscombe et al., 1999), the present study found that this proposition is not always true by revealing the moderating effect of internalized stigma on the relationship between perceived discrimination and group identification. The results showed that the positive association between perceived discrimination and group identification was stronger among sexual minority individuals who had low levels of internalized stigma. On the other hand, the association between perceived discrimination and group identification was not significant among those with high levels of internalized stigma. For those who strongly endorsed negative beliefs and attitudes toward sexual minorities, the rejection-identification hypothesis did not hold, and their discriminatory experiences were not significantly related to group identification. It may be because when they espouse high levels of stigmatizing beliefs toward sexual minorities, they may perceive discrimination as legitimate and inevitable (Corrigan & Watson, 2002) and may not value the bonding and connection with the LGBT community (Chan & Mak, 2021a), which make them unlikely to identify with the ingroup when facing discrimination and rejection.
Furthermore, the indirect effects of perceived discrimination on collective action were moderated by internalized stigma, which provided support for Hypothesis 3. Specifically, the positive indirect association between perceived discrimination and public collective action through group identification was only found among sexual minority individuals with low levels of internalized stigma. For those with high levels of internalized stigma, the indirect association between perceived discrimination and public collective action was not significant. In other words, discriminatory experiences would not facilitate their identification with the ingroup or motivate their participation in public collective action (e.g., organizing educational activities, community events, and protests) because they internalized societal stereotypes about sexual minorities and developed negative views about their ingroup (Major & Crocker, 1993;McNamara et al., 2013). Similarly, the indirect association between perceived discrimination and private collective action was stronger among sexual minority individuals with low levels of internalized stigma, whereas this association was not observed among those with high levels of internalized stigma. The results imply that sexual minority individuals with high levels of internalized stigma would not resort to the LGBT community as a source of solidarity for coping with discrimination or become aware of the oppression directed toward their ingroup. Therefore, they may not engage in private collective action (e.g., discussing LGBT issues with family and/or friends) to foster acceptance of sexual minorities in their immediate environment.
Limitations and Future Research Directions
Despite the strengths of the study, there are a few limitations that may affect the generalizability and interpretation of the findings. First, the present study adopted a non-probability sampling, and the participants were recruited from LGBT organizations and spaces. Thus, the sample may be subject to self-selection bias and have greater identification with sexual minorities, which limits the generalizability of the results. Second, this study did not take baseline collective action into account, and therefore the findings do not provide insights into whether prior experiences of discrimination may affect changes in collective action. To strengthen conclusions about the temporal ordering of effects, future studies may consider adopting a cross-lagged panel design to disentangle the relationships. Third, the present study draws on the rejectionidentification model to examine how different minority stress processes interact to affect group identification and collective action, but it does not account for other related variables in the collective action literature. Future work should build on the current research to further examine other personality characteristics and life experiences (e.g., developmental stage, family background) and group consciousness variables (e.g., efficacy) (Duncan, 2018;van Zomeren et al., 2008), so as to provide a more comprehensive picture of collective action among sexual minorities. Fourth, the internal consistency estimate for private collective action was below the acceptable threshold of 0.70, which could be attributed to the small number of items in the subscale. Given the inadequate internal consistency of this measure, the results should be interpreted with caution. Fifth, the current study included a sample of cisgender sexual minority individuals, and the experiences of transgender and non-binary individuals were not examined. As members of transgender and gender diverse communities may face more overt and blatant forms of oppression, their group identification and motivation for collective action may differ from those of cisgender sexual minorities and merit further research attention.
Practice Implications
The present findings revealed the importance of group identification among sexual minorities (Bourguignon et al., 2020). In response to stigma, collective action may provide a sense of belonging and acceptance that would buffer the damaging effects of discrimination and victimization (Branscombe et al., 1999;Chan & Mak, 2021b). Given the benefits of group identification and collective action, psychologists and human service professionals working with sexual minority clients should facilitate the building of support systems with peers with similar backgrounds and experiences. By offering a safe environment to share and discuss their challenges in a support group, sexual minority individuals may relate to others who have similar experiences with discrimination as they do. The consistent interaction through relationships with other ingroup members is integral not only to the acceptance of their sexual identity, but also to collective action for positive social changes because they can recognize that the challenges are commonly experienced by others in the LGBT community (McCullough et al., 2017). By educating themselves about the problems facing the community, they are more empowered to act with and on behalf of the LGBT community to undertake collective action to resist stigmatization. Such a social justice approach in psychological and human services is essential to improve wellness and equity for sexual minorities.
Nevertheless, it is important to note that collective action does not naturally stem from discriminatory experiences, especially among sexual minority individuals who endorse self-stigmatizing beliefs. To mitigate internalized stigma, psychologists and human service professionals should be ready to address the thoughts and concerns that their clients have about the ingroup and identities. In particular, practitioners need to help their clients explore how oppressive experiences affect the internalization of stigma and recognize that internalized stigma is a normal and expected reaction to living in an oppressive environment (Puckett & Levitt, 2015). By bridging their internal experience and external oppression, the clients can detach from negative evaluations that pervade their sense of self.
Conclusion
In summary, the results of the present study extend the literature by identifying differential effects of minority stress on group identification and collective action among sexual minorities. The prospective research design allows for a better understanding of how prior discriminatory experiences are related to subsequent group identification and collective action participation. The results also indicated that internalized stigma moderated the effects of perceived discrimination on group identification and collective action, such that perceived discrimination was not significantly related to group identification and collective action among those who had high levels of internalized stigma. These findings highlight the importance of fostering group identification, strengthening collective action, and mitigating internalized stigma among sexual minorities in psychological practice (Bourguignon et al., 2020) and support the role of grouplevel coping (e.g., community connectedness and collective action) in the face of discrimination.
Authors' Contributions RCHC was primarily responsible for study conceptualization, data collection, data analysis, and the writing of the manuscript.
Data Availability
The datasets generated and/or analyzed during the current study are not publicly available due to concerns regarding confidentiality and data protection.
Code Availability Not applicable.
Compliance with Ethical Standards
Ethics Approval The study was approved by the Human Research Ethics Committee of the Education University of Hong Kong. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amend-ments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.
Conflicts of Interest
The author has no conflicts of interest related to this study.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. | 2022-08-19T15:15:02.207Z | 2022-08-17T00:00:00.000 | {
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15571057 | pes2o/s2orc | v3-fos-license | Health promotion interventions in social economy companies in Flanders (Belgium)
Background Disadvantaged groups are often not reached by mainstream health promotion interventions. Implementing health promotion (HP) interventions in social economy companies, can be an opportunity to reach those people. The implementation of these interventions in social economy companies was studied. Factors that could be related to the implementation of HP and being supportive towards implementation in the future, were investigated. Methods An online, quantitative survey was sent to all 148 sheltered and social workshops in Flanders. In the questionnaire, the status of HP interventions and characteristics of the workshop were explored. Personal factors (such as attitudes towards HP, behavioural control, social norms and moral responsibility) were asked to the person responsible for implementation of HP interventions. Univariate and multivariate logistic regressions were performed. Results Respondents of 88 workshops completed the questionnaire. Almost 60 % of the workshops implemented environmental or policy interventions. Having a positive attitude towards HP, being more morally responsible, and having the subjective norm that employees are positive towards health promotion at work, were related to being more supportive towards the implementation of HP in the univariate analyses. Only attitude stayed significantly related to being more supportive towards the implementation of HP in the multivariate analyses. Conclusions Sheltered and social workshops are open to HP interventions, but more can be done to optimize the implementation. To persuade persons responsible for the implementation of HP to invest more in HP, changing attitudes concerning the benefits of health promotion for the employee and the company, is an important strategy.
Background
People with intellectual and physical disabilities, or psychiatric problems are more likely to be at risk of an unhealthy lifestyle. They are more likely to have obesity and to be less physically active [1][2][3], more at risk for depression [4] and more likely to be a smoker [2]. Research concerning Health Promotion (HP) for people with disabilities or psychiatric problems is underdeveloped and this population is hard to reach by mainstream HP initiatives [5,6]. Therefore, HP for people with disabilities should be implemented in their natural settings such as in supported living facilities and day care centers [5].
One of these natural settings can be the social economy companies. These companies "seek to serve the community's interest rather than profit maximization" [7]. They employ society's most fragile members, and in that way, contribute to social cohesion, employment and the reduction of inequalities. Also in Flanders-Belgium, social economy companies employ a diverse group of people who are (yet) unable to work in the regular economy. Most of them have a low educational level and are living in precarious life conditions [8].
The focus of this study is on HP interventions in social economy companies, defined as the promotion of healthy nutrition, physical activity, better mental health, and the prevention of smoking and alcohol (ab)use, themes also found in the health targets of the Flemish Government [9]. An unhealthy lifestyle is one of the major risk factors for noncommunicable diseases (e.g. cardiovascular diseases, cancers) [10]. Employees with an unhealthy lifestyle are more likely to be absent due to sickness, the length of their absenteeism is longer and their productivity lower [11].
Research has shown that HP at the workplace has positive effects on the health of employees and has advantages for the company. Verweij et al. found evidence that physical activity and nutrition interventions at the workplace had a positive effect on body weight, BMI and body fat [12]. While for the company, the promotion of health behaviour had a positive influence on absenteeism, job performance [13], productivity and presenteeism [14].
But not all interventions are equally effective. Interventions with an environmental component that include environmental modifications were found to be more effective, than those without environmental changes [12,15]. Also policy measures (e.g. smoking or alcohol regulations) had more chance to have long-lasting results [16]. These interventions (further called 'environmental HP interventions') influence both the conscious and unconscious behaviour and habits of the employees [17]. Other interventions such as temporary educational group sessions, individual counseling and short running actions were less effective in the long-term [18].
In order to promote HP interventions in social economy companies, it is important to get insight into the determinants that are related to the implementation of these interventions. These factors can be characteristics of the company (such as size and sector), but also individual factors of the person responsible for the implementation of HP interventions. The Theory of Planned Behaviour [19] can be used to explain the implementation of HP interventions at an individual level. In this model, the three constructs 'attitude' , 'subjective norm' and 'behavioural control' , predict the intention to implement HP interventions, while the intention predicts the implementation. Besides these three 'classical' constructs, Ajzen [20] argued that in some contexts, personal feelings of moral responsibility could add power to the model.
In this study, three aims were formulated. The first aim was to investigate the current status of the implementation of HP interventions in social economy companies in Flanders-Belgium. The second aim was to investigate which characteristics of the company and factors of the person responsible for implementing HP, were related to the implementation of environmental HP interventions. The third aim was to investigate which characteristics of the company and which personal factors of the person responsible for implementing HP, were related to being supportive towards investing more in HP in the future.
Design
An online, quantitative survey was organized. An email with an invitation to participate was sent to all sheltered and social workshops in Flanders (n = 148). The social economy in Flanders comprises four types of companies, each with their own target population [8]. In this study, two types were included which employ the largest group of disadvantaged people. Sheltered workshops employ mainly people with disabilities (intellectual and physical). Social workshops provide employment to people with physical, social or psychological problems (e.g. people with psychiatric problems, people reintegrating into society after prison, immigrants). The two excluded types of social economy companies were the local service economy who employ older people who are already long-term unemployed, and the insertion companies who provide a job for people with a low education level together with a history of long-term unemployment. The email-list was provided by the umbrella-organization for the social economy (CollondSe). The person who would normally be responsible for implementing HP interventions, completed the questionnaire. After two weeks, a reminder was sent. The study was executed from February to April 2013. The study was approved by the ethical committee of the Ghent University Hospital (2013/076). The respondents gave their informed consent for participation in the study by clicking on the link to the questionnaire.
Questionnaire
In the first part of the questionnaire, the current status of HP interventions and the characteristics of the workshop were questioned. The current status of HP was assessed by: "Does the company organize HP actions, besides the obligatory smoking ban at the workplace? Yes or no" and "If so, for which themes and how did the company organize HP?". Examples were given for each HP action to make sure respondents knew what is understood under HP. The examples were: policy changes (e.g. an alcohol ban during lunch), environmental changes (e.g. providing fruit for free), education in groups (e.g. group session on healthy food), individual guidance (e.g. counseling at the social department), and short running actions (e.g. a smoke-free day). The themes were: nutrition, physical activity, smoking, alcohol use and mental health. Five new variables were constructed by recoding per theme the options 'policy changes' or 'environmental changes'. The sum of these five variables was made and recoded into a new variable with categories 'implemented an environmental intervention' and 'no environmental intervention implemented'.
Three characteristics of the workshop were assessed. First, the type of workshop (sheltered or social workshops) was asked. Second, the size of the workshop was asked and recoded into small (less than 49), medium (between 50 and 249) and large (250 employees or more) companies. Finally, the economical sector was asked including: primary (agriculture, retrieval of raw materials), secondary (industrial sector), tertiary (supplies commercial services) and quaternary sector (not-commercial service sector e.g. hospitals, education, social work, cultural sector).
In the second part, the personal opinion of the respondent about HP was questioned. This respondent was the person responsible for implementing HP in the company or the person that could have that task (if no HP was already implemented).
Being supportive towards implementing HP in the future, was asked by the question: ' Are you a supporter to invest more in HP at your company in the future?'. A 5point Likert scale was used ranged from '1-totally disagree' to '5-totally agree'. Because of a skewed distribution, the variable was dichotomized into 0 'no supporter or neutral' (scores 1-3) and 1 'being a supporter' (scores 4 and 5).
A second question assessed the perception of the respondent on the statement if employees with a disability benefit from health promotion initiatives. The answer possibilities to that question were: 1) yes and there are enough suitable interventions for this specific group, 2) yes but the existing interventions are not adapted to people with a disability and therefore the results are limited, 3) no because the target group is not open for health messages, 4) no because the health and social issues of the target group are too big for the means that are available in the company.
The questions about the personal factors, derived from the Theory of Planned Behaviour, were based on the questionnaire developed by Downey and Sharp [21]. To be in line with the other questions in the questionnaire, all answers were rated on a 5-point Likert scale, instead of the 7-point Likert scale used in the original questionnaire [21]. The questionnaire was adapted to the Belgian situation, such as the inclusion of the trade union in the subjective norm scale and the exclusion of questions about discretionary spending on health care. Clarity of the questions and exhaustiveness of the questionnaire were tested in people working in the umbrella organization of the workplaces and some employees of the social department of the workplaces.
Attitude was measured by two constructs: behavioural beliefs (the perception of the respondent concerning the benefits of HP, e.g. investing more in HP will increase the moral of employees) and outcome evaluations (the importance the respondent gives to these benefits, e.g. trying to improve employees' morale is desirable). Five different beliefs and their accompanying evaluations were assessed. The attitude-score was calculated by multiplying beliefs with the outcome evaluation and dividing them by 5 (see Table 1 for the 5 attitudes asked). The higher the attitude-score, the more likely the respondent beliefs that HP is beneficial and that the outcome is desirable. A total attitude-score was calculated by taking the mean of the 5 attitudes. The Cronbach alpha for the scale was 0.82.
Behavioural control was measured using three questions on control over implementation, resources and budgets. For the total scale, the mean of the three questions was calculated. The Cronbach alpha for the scale was 0.87.
The subjective norm was measured by two constructs: normative beliefs (the perception of the (dis)approval of a reference group concerning HP, e.g. how likely is it that your colleagues believe that you must invest resources in HP?) and the motivation to comply (the importance of this reference group for the respondent, e.g. how much do you care whether your colleagues approve that you invest in HP?). Normative beliefs and motivation to comply were assessed concerning eight reference-groups: the seven reference-groups from the questionnaire of Downey and Sharp [21], plus the reference-group 'trade union' ( Table 1). As with the attitude scale, the scores of the normative beliefs and their accompanying motivation to comply were multiplied and divided by 5. The higher the score on the subjective norm, the more likely the respondent believes that HP should be implemented according to a relevant reference-group.' A total score was calculated by taking the mean. The Cronbach alpha for this scale was 0.77.
Moral responsibility was measured using a scale developed by Hart [22]. Three dimensions of moral responsibility were included (see Table 1 for the themes). The mean was used as total score on moral responsibility. The Cronbach alpha for this scale was 0.60.
Data analysis
To investigate the current status of the implementation of HP interventions (aim 1), percentages were given of the currently implemented HP themes and actions. Chi 2 -test were used to analyze if these results differed by the characteristics of the workshop.
To investigate which characteristics of the workshop and individual factors of the respondent were related to the implementation of environmental HP (aim 2) and to being supportive towards implementing HP in the future (aim 3), univariate logistic regressions were used. A multivariate logistic regression was performed with all significant factors from the univariate analyses.
SPSS 21 was used to analyze the data. P-values lower than 0.05 were considered to be statistically significant.
Descriptives
Eighty-two workshops completed the online questionnaire (response rate 55.4 %), of which 65.9 % social workshops and 34.1 % sheltered workshops. Half of them were medium-sized (48.1 %), followed by largesized (27.8 %) and small companies (24.1 %). Concerning the economical sector, 11.2 % could be categorized into the primary sector, 31.2 % into the secondary sector, 40 % into the tertiary sector and 17.5 % into the quaternary sector.
The means and standard deviations of the personal factors of the respondent can be found in Table 1. Of the 82 respondents, 24 were directors of the company, 32 were working at the social department, 15 were working on the personnel department, 7 were department coordinators and 4 were prevention advisors.
Aim 1: Current status of HP in sheltered and social workshops
Of the workshops, 64.6 % indicated that they organized HP. The theme that was most frequently chosen to work on in a HP intervention was alcohol use (58.5 %), followed by nutrition (50 %), mental health (37.8 %), tobacco use (36.6 %) and physical activity (28 %). The kind of interventions that were already implemented were most policy changes (43.1 %) and individual guidance (43.1 %), followed by education in group (31.5 %), environmental changes (26.2 %) and short running actions Normative beliefs ('How likely is it that following persons believe that you should invest in HP' with '1-very unlikely to approve' to '5-very likely to approve') * Motivation to comply ('How important is the opinion of following persons' with . The information about which actions per theme were implemented, can be found in Table 2. No significant differences in size of the company (chi 2 = 0.997, df = 2, p = 0.607), type of workshop (chi 2 = 0.002, df = 1, p = 0.962) or sector (chi 2 = 4.985, df = 3, p = 0.173) were found between those who had implemented HP and those not.
Also, no significant differences were found between the themes and kind of interventions by type of workshop or by sector. One significant difference was found by size. Large workshops were more likely to facilitate group education (54.8 %), compared with mediumsized (36.8 %) and small workshops (15.8 %) (chi 2 = 6.592, df = 2, p = 0.037).
Environmental HP interventions were more commonly implemented for alcohol (45.1 % of the workshops), followed by nutrition (31.7 %), and tobacco (23.2 %). Only 9.8 % of the workshops implemented an environmental intervention concerning physical activity and 7.3 % concerning mental health.
Only twelve percent of the respondents indicated that employees with a disability benefit from HP initiatives and that there are suitable interventions available for this specific group. More than half of the respondents (55 %) answered that employees with a disability could benefit from HP interventions but that there are no interventions available that are adapted to the target group. The other 33 % of the respondents answered that employees with a disability do not benefit from HP initiatives: 26 % indicated that the target group is not open for health messages, and 7 % indicated that the health and social issues of the target group are too big for the means that are available in the company.
Aim 2: Factors of implementing environmental HP interventions
Almost 60 % (59.8 %) of the workshops had one or more environmental HP intervention implemented. In univariate logistic regressions, none of the characteristics of the workshop and none of the personal factors of the respondents were related to having an environmental intervention implemented.
Aim 3: Factors of being supportive towards investing more in HP in the future
Half of the respondents (50 %) were supportive towards investing more in HP in the future. In Table 3, the results of the univariate logistic regressions with the characteristics of the workshop and the personal factors, can be found.
None of the characteristics of the workshop were related to being supportive towards more HP in the future. All attitude variables were positively related to being supportive. Respondents scoring one point higher on the attitude scale were even 5 times (OR = 5.15) more likely to being supportive to more HP than those scoring one point less. Respondents believing that employees are expecting that the workshop invest in HP, were more likely to be a supporter of more HP in the future. Respondents agreeing that the benefits of HP exceeds the costs, and those who had the belief that HP is an obligation, were more likely to be supportive. Also, the total moral responsibility scale was positively related to being supportive towards more HP.
A multivariate logistic regression was performed with all significant results. As all attitudes and the total scale was significant related to the dependent variable, the total scale was chosen to be included in the analyses (Table 3). Only the attitude-scale stayed significantly related to being supportive towards more HP in the workshop in the future.
Discussion
Sheltered and social workshops in Flanders (Belgium) employ people from disadvantaged groups who are less likely to be reached by mainstream HP interventions. Therefore, these companies might be a good channel to provide HP in disadvantaged people. In this study, the current status of HP interventions in these companies and factors related to the implementation were studied.
Companies can organize HP in different ways, but environmental and policy changes are seen as most effective and long-lasting [12,15,16]. In this study, almost 60 % of the workshops had an environmental or policy HP intervention implemented. Besides environmental and policy HP interventions, individual HP strategies can be used to increase the knowledge and positive attitudes concerning a healthy lifestyle, or to improve the self-efficacy to perform the healthy behaviour [23]. Workshops also invest in these strategies: 40 % gave individual guidance to their employees and one third organized educational group sessions. In 2012, the Flemish Institute for Health Promotion and Disease Prevention organized a survey on health indicators in general Flemish companies [24]. In their report, percentages of HP ranged from 15 % for nutritional topics to 40 % for tobacco interventions. Although they used other measurements to assess HP, our results indicate that workshops have almost equal or more attention to HP themes compared with normal economy companies. The daily confrontation with health-related problems of their employees, such as high sickness rates due to unhealthy lifestyle, and alcohol and tobacco [25]. Also in the Flemish report on health indicators, the same was observed [24]. In addition, Flemish companies working in the tertiary sector, were less likely to have HP implemented compared with companies from the secondary and quaternary sector (primary sector not included in this study). But in the present study, none of these characteristics were related to having an environmental HP intervention or to being supportive towards investing in HP in the future.
According to the Theory of Planned Behaviour, this study found that having a positive attitude towards HP was related to being a supporter of investing more in HP in the future. Also, in the study of Downey & Sharp in normal economy companies, a positive attitude was the best predictor of the intention to implement WHP [21]. They also found that the perception of control over resources and budget in human resource managers and moral responsibility in general managers predicted the intention to implement WHP. In our study, control was not related to being a supporter of investing more in HP. Moral responsibility was significantly related in the univariate analyses but the significance disappeared when controlling for the other significant factors. Subjective norm was not a significant predictor in both studies.
However, none of these factors were related to the current implementation of HP in the workshop. This can partly be explained by the intention-behaviour gap. Some barriers may exist between having a positive intention to invest in HP and the actual behaviour of implementing HP. Known barriers for the implementation of HP are a lack of time and logistical challenges [26]. Also in workshops, these barriers may exist as mostly only one or two persons are responsible for the implementation of HP. Another barrier can be the lack of tailored interventions for this special group [5]. In our study, 55 % of the respondents indicates that there are no suitable interventions for the target group. More research is needed to find better individual predictors of (the intention to invest in) the implementation of HP at the workplace.
But also methodological problems can explain this result. The question we asked was if the company had already implemented HP interventions and not if the respondent had experience in implementing HP in the company. Therefore, other persons could have been responsible for the implementation of the current HP interventions. Also, no questions were asked about the socio-demographic characteristics (such as age and gender) and the respondents' knowledge on and skills for implementing HP interventions, while these factors can be important in predicting the implementation of HP at the workplace. If the respondents have limited knowledge on HP and have not much expertise in implementing HP, they will be less aware about possible HP actions and their implementation. In addition, we have no information about details and quality of specific interventions that were implemented, such as duration of intervention, type of education sessions and dissemination of the intervention. This lack of information can be seen as a limitation of this study.
Another limitation is the low response rate and relatively small sample size. Only 55 % of the workshops participated in the study, which limits the generalization of the results. Of all social workshops, 57.4 % participated in the study compared to 51.9 % of all sheltered workshops. This low response may be associated with the reconstruction of sheltered and social workshops in 2013, leading to an overload of work and insecurities for the companies. Therefore, it may be that they were less motivated to cooperate to this project.
Finally, only univariate analyses could be performed to study aim 2 as none of the included variables were significantly related to implementation of HP at the workplace.
Implications
Despite these limitations, some implications can be formulated. To persuade HP implementers in workshops to invest more in HP in the future, disseminators of HP interventions have to influence the attitudes towards HP in these implementers. This can be done by increasing the knowledge of implementers about HP interventions in workshops, to show them the advantages of HP interventions, and to discuss negative consequences of the absence of HP in this specific group of employees [23]. However to our knowledge, until now, the effectiveness of HP interventions for disadvantaged groups in the setting of workshops is not studied. Therefore, research is needed on the effectiveness of HP on e.g. decreasing absenteeism and increasing the moral of employees working in workshops. Also cost-effectiveness studies are needed to prove that the benefits of HP outweigh the costs.
Besides working on the attitude of the implementers, it is also important to increase their skills to plan, implement and evaluate HP initiatives. In most workshops, only one or two persons are responsible for the implementation of HP. Therefore, tools should be developed to help them and guide them through the different stages of implementation. An important topic in these tools is how to make the HP project a team effort instead of the project of one or two individuals.
But to have sufficient effect, interventions should be tailored to the specific target group and setting. As HP in people with disabilities is still underdeveloped [5], workshops are using HP interventions that are not adapted to the needs of these employees or to the setting of workshops. One of the elements of structural feasibility for the implementation of HP in workplaces are the literacy levels, which can be low in workshops [27]. Therefore, interventions such as choice architecture, that alter the properties or placements of objects in the workplace with the intention to change health-related behaviour, should be investigated as these interventions require minimal conscious engagement, and can change the behaviour of many people simultaneously [17]. As 55 % of the persons responsible for implementing HP indicated that existing interventions are not adapted to people with a disability, more research is needed on which HP interventions are suitable for people with disabilities (in terms of attainability) and which HP interventions are effective in this target group (in terms of behavioural change and health outcome).
Conclusion
Although this study described the specific situation of sheltered and social workshops in Flanders (Belgium), the results of this study could be used to optimize dissemination of HP in companies working with employees of so-called disadvantaged groups. Creating positive attitudes towards HP could make the implementer more supportive towards investing in HP in the future.
Abbreviations HP: Health Promotion; WHP: Workplace Health Promotion.
Competing interest
The authors declare that they have no competing interests.
Authors' contributions AH contributed to the conception, design, acquisition of the data and outlined the manuscript, LM contributed to the design and interpretation of the data, JM, BD and ID contributed to the analyses and commented on the interpretation of the data. All authors read and approved the final manuscript. | 2016-05-12T22:15:10.714Z | 2015-12-01T00:00:00.000 | {
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140626776 | pes2o/s2orc | v3-fos-license | Isotopes as Tracers of Water Origin in and near a Regional Carbonate Aquifer: the Southern Sacramento Mountains, New Mexico
High-elevation groundwater sampled in 2003 in the Sacramento Mountains defines a line resembling an evaporation trend in δD-δ 18 O space. The trend results from recharge of winter precipitation into fractured limestone, with evaporation prior to recharge in broad mountain valleys. The same trend occurs in basin groundwater east and west of the range, indicating the high Sacramento Mountains as the principal regional water source, either direct from the limestone aquifers or from mountain-derived surface water. Tritium and carbon-14 indicate bulk residence times of a few decades in the high Sacramento Mountains and at Alamogordo, and of thousands of years south of Alamogordo and in the artesian aquifer near Artesia. Stable O, H isotope data fail to demonstrate the presence of Sacramento Mountains water in a saline aquifer of the Hueco Bolson (Texas).
Introduction
In the Basin and Range province of the southwest USA, stable isotope studies have proved useful in distinguishing sources of recharge where altitude effects are large, e.g., Tucson Basin [1], or where OPEN ACCESS isotope effects due to latitude/altitude and evaporation generate river water that is distinctive beside native basin groundwater [2,3].
The Sacramento Mountains of south-central New Mexico (Figure 1) include a broad area of forested, well-watered terrain, the source of perennial streams flowing east toward the Roswell Basin and the Pecos River and of intermittent streams flowing west into the Tularosa Valley.Pre-development and recent water level data indicate groundwater movement from Tularosa Valley to the Hueco Bolson [4][5][6].Mayer and Sharp [7] suggested regional flow of groundwater from the Sacramento Mountains to the Texas-New Mexico border, through karst aquifers.Groundwater samples for this study were collected in the Sacramento Mountains and the flanking basins from 2003 to 2008.The aim of the study was to use environmental isotopes to determine the relationship between water from the Sacramento Mountains and the adjacent basin aquifers.The relationship between groundwater in the high mountains and that in the Tularosa Valley, the deep alluvial basin to the west, is the first topic to be addressed.The relationship between groundwater in the high mountains and that in the hard-rock Roswell artesian basin to the east is the second topic to be addressed.In both cases, we also attempt to constrain groundwater residence times, and to determine the seasonality of recharge.Finally, we discuss whether the isotope signature of Sacramento Mountains groundwater can be recognized as far south as the Hueco Bolson in Texas (Figure 1).
Background
Figure 1 shows the location of the study areas.Area 1, encompassing the Sacramento Mountains near Cloudcroft and Weed, and the freshwater lens on the western flank of the mountains, encompasses sites sampled for the first topic described above.Area 2 stretches from the eastern flank of the mountains to the Pecos River at Artesia, and encompasses sites samples for the second topic.
Topography, Climate and Vegetation
The Sacramento Mountains rise to 2500-2800 m above sea level (m.a.s.l.) in the study area.A steep western escarpment with deep canyons abuts the Tularosa Valley, a typical fault-bounded basin of the Basin-and-Range province.Tularosa Valley continues southward into Texas where the valley is named the Hueco Bolson; Neogene alluvium fills the entire extent of the combined basin to depths of 600 to 3000 m in the basin center [6].The eastern flank of the Sacramento Mountains approximates a dip-slope, and descends gradually toward the Pecos River.No deep alluvial basin is present on the east side of the range.The climate in the basins is semi-arid; average annual precipitation is 335 mm at Alamogordo and 340 mm at Artesia.In the high mountains, precipitation is higher, e.g., 715 mm at Cloudcroft near the range crest [8].There are two wet seasons, a weak summer monsoon (June to October) providing 65%-70% of the precipitation, and a winter season of rain and snow from frontal weather systems [8].The amounts of both winter and summer precipitation vary greatly from year to year (Figure 15 of [9]).Vegetation consists of coniferous forest interspersed with grassy valleys above 2300 m.a.s.l..At lower elevation, scrubby oak forest and desert scrub predominate, except along perennial streams where riparian forest is present.Much of the study area is dry ranch land on which groundwater pumping is essential to the survival of cattle herds.Large-scale irrigated agriculture, using quarter-section and larger center-pivot and side-roll equipment, is practiced on the Pecos River flood plain near Artesia.Scattered irrigated plots are present near Tularosa.
Geology
The Sacramento Mountains constitute a tilted horst with range-bounding faults on the western side (Figure 2), and consist of Paleozoic marine sedimentary rocks, mostly Permian-Mississippian limestone and evaporite, overlying concealed Precambrian basement [9,10].The surface east of the range crest approximates a dip slope, with a discontinuous veneer of lower members of the San Andres formation overlying dolomite and anhydrite of the Yeso formation, both overlain to the east by the Queen-Grayburg anhydrite and limestone and dolomite of the San Andres formation.Thin (40 m) Quaternary alluvium overlies Paleozoic strata on the Pecos River flood plain.West of the range crest, the entire Paleozoic section of the region, mainly carbonate strata, is exposed.Neogene alluvium fills the Tularosa Valley to depths of 230 to 300 m at Alamogordo.
Geohydrology
The carbonate strata constitute a regional aquifer system conveying water from the mountains to the basins, eastward from the range crest through the Yeso formation, and westward through the highly fractured Paleozoic carbonate section.A map of the potentiometric surface east of the range crest is available in ([9], Figure 18), and indicates general eastward groundwater flow.West of the range crest, groundwater levels are less precisely known within area 1, but they decline steeply towards the west and southwest elsewhere on the escarpment [9].In the high mountains, the geohydrology is complex and governed by the detailed lithology of the Yeso formation.The following geohydrologic features are present ( [9], Figure 25): a regional aquifer, locally confined beneath impermeable interbeds of the Yeso formation, and probably continuous with regional aquifers east of the range; multiple perched aquifers overlying the regional aquifer, some discharging in small springs controlled by impermeable strata; and vadose zones above and between the perched aquifers.Large summer rain events in 2006 and 2008 caused rapid water-level response in the perched aquifers, but slower response in the regional aquifer.In the Roswell artesian basin, a shallow unconfined aquifer is present in carbonate strata overlying the Queen-Grayburg anhydrite, at depths less than 100 m below the surface at Artesia.The regional aquifer in this area, at 200-300 m below the surface, is confined beneath the Queen-Grayburg anhydrite and was artesian at the time of first development; subsequent pumping has lowered static water levels by tens of meters (Table 1) [11].Groundwater is present in an unconfined basin-fill aquifer in the alluvium of the Tularosa Valley, where supply wells pump water from the upper 50 m.
Figure 2.
Cross section X-X' (see Figure 1 for location), after Roswell Geological Society (1956).SL = sea level.The east slope of the Sacramento Mountains is a dip-slope with widespread veneer, too thin to depict here, of the Glorieta Sandstone and overlying members of the San Andres formation.
Previous Isotope Studies
Stable oxygen and hydrogen isotope and tritium data were collected for rainwater, surface water and groundwater from Roswell Basin in the late 1970s [12,13], in order to identify sources of recharge and groundwater residence times.The authors concluded that more detailed sampling was required, but were able to identify loci of local, rapid recharge using tritium data in the mountain areas and near Roswell [13].Stable oxygen and hydrogen isotope data for surface water in the Pecos River [14,15], have been used to determine the relative contributions of winter snow and monsoon precipitation to the river in Texas, the authors concluding that the latter predominates [14].Sulfur isotopes in Sacramento Mountains groundwater have been utilized to determine the relative inputs of evaporite gypsum, oxidized sulfides and rain sulfate to the dissolved sulfate inventory [16].Reference [17] provided stable O and H isotope and 14 C data for the well-field supplying water to the air-force base in Tularosa Valley, and interpreted the data to indicate water residence times greater than 1000 years.The most detailed recent work is in reference [9], which presented detailed stable isotope data for precipitation and groundwater collected in 2006-2009 between the range crest and Hope, New Mexico.The authors identified predominant summer recharge in years of heavy summer rainfall, and used tritium, 14 C and CFCs to estimate groundwater residence times of decades in the high mountains, to thousands of years in the aquifer extending east of the range.The pattern of stable O and H isotope data in groundwater presented in reference [9] differs markedly from that in our dataset, allowing for an improved understanding of the hydrology of the mountain range when both datasets are taken into account.Our study also complements reference [9] in extending spatial coverage into flanking basins east and west of the Sacramento Mountains.
Analytical Methods
Samples were taken from domestic, agricultural and municipal production wells, springs, and surface water in the Peñasco and Pecos Rivers, and from rain gauges near Weed.Isotope measurements (except accelerator mass spectrometry carbon-14) were performed at the Environmental Isotope Laboratory, University of Arizona.Stable O, H and C isotopes were measured on a Finnigan Delta S ® dual-inlet mass spectrometer equipped with an automated CO 2 equilibrator (for O) and an automated Cr-reduction furnace (for H).Stable S isotopes were measured on a Thermo Electron Delta Plus XL ® continuous flow mass spectrometer equipped with a Costech ® elemental analyzer for preparation of SO 2 .Carbon-14 was measured by accelerator mass spectrometry at the NSF-Arizona Accelerator Facility, University of Arizona.Data generated for this study are listed in Table 1.Analytical precisions (1σ) are 0.08‰ (O), 0.9‰ (H), 0.15‰ (C) and 0.15‰ (S).Detection limits are 0.6 TU ( 3 H) and 0.2 pMC ( 14 C).
Correction of Raw Carbon-14 Data
The data lack sufficient detail for chemical balance modeling; therefore a simpler method based on δ 13 C values is used, following ( [18], p. 210).Values of δ 13 C for soil gas are assumed to be −23‰ (corresponding to 100% C3 plant matter input) for the forested mountains, and −19.9‰ (corresponding to 75% C 3 input) for desert areas."Dead" rock carbonate of Guadalupian age has δ 13 C values from +1 to +5‰ [19,20]; for these strata corrections were calculated for +1‰ and +4‰.Corrected ages are given in Table 1.In the basin-fill aquifer near Alamogordo, corrections were calculated for rock δ 13 C from 0‰ to +3‰, representing the entire Paleozoic section.
Area 1: High Sacramento Mountains
Samples were collected from wells near La Luz and Fresnal canyons and areas near New Mexico Route 24 east of the range crest (Figure 3).All samples are from fractured limestone except for 1-4, which is from shallow alluvium in Fresnal Canyon. 1, where the corresponding number is 1-1 for area 1, or 2-1 for area 2. Black circles: sample sites for this study; white circles: sample sites from reference [17].
O and H Isotopes
On a plot of δD vs. δ 18 O, most of the data fall on a straight line with a slope near 5.6 (Figure 4A), henceforth called the Sacramento Mountains Trend (SMT).The straight line intersects an estimate of average winter precipitation at a station at 2790 m.a.s.l.(calculated as arithmetic means (because amount data are not available) of δ 18 O and δD for three bulk collections in March 2007, 2008 and 2009, and representing the prior 3 months; data from [9]), but does not intersect mean summer precipitation [9] for that station.In Figure 4B, three groups of δ 18 O values emerge in relation to site altitude.For the wells, collar altitude is used because static water levels are not available in all cases.Values of δ 18 O of group W (western slopes) overlap those of group H1 (high elevations), despite the large altitude difference between the two groups.The difference between groups H2 (high elevations, but generally lower than H1) and H1 is too great to attribute to altitude.Group H2 sites (1-13, 1-14, 1-16, 1-17) are adjacent to broad, flat, canyon bottoms, a typical geomorphic feature of the Sacramento Mountains.In such places, deep soil (more than 1 m near site 1-16) overlies carbonate strata, while elsewhere carbonate outcrop is widespread.The data for groundwater in 2003 differ from data for groundwater in 2006-2009 [9].The latter occupy a field between the SMT and summer rain for 2006 and 2008 (Figure 4A), and reflect rapid recharge from heavy monsoon rains in 2006 and 2008.Prior to 2003, there had been no large monsoon rain totals since 1997.[21], and 1.8‰/1000 m [13].Site numbers (e.g., 3) correspond to entries in Table 1, where the corresponding number is 1-3.
Other Parameters
Groundwater in this area generally has δ 34 S values of 10‰ to 13‰, tritium concentrations of 1 to 3 TU, and 14 C in the range 72 to 93 pMC (cf.0 to 7 TU, and 83 to 93 pMC, for samples from 2006 to 2008 [9]).Corrected 14 C data indicate post-bomb water in the west-slope canyons and in two high-elevation springs, with older groundwater (300-4500 years) at sites 1-15 and 1-18 (Table 1).
Interpretation
The SMT can be explained as an evaporation trend originating in winter precipitation.Evaporation prior to infiltration varies in degree, and is greatest in groundwater near the broad canyon bottoms, (sites 1-13, 1-14, 1-16 and 1-17), where standing water and wet soil are likely to undergo partial evaporation.Well-mixed high altitude groundwater will plot between groups H1 and H2, and this isotope signature will be found in groundwater of the limestone aquifer at lower elevations unless water of different isotope composition is added downgradient.Evaporated runoff from high elevations may plot on the SMT to the right of group H2.Addition of water from local low-elevation precipitation would shift groundwater isotopes towards the GMWL.
The difference between the 2003 and 2006-2009 data sets indicates two modes of recharge.In years with unusually wet summers, (e.g., 2006 and 2008), summer recharge with little evaporation is the dominant source of recharge.The local meteoric water line (LMWL) in Figure 4A is governed by rainfall from those years, and may not apply under drier conditions.Following a succession of dry to average summers, however, winter recharge predominates, even though there is more precipitation in summer than in winter rain on average.Such was the case from summer 1998 to 2003 when sampling for this study occurred.Under these conditions, evaporation of the infiltrating water occurs in the broad canyon bottoms east of the range crest, but is not observed between the range crest and the canyons on the steep west escarpment.
Tritium and corrected 14 C contents of high-elevation groundwater indicate the presence of post-bomb recharge, but tritium levels in 2003 were predominantly lower than average tritium in post-1992 precipitation (4-7 TU, see Table 1 and [9]; compare a better-constrained average of 5.3 TU for Tucson [22]), indicating mixing with pre-bomb meteoric water.By 2006-2008, more post-bomb recharge was present, tritium-helium dates were mainly 1-15 years, and CFC ages were largely 20-30 years [9].Values of δ 34 S indicate Permian marine gypsum (+12‰ to +13‰) as the main source of sulfate; lower values most likely reflect oxidation of sulfide present in these strata [16].
Area 1-Alamogordo and Tularosa
Sampling from supply wells in basin-fill alluvium represents lower-TDS water suitable for human consumption; brackish water is also present >5 km west of the range front.Groundwater in this area flows west at Alamogordo and Tularosa [4,5], but parallel to the range front south of Alamogordo, where no major canyons contribute water to the basin.
O and H Isotopes
Most data plot on the SMT (Figure 5), to the right of group W. Samples from Tularosa include the most and least evaporated of the set.Data for wells south of site 1-33 (δ 18 O between −9.9‰ and −9.5‰ in reference [17]) differ from data collected for the present study in the same area (δ 18 O between -9.5‰ and -9.0‰).Actual variation in δ 18 O (as opposed to measurement error) is unlikely in such old groundwater (see below); the earlier data are not used here.In southeastern Tularosa Valley, sites 1-39 and 1-40 (Figure 1) have groundwater that plots below the SMT Figure 5. Plot of δ 18 O vs. δD for groundwater samples from basin sediments near Alamogordo and Tularosa, in relation to samples from La Luz and nearby canyons and the high Sacramento Mountains.Samples 1-39 and 1-40 are from basin fill more than 40 km south of Alamogordo (see Figures 1 and 3).
Other Parameters
Tritium is present (1-3 TU) north of site 1-33, and is generally absent (below detection to 0.5 TU) south of 1-33. 14C generally decreases from near 80 pMC near Tularosa to 20 pMC south of Alamogordo (Figure 6).Corrected 14 C data indicate young groundwater (post-bomb to a few hundred years) north of Alamogordo and in La Luz canyon, and much older water (500-7500 years, considering also corrected data from [17]) south of Alamogordo.Values of δ 34 S are near +12‰.At sites 1-39 and 1-40, tritium is below detection, 14 C levels are 3 pMC, and corrected ages are 12,000 to 21,000 years (Table 1).
Interpretation
O and H isotope data plotting on the SMT indicate high-elevation precipitation as the source of groundwater in basin alluvium near Alamogordo and Tularosa.Groundwater from the high Sacramento Mts.flows to La Luz Canyon sample sites without isotopic shift.The higher degree of evaporation in samples from the alluvial aquifer could be explained: (1) as mountain-block recharge combining more-evaporated and less-evaporated recharge from high elevations; or (2) as mountain-front recharge of surface water supplied from high elevations by way of the mountain canyons.The absence of an evaporation signature in groundwater from carbonate strata in La Luz Canyon, between the range crest and the basin) argues against the first possibility, while the presence of evaporated water in the alluvium argues for the second.The higher degree of evaporation of groundwater farther from the range front (Tularosa, 12-15 km from the range front), in contrast to groundwater nearer to the range front (Alamogordo, within 6 km), suggests that the sites of infiltration of surface water extend into the basin, rather than being confined to a narrow zone at the range front.This is particularly evident in the case of site 1-22 at Tularosa, (Figure 5), where the coincidence of low δD and δ 18 O with high 14 C indicates recharge of very recent runoff at a distance of up to 15 km from the range front.Both mountain-front and mountain-block recharge seem likely, but the data do not indicate the relative amounts.In the basin fill south of Alamogordo, tritium and 14 C data are consistent with slow southward flow of groundwater, with little recharge from nearby mountain canyons.
Area 2: Peñasco to Artesia
Samples were collected between Peñasco and Artesia (Figure 3).Near Peñasco, groundwater samples were taken from a spring and a windmill in limestone, and from wells in the Rio Peñasco flood plain.East of the range front, as far as site 2-10, an unconfined aquifer (the principal aquifer of [11]) is present near the boundary of the Yeso and San Andres formations.Recharge to these strata may occur near the range front.Samples are from domestic and agricultural wells up to 260 m deep, with static water levels (SWLs) near 190 m below the surface.East of site 2-10, beneath a broad plain west of the Pecos River, two major aquifers were sampled.An unconfined aquifer with SWLs from 30 to 60 m below the surface exists in flood-plain sediments near Artesia.The eastward continuation of the principal aquifer, 275 to 300 m below the surface, is confined beneath the Queen-Grayburg anhydrite (Figure 2).It was artesian at the time of first exploitation; SWLs at present range from 20 to 60m below the surface.Surface water samples were collected from the Peñasco and Pecos Rivers.
From Peñasco to Hope, groundwater flow is east-southeast (Figure 18 of [9]).Allowing for variation due to pumping, SWLs in the principal aquifer east of Hope are close to 1000 m.a.s.l.(Table 1).Southward flow is likely in this area.
O and H Isotopes
All groundwater and surface water samples plot close to the SMT (Figure 7A).Most data cluster at the intersection of the SMT with the GMWL, where the separation of the lines is less than 2‰ in δD, and therefore impossible to resolve within measurement error.Data for the principal aquifer from Mayhill to Hope [9] match the present data set in δD but include lower values of δ 18 O.Two groundwater samples from near Artesia (2-13, 2-19) plot to the right of the main data cluster.Surface water from the Pecos River in the reaches between Artesia and Red Bluff ( [15] for 1984-1987, [14] for 2005, and data from this study) largely plot as a linear trend, close to an extrapolated SMT (Figure 7A,B).
Previous data for the principal aquifer [13] pre-date automated isotope methods, and partially overlap the main data cluster from the present study.The two data sets correspond in δ 18 O, but the older δD data have a spread >20‰, apparently spurious, and appear not to be useful.Weighted precipitation averages from [12] are for δ 18 O alone, and have been plotted on the GMWL in Figure 7.
Tritium
In 1977-1978, when bomb tritium averaged about 35 TU in local precipitation, surface water and alluvial groundwater from the Peñasco River flood plain contained about 10 TU, and tritium in the principal aquifer near Artesia was below detection [13].In samples collected for this study, tritium is present at low levels (<2 TU) in groundwater from near the range front (sites 2-2, 2-3, 2-5), at site 2-19 in the shallow aquifer, and in one deep aquifer sample (site 2-21, 0.5 TU); at other sites it is below detection (Figure 8).Groundwater from the alluvial aquifer beneath the Peñasco River (site 2-3) contains 1.5 TU, distinctly lower than the average for precipitation, and consistent (cf.[13]), with a large pre-bomb groundwater contribution to the surface water of the river.Reference [9] listed tritium contents <2 TU in groundwater between Elk and Hope. 1O vs. δD for groundwater and surface water samples from study area 2. (A) All data from this study.The field of data from [13] is for the principal aquifer from Artesia to Roswell, and encompasses all but three outlying data points.The inset shows a magnified view of clustered data.Site numbers (e.g., 1) correspond to entries in Table 1, where the corresponding number is 2-1; (B) Plot of δ 18 O vs. δD for the Pecos River between Artesia and Red Bluff, from other studies [14,15], relative to the SMT.showing well depths and static water levels (SWL) in relation to the surface."Shallow" refers to the shallow aquifer at Artesia, and "deep" to the deeper artesian aquifer.Also shown are measurements of carbon-14 (pMC) and tritium (TU).Tritium below detection is indicated as "bd".
Interpretation
Groundwater in the Pecos Slope and artesian aquifers is largely uniform in isotope content over an east-west extent of about 100 km, and lies on or close to the SMT.A dominant water source in the high Sacramento Mountains is therefore likely.East of Peñasco, a few samples (2-13, 2-19, 2-24 and 2-25) have isotope data plotting on the SMT, but to the right of the main data cluster; these may reflect local recharge of evaporated surface water.Site 2-13 is close to the ephemeral lower reach of the Rio Peñasco, where recharge of evaporated surface water may occur.The other three samples are from the shallow aquifer beneath irrigated fields, where reflux of evaporated irrigation water is probable.Addition of local rainwater is likely for site 2-10 (Figure 7).
Bulk groundwater residence times (the corrected versions of the data shown in Figure 8) in the principal aquifer are 1300 to 5600 years east of Hope, greater than those suggested in [13].
Most of the δ 18 O and δD data for the Pecos River between Artesia and Red Bluff plot on a linear trend close to an extrapolated SMT, regardless of season (Figure 7B), and can be therefore be generated as a result of evaporation of water like that in the principal aquifer at Artesia.The principal source of river water in this area is therefore most likely the Sacramento Mountains, either by natural recharge from the aquifer, or by way of irrigation on the Pecos flood plain.If this is true, mountain-derived water is discharged with an isotope signature of evaporation into the river near Roswell and Artesia.This suggests a modification to the modeling, based on deuterium excess, of river water sources in reference [14].
Water Sources in Study Area
Most water sampled for this study plots on the Sacramento Mountains trend (SMT) in δD-δ 18 O space.The SMT originates in high-altitude winter precipitation.Such precipitation is therefore the principal and ultimate source of groundwater in the area between Alamogordo and the Pecos River.Most water in the Pecos River near Artesia also appears to be of that origin.There is scant evidence for recharge of local meteoric water at low altitudes.The few exceptions include groundwater from the southeastern part of Tularosa Valley (where ancient water from high elevations appears to be present), and some unconfined-aquifer samples from Artesia (where local recharge probably occurs).
Seasonality of Recharge
The heavy monsoon rains of 2006 and 2008 generated recharge of distinctive δD-δ 18 O signature in groundwater of the high Sacramento Mountains, but in drier years, 2007 and 2009, groundwater isotopes shifted towards the SMT [9].Monsoon rainfall comparable to that in 2006 had not occurred since 1997, and in the 2003 sampling for this study, winter recharge, plotting on the SMT as a result of local evaporation prior to infiltration, was predominant.Where old groundwater is present (south of Alamogordo and in the principal aquifer of Roswell Basin), δD and δ 18 O conform largely to the SMT.In the long term, therefore, recharge in dry to average years contributes the larger volume to low-elevation aquifers around the Sacramento Mountains.Years with unusually wet summers lead to a transient (a few years) response in the high-altitude aquifers, but make little contribution to the old groundwater in basins at the foot of the mountains.
The Sacramento Mountains are therefore an unusual example of a mountain block in which the dominant season of recharge can change in response to seasonal precipitation amounts, although winter precipitation, only 35% of total precipitation on average, predominates in the long-term.Winter recharge is considered predominant in a number of other mountain ranges in the arid western USA.In the Spring Mountains, Nevada, another carbonate-rock range, winter precipitation is dominant; summer rain contributes about 30% of annual precipitation, but only about 10% of recharge [23].Winter recharge also predominates in the Huachuca Mountains, Arizona, where summer precipitation contributes 54% of the annual total on average, but winter precipitation accounts for 65% ± 25% of recharge [24].In the Santa Catalina Mountains, Arizona [25], and the ranges delimiting the Verde River watershed, Arizona [26], winter recharge is considered predominant, contributing 98% of recharge in the latter case.
Sacramento Mountain Carbonate Strata as a Karst Aquifer
A continuum of aquifers exists in carbonate rock [27].At one extreme, carbonate dissolution leads to wide solution cavities that self-organize into dendritic drainage networks discharging through large springs; water flow rates are commonly 10 2 to 10 4 m/day over distances of 10 3 to 10 4 m.At the other extreme, solution cavities are narrow and of limited interconnection, generating an aquifer with lower flow rates and discharge through many small springs.Although small-scale collapse structures are recognized [9], cavern networks are not developed in the thinly bedded strata, some impermeable, of the study area.A flow rate of 10 m/day over 30 km between the range crest and the eastern range front would result in water travel times of about 8 years.The tritium and 14 C data imply residence times >60 years in the mountain aquifers several cases, while surface water in the Rio Peñasco and associated flood-plain groundwater also contain some pre-bomb precipitation.The isotope evidence indicates widespread persistence of pre-bomb precipitation in groundwater, and flow rates typically much lower than 10 m/day.The Sacramento Mountains therefore fall at the latter end of the continuum of karst aquifers as described above.
Nonetheless, the carbonate strata in and east of the Sacramento Mountains compose a regional aquifer system over a distance of 130 km.Regional carbonate aquifers of similar extent have been demonstrated elsewhere in the region on the basis of geochemical modeling, east of the Salt Basin in West Texas [28], and in the region southwest of the Cuatrocienegas Basin of Coahuila, Mexico [29].
Mode of Mountain-Front Recharge to Basin Alluvium
The location of mountain-front recharge relative to the interface between hard-rock mountain blocks and basin alluvium in the southwest USA has been addressed in several studies.In the middle Rio Grande Basin (New Mexico), infiltration is thought to occur in a narrow zone along the range front of the Sandia and Manzano Mountains [30].In Chino Valley (Arizona) [26] and Tucson Basin (Arizona) [1], evidence indicates infiltration from stream beds downstream of the mountain fronts, at distances of 6 to 10 km in the case of Tucson Basin.Groundwater isotope data also indicate recharge of ponded surface water in the center of the Hueco Bolson (Texas) [31].The present study concurs with the possibility of infiltration as far as 15 km downstream of the range front.
Source of Hueco Bolson Groundwater
The question addressed here is the source of saline water in the center of the Hueco Bolson, an alluvial basin near El Paso, Texas, 100-130 km southwest of Alamogordo (Figure 1).Subsurface movement of groundwater from the Tularosa Valley to the Hueco Bolson is physically possible according to piezometric data [4,6].An alternative source is recharge from the Organ and Franklin Mountains (Figure 1), which supply a freshwater aquifer, the Franklin Mountains freshwater lens (FMFWL) in ancient fluvial deposits at the western edge of the Hueco Bolson [6].The catchment for the FMFWL is largely at altitudes between 1300 and 2400 m.a.s.l. in the Organ Mountains, in contrast to a catchment at 2400-2800 m.a.s.l. for the four large canyons that focus fresh water from the Sacramento Mountains into basin sediment near Alamogordo.H and O isotopes might therefore discriminate between the two sources, as discussed inconclusively in [31].
Groundwater from the FMFWL plots along the global meteoric water line with δ 18 O values between -9‰ and -11‰ (Figure 9).The upper end of the data array corresponds to groundwater of short residence time, while the lower end corresponds largely to groundwater resident for thousands of years [2].The SMT and the suggested paleo-SMT (based on samples 1-39 and 1-40) intersect the FMFWL trend near -9‰ and -11‰, respectively.On the one hand, the δ 18 O and. δD values of the saline, evaporated water in the center of the Hueco Bolson plot between the SMT and the paleo-SMT, and could therefore represent mixtures of older and younger water from the Sacramento Mountains.On the other hand, δ 18 O and. δD values define an evaporation trend that could originate in older FMFWL water, so that the water could have originated in the Frankin and Organ Mountains, perhaps as surface water ponded and evaporated in the basin center at a time of cooler, wetter climate.The stable isotopes fail to distinguish the two possibilities because of the likely presence of ancient groundwater.4) and data for groundwater at Alamogordo; (b) Data for groundwater in the Hueco Bolson in Texas, from [2] and [24], distinguished according to salinity (HB-saline at the basin center, and HB-fresh from the Franklin Mountains fresh water lens on the western side of the basin); (c) A suggested paleo-SMT based on two samples of ancient water in the southeastern part of Tularosa Valley.
Implications for Groundwater Management
High-elevation winter recharge is the principal source of groundwater over the long term in the aquifers of the Sacramento Mountains and the flanking basins.If winter precipitation declines, for instance in response to global climate change, groundwater supply will decrease.The effect would be felt initially in the high mountain communities such as Weed and Cloudcroft (but might be mitigated if occasional wet summers persist) and in areas from La Luz Canyon to Alamogordo and Tularosa where groundwater is of post-bomb age (Table 1).In the Roswell basin, where artesian water has been resident for thousands of years, there would be no short-term effect of diminished winter recharge; over-pumping for irrigation would be of more immediate concern.
Figure 4 .
Figure 4. (A) Plot of δ18 O vs. δD for groundwater samples from the high Sacramento Mountains.The green line encloses groundwater isotope data from[9].Seasonal means for precipitation and the local meteoric water line (LMWL) are for years 2006-2009[9].Data plotted as individual points were collected for this study in 2003; (B) Plot of elevation of well collars vs. δ18 O for sample sites in the high Sacramento Mountains.The diagonal lines show the long-term δ 18 O lapse-rates of −1.2‰/1000 m (Tucson Basin[21], and 1.8‰/1000 m[13].Site numbers (e.g., 3) correspond to entries in Table1, where the corresponding number is 1-3.
Figure 7 .
Figure 7. (A) Plot of δ18 O vs. δD for groundwater and surface water samples from study area 2. (A) All data from this study.The field of data from[13] is for the principal aquifer from Artesia to Roswell, and encompasses all but three outlying data points.The inset shows a magnified view of clustered data.Site numbers (e.g., 1) correspond to entries in Table1, where the corresponding number is 2-1; (B) Plot of δ18 O vs. δD for the Pecos River between Artesia and Red Bluff, from other studies[14,15], relative to the SMT.
Figure 8 .
Figure 8. A. East-west profile of Area 2 (refer to Figure2for location) showing well depths and static water levels (SWL) in relation to the surface."Shallow" refers to the shallow aquifer at Artesia, and "deep" to the deeper artesian aquifer.Also shown are measurements of carbon-14 (pMC) and tritium (TU).Tritium below detection is indicated as "bd".
Figure 9 .
Figure 9. Plot of δ 18 O vs. δD showing: (a) The Sacramento Mountains Trend (SMT, as in Figure4) and data for groundwater at Alamogordo; (b) Data for groundwater in the Hueco Bolson in Texas, from[2] and[24], distinguished according to salinity (HB-saline at the basin center, and HB-fresh from the Franklin Mountains fresh water lens on the western side of the basin); (c) A suggested paleo-SMT based on two samples of ancient water in the southeastern part of Tularosa Valley.
Table 1 .
Site information and isotope data.
Notes: S = shallow aquifer, D = Deep (Principal) aquifer in Artesia area; na = not available; A = Apparent tritium; * meters below surface. | 2016-03-01T03:19:46.873Z | 2014-01-28T00:00:00.000 | {
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204163577 | pes2o/s2orc | v3-fos-license | Mutation of CFAP57 causes primary ciliary dyskinesia by disrupting the asymmetric targeting of a subset of ciliary inner dynein arms
Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis, and is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious TEM structural phenotype for pathogenic variants using whole exome capture and next generation sequencing. The population sampling probability (PSAP) algorithm identified one subject with a homozygous nonsense variant [(c.1762C>T) p.(Arg588*) exon 11] in the uncharacterized CFAP57 gene. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Analysis of cells from the PCD patient shows a loss of CFAP57, reduced beat frequency, and an alteration in the ciliary waveform. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is conserved in organisms that assemble motile cilia, and CFAP57 is allelic with the BOP2 gene identified previously in Chlamydomonas. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass spectroscopy showed that CFAP57 is missing, and the “g” inner dyneins (DHC7 and DHC3) and the “d” inner dynein (DHC2) are reduced. Our data demonstrate that the FAP57 protein is required for the asymmetric assembly of inner dyneins on only a subset of the microtubule doublets, and this asymmetry is essential for the generation of an effective axonemal waveform. Together, our data identifies mutations in CFAP57 as a cause of PCD with a specific defect in the inner dynein arm assembly process. Significance Motile cilia are found throughout eukaryotic organisms and performs essential functions. Primary ciliary dyskinesia (PCD) is a rare disease that affects the function of motile cilia. By applying a novel population sampling probability algorithm (PSAP) that uses large population sequencing databases and pathogenicity prediction algorithms, we identified a variant in an uncharacterized gene, CFAP57. This is the first reported example of PCD caused by a mutation that affects only a subset of the inner dynein arms, which are needed to generate the waveform. CFAP57 identifies an address for specific dynein arms. These findings demonstrate the effectiveness of the PSAP algorithm, expand our understanding of the positioning of dynein arms, and identify mutations in CFAP57 as a cause of PCD.
Introduction
Motile cilia are complex organelles that project from the surface of cells and are essential for propelling fluids (e.g., in the airways, ventricles) or for providing cell locomotion (e.g., sperm, Chlamydomonas). The microtubule-based axoneme shows the classic structure of 9 doublet and 2 central pair microtubules, which is conserved throughout the eukaryotic lineage. Motility comes from the coordinated activity of inner and outer dynein arms (IDA and ODA, respectively) that are attached to the A tubule of the microtubule doublets. Defects in motile cilia cause primary ciliary dyskinesia (PCD), which is a genetically and phenotypically heterogeneous disorder that is characterized by chronic and debilitating respiratory disease, and is frequently accompanied by laterality defects (~50% of patients) due to abnormal left-right body asymmetry (1). The most common genetic lesions that cause PCD are those that affect components of the ODA, including DNAH5, DNAI1, and DNAH11 (2)(3)(4). Another group of PCD associated genes encode ODA-docking complex components (CCDC114 and CCDC151) (5)(6)(7), or the proteins that play a role in the cytoplasmic assembly of dynein (SPAG1, DNAAF1-3, HEATR2, and DYX1C1) (8)(9)(10)(11)(12)(13)(14). In addition, mutations in two genes, CCNO (15) and MCIDAS (16), cause a PCD-like phenotype by greatly reducing the number of motile cilia. Although genetic variants that cause PCD have been identified in over 40 genes (2,(17)(18)(19)(20)(21)(22)(23), there are individuals with confirmed clinical features of PCD but normal axonemal structure as determined by transmission electron microscopy, for whom the genetic basis of their disease is unknown.
In an ongoing effort, we have performed whole exome capture sequencing on more than 400 unrelated cases in order to identify genetic causes of PCD. Within this cohort, there were 99 unrelated cases with clinical features of PCD that include chronic otosino-pulmonary symptoms, and low levels of nasal nitric oxide who presented with no obvious axonemal defects by transmission electron microscopy (TEM). Using a new algorithm to analyze exome sequencing data (24), we identified an apparently homozygous stop-gain variant in ciliary and flagella associated protein 57 (CFAP57; MIM: 614259; NM_152498.3) in a patient with classical symptoms of PCD that included bronchiectasis, neonatal respiratory distress, otitis media, and sinusitis.
CFAP57 is allelic with the BOP2 gene identified previously in Chlamydomonas.
The BOP2 locus was first identified as a suppressor of the swimming defect of the pf10 mutant (25). Double mutant analysis, using IDA and ODA mutants, suggested that the mutation in the BOP2 locus affects only the IDAs. Electron tomography of the bop2 mutant showed that a subset of dynein arms were missing on only certain microtubule doublets (26). Specifically, electron tomography revealed reduced intensity on doublets 5, 6, and 8, while doublet 9 showed an intermediate loss of intensity (26).
To date, no pathogenic variants have been identified in proteins that affect only the IDA complexes in PCD patients. The IDAs play a crucial role in determining axonemal waveform and are more heterogeneous than the ODAs. For example, Chlamydomonas reinhardtii has six single headed IDA (a, b, c, d, e, and g) and one double headed IDA (I1/f) that span the 96 nm repeat, as determined by cyro-EM tomography using mutant strains affecting these proteins (27,28). In addition, there are three unique minor inner dynein arms that are only found in the proximal regions and replace the major IDAs; these include DHC3, DHC4, and DHC11 (29). Mutations in IDAs are difficult to detect by TEM cross sections since there are 7 dynein arms in 96 nm, and TEM sections are often 60 nm deep. Thus, TEM lacks the resolution necessary to detect changes in individual IDAs, as a single IDA would be obscured by the other IDAs in the section. Our data suggests that homozygosity of a pathogenic variant of CFAP57 causes PCD, likely by a failure to assemble a subset of inner dynein arms, and should be considered a candidate gene in cases of PCD with apparently normal axonemal structure by TEM and significantly reduced CBF.
Results
Whole exome capture and sequencing identifies a CFAP57 pathogenic variant in a PCD subject: We studied a 38 year old male from family UNC-1095 (Fig. 1a). The subject has situs solitus with a classical PCD phenotype that includes neonatal respiratory distress, otitis media, sinusitis, bronchiectasis, and a low rate of nasal nitric oxide production (40 nl/min; cut-off 77 nl/min) (2), but no axonemal defect was detected by TEM (Fig. 1b) (30), DNA from the subject, who had been previously screened for mutations in genes known to be associated with PCD, was sequenced using the IDT capture reagent (31). We analyzed the resulting genotype data using population sampling probability (PSAP), a statistical framework for assessing the significance of variants from n=1 cases of rare genetic disease (24) (Table S1). A new candidate gene, CFAP57, was identified and confirmed by direct Sanger sequencing ( Fig. 1c and 1d).
Analysis revealed a homozygous stop-gain variant [(c.1762C>T) p.(Arg588*)] in exon 11 of CFAP57, which is predicted to result in complete loss of function due to nonsensemediated decay of the primary CFAP57 transcripts. Two unaffected siblings were determined to be wild-type and a carrier of the pathogenic variant, consistent with an autosomal pattern of inheritance. CFAP57 is ~144 kD and contains 10 WD repeats along with 3 predicted coiled-coil domains. PSAP analysis identified the homozygous stop-gain in CFAP57 as the second most deleterious change in the subject's genome, following a homozygous missense change in CD2, a T-cell surface antigen. Mutations in the latter do not explain the subject's symptoms (Table S1).
Expression of CFAP57 in ciliated human airway epithelial cells: Normal primary
human tracheal epithelial cells (hTEC) cells were cultured at an air-liquid interface as previously described (32,33). Under these conditions, the cells first proliferate as an undifferentiated monolayer and then undergo ciliated cell differentiation. In hTEC cells, the expression of CFAP57 increased in parallel to the expression of FOXJ1, a key gene that drives ciliogenesis (Fig. 2a) and to the expression of DNAI1, a known ciliary specific gene (Fig. 2b). Similarly, the levels of CFAP57 protein increased as ciliated airway cells underwent differentiation, as determined by increased levels of FOXJ1 detected by immunoblot (Fig. 2c). This signal was strongly enriched in samples of detergent isolated ciliary axonemes (Fig. 2d). Immunofluorescent staining of CFAP57 in both intact hTEC cultures (Fig. 2e) and isolated cells demonstrate strong positive reactivity throughout the length of the ciliary axoneme (Fig 2f). These results demonstrate that CFAP57 is an axonemal protein, as suggested previously by proteomic analysis of both human and Chlamydomonas axonemes (34,35).
A pathogenic variant of CFAP57 causes defective ciliary beating: To characterize the effects of the genetic variant in CFAP57 on the function of motile cilia, we obtained human nasal epithelial (HNE) cells from the PCD subject and healthy controls. Cells were expanded in culture as conditionally reprogrammed cells (CRCs) (36), then allowed to differentiate using air-liquid interface cultures as previously described (22). Immunofluorescent staining of isolated ciliated cells from the control cultures showed CFAP57 reactivity along the entire length of the axoneme, while no positive staining was observed in cells from the PCD subject (Fig. 3a). Immunofluorescent intensities for the ODA protein DNAH5 and the radial spoke component RSPH1 were not different between PCD and control cells (Fig. S2), in agreement with the TEM analysis that showed no obvious axonemal structural defect (Fig. 1b). Although the fluorescent intensity of the IDA component, DNALI1, was not obviously different in cells obtained from the PCD subject compared to control cells, it appeared that there was a reduction in intensity at the tip ( Fig. 3b; Fig. S1). The lack of CFAP57 resulted in a significant reduction of ciliary beat frequency (CBF) in the PCD cells. n=21) was significantly reduced (p < 0.00001). Thus, the PCD cells exhibited shorter cilia, an altered waveform, and a reduced CBF.
CFAP57 silencing in hTEC results in reduced motile cilia motility: To confirm these results, the role of CFAP57 in motile cilia function was further defined by silencing expression using an RNAi approach in primary hTEC that were transduced with a control plasmid expressing a non-targeted shRNA sequence and a green fluorescent tag (37) or a CFAP57-specific shRNA plasmid together with a recombinant lentivirus that contains a cassette that confers puromycin resistance (33). In three biological replicates, CFAP57 expression was reduced by three out of the four CFAP57-specific shRNA sequences when compared to cells transduced with non-targeted shRNA sequences using RT-PCR (Fig 4a). Immunoblot analyses (Fig. 4b) confirmed the absence of the protein from total cell lysates (Fig. 4b), and immunofluorescent staining showed an absence of CFAP57 in ciliated cells. Silencing CFAP57 did not affect the degree of ciliogenesis in cultured airway cells (Fig.4). High-speed video microscopy analysis of ciliary motility (38) of the CFAP57-silenced cultures showed significantly reduced CBF when compared to control cells. (Fig. 4d). Analysis of ciliary beat using high speed video microscopy showed subtle changes in the ciliary waveform, which results in a slightly reduced curvature in CFAP57-silenced cells compared to control cells (Movies S3-S6).
CFAP57 is conserved in organisms with motile cilia: To evaluate the conservation of CFAP57 across species, we constructed a phylogenetic tree for CFAP57 (Fig. S3).
CFAP57 is found in most organisms with motile cilia, except in the cycads, gingkos, and the water fern, Marselia. As expected, CFAP57 is missing in organisms that lack motile cilia (flowering plants, nematodes, most fungi). The N-terminus of the protein is predicted to be composed of WD40 repeats that form beta-sheets and the C-terminus is predicted to be -helical (39).
Effect of CFAP57 mutations in Chlamydomonas:
Chlamydomonas reinhardtii is an important model for identifying ciliary genes and probing their functions (40). To further investigate the function of CFAP57, we carried out additional studies in Chlamydomonas. We obtained three insertional mutant strains (LMJ.RY0402.157050, LMJ.RY402.107706 and LMJ.RY0402.211005) from the CLiP collection, which is an indexed library of insertional mutations in Chlamydomonas (41,42). Insertions in two of the three strains were verified by PCR (Fig. S4). The insertion and drug resistance cosegregate with the swimming phenotype in 21 tetrads for LMJ.RY402.107706 and 7 tetrads for LMJ.RY0402.157050. LMJ.RY402.107706 carries an insert in exon 2 and LMJ.RY0402.157050 carries an insert in intron 7. We examined the mRNA for the two mutants. The transcript in LMJ.RY402.107706 could not be amplified across exons 2 and 3. In strain LMJ.RY402.107050, the transcript for exons 1-7 is present, but we were unable to amplify the remaining transcript (Fig. S4). These alleles failed to complement the bop2-1 allele in diploid strains and were tightly linked to the locus (n=89 tetrads).
Defective swimming and ciliary waveform in fap57 Chlamydomonas. We analyzed the swimming behavior of these mutants. Swimming velocity analysis shows that fap57-050 and fap57-706 swim significantly slower than wild-type cells (CC-125). fap57-050 was also slower than fap57-706 ( Fig. 5a; Table 1). Ciliary beat frequency extracted from the trajectory was unchanged compared to wild-type cells (Fig. 5b).
To characterize the waveform of mutants, both the fap57-050 and fap57-706 mutants were crossed with the uniciliate mutant uni1-2 (3). Fifty movies of fap57-050; uni1 and 51 movies of fap57-706; uni1 were compared to 153 movies of uni1-2 (wild-type samples previously described by Bottier et al. (44) (Fig. 5a). Body motion analysis shows that both mutants have a significantly slower body rotation rate compared to wild-type cells (Fig. 5c), which agrees with the reduced velocity. Both fap57 strains have a significantly reduced bend amplitude (Fig. S5b) and an increased average curvature compared to wild-type cells (Fig. S5c). The fap57-050 mutants also have an average curvature significantly smaller than fap57-706, which suggests that the fap57 mutant waveforms are less wavy, and straighter. Both the amplitude of power and torque are significantly reduced for the fap57 mutants compared to wild-type (Fig. S4d). Those results are compatible with a reduced swimming velocity as well as a reduced body net rotation. Both alleles displayed waveforms with a shorter stroke.
Protein composition of fap57 mutant cilia show a partial phenotype: Using isobaric tags (TMT) for tandem mass spec, we analyzed isolated cilia from four wild-type strains and two technical replicates of two different fap57 meiotic progeny. There was an average of 107 FAP57 peptides in wild-type cilia, but only 6 peptides in mutant cilia, which suggests a strong loss of function ( Table 2). Several of the inner dynein arms (DHC7, 3, 2) are reduced ( Table 2) compared to the other dynein heavy chains (Table S3; Fig. S6). The translational elongation factor 1 alpha (EEF1A1) is also reduced in the fap57 mutant strains to the same extent as the inner dynein arms. This elongation factor is present in proteomic analyses of both Chlamydomonas and human axonemes (34,35,(45)(46)(47), which suggests it is not a cytoplasmic contaminant. It can be extracted with KCl and is found in the membrane matrix fraction (34). However, its role in the axoneme is unknown. Four additional proteins are significantly reduced but have few spectral counts ( Table 2). Cre12.g540050 and Cre10.g438500 have no orthologs outside of the green algae. Cre13.g562800 has a paralog in Chlamydomonas (Cre07.g313850) and both are likely orthologs of WDR49. Cre10.g438500/FAP264 is an ortholog of LRRC74.
In humans, WDR49 is expressed in the fallopian tubes and lung and FAM74A is highly expressed in the testes based on the GTEx project (48). They have no known functions.
To further examine these additional proteins, we tried a commercial antiserum to WDR49 (Table S5), and it failed to show specific staining in hTECs or Chlamydomonas.
We examined an insertional mutant strain from the CLiP collection (41) (Table S4).
Taken together, the above data demonstrates that CFAP57 plays an important role in the assembly of a specific subset of IDAs, and the proper positioning of these IDAs is important for the generation of normal waveforms. This is consistent with the phenotype observed in the PCD subject, in which absence of CFAP57 results in reduced CBF and altered waveform, in the absence of a structural defect detectable by TEM.
Discussion
Mutations in over 40 genes have been reported to cause PCD, and these account for the majority of disease cases (2,49). However, many cases of PCD (~30%) remain unsolved at the genetic level. The large number of causative genes are a direct reflection of the complexity of ciliary assembly and structure. Many of the known mutations occur in genes that encode structural proteins of the ODAs or preassembly factors for ODAs and IDAs. To date, no mutations have been reported that cause a defect that affect only the IDAs. We performed exome capture and sequencing on a patient with a clinical diagnosis of PCD who had no known genetic mutation, and no obvious structural changes visible by TEM. Using PSAP, a newly developed algorithm to analyze exome sequence together with large genome databases, we identified a homozygous stop-gain mutation in CFAP57. The PSAP algorithm uses large population sequencing databases and pathogenicity prediction algorithms to calculate the probability of sampling a particular genotype, or set of genotypes, observed in a single n=1 case. PSAP is most useful in rare disease studies where families are unavailable, and/or the cohort size is modest (dozens to hundreds). We currently use PSAP on cases of idiopathic nonobstructive azoospermia to identify new causative genes (50), and expect that PSAP will become a useful tool to identify causative genetic variants in other rare diseases. These may include diseases similar to azoospermia and PCD that are characterized by large effect mutations and extensive locus heterogeneity.
In cultured nasal epithelial cells from the PCD patient, we observed a complete absence of CFAP57 by immunofluorescence and the cells exhibited a significantly reduced CBF.
Knockdown of CFAP57 in hTEC cells using shRNA, confirmed by immunofluorescence and immunoblotting, also show a reduced CBF. In contrast, TEM analysis of nasal epithelial cells from the subject appear normal and immunostaining for the ODA protein DNAH5 was unchanged. However, immunostaining for DNALI1 suggested a defect in the localization along the axoneme, with an accumulation at the base and a reduction at the tip. The ortholog of DNAL11 in Chlamydomonas is p28/DII1 and it is found in three of the inner dynein arms, including the d dynein. Although the mechanisms that regulate basal CBF have been studied extensively, they remain unclear. These results suggest that a proper balance between ODAs and IDAs is also required to maintain normal CBF. Waveform analysis revealed subtle differences between the PCD or knockdown cells and controls that varied among the cells. The heterogeneous waveform observed between individual cilia in the PCD samples could be related to the maturity of individual cilium. It has been shown that the variation in ciliary waveform is associated with progression of ciliary length and the differentiation of ciliated cells (52).
To characterize the function of CFAP57 in more detail, we utilized the well-studied model organism Chlamydomonas. We show here that CFAP57 encodes a WDR protein that plays a role in the docking/assembly of a subset of IDAs. Although the ciliary axoneme is described as showing nine-fold symmetry, there are many structural asymmetries in the cilium (reviewed in Dutcher, in revision). The generation of waveforms requires the spatial and temporal regulation of dyneins, and this is likely to require structural asymmetries. Multiple approaches have helped to catalog the asymmetric structures and proteins in Chlamydomonas cilia. These asymmetries have been identified through analysis of mutant Chlamydomonas flagella by electron tomography and proteomics and are beginning to provide a wealth of information to use for understanding how asymmetric and symmetric waveforms are generated and propagated (27,28,45). If all of the ciliary dyneins were active at one time, the cilia would be in a rigor state, resulting in no net movement or bending. In order to generate an effective bend, dynein motor function must be tightly controlled both along the length of the cilium and around the circumference of the axoneme across a defined axis.
In elegant cryo-EM tomography studies, Lin and Nicastro unexpectedly found that most dyneins are in an active state confirmation with a smaller population of inactive dyneins (53). The locations of the inactive dyneins are asymmetric and bend-dependent. They propose a switch-inhibition mechanism in which the bend is generated by inhibiting, rather than activating, dyneins on one side of the cilium. The data suggest that the initiation of a bend starts with the inhibition of the inner dynein arms (a, d, and g) on specific doublet microtubules (DMT). DMT 2 to 4 is needed for the asymmetric waveform and DMT 7 to 9 for the symmetrical waveform that promotes a bend by the dyneins on the other side of the axoneme. CFAP57 is likely to play a key role in regulating the waveform.
The electron tomographic analysis of the Chlamydomonas bop2-1 mutant showed that CFAP57 is required for the assembly of dynein arms on only a subset of the nine doublet microtubules (26). A loss of IDAs on doublets 5, 6, and 8 was observed. In recent work using cryo-EM tomography, there is a loss of a subset of inner dynein arms on doublet microtubules 5-8 with a partial loss on doublets 1 and 9 (Lin et al., (Table 3).
In comparison to Chlamydomonas, less is known about the detailed structure of the IDA in human cilia. Based on the high level of conservation between species, it is likely that CFAP57 plays a similar role in human cilia as it does in Chlamydomonas. However, because the planar waveform of human cilia is inherently different from the waveforms of Chlamydomonas, the exact positioning and regulation of IDA activity is also likely different. Additional studies using advanced techniques to culture and study human respiratory cells are needed to address these questions.
In summary, our results show that a genetic variant in CFAP57 reduces CBF and alters waveform, likely by affecting the assembly of a subset of IDA, resulting in PCD. This is the first reported example of PCD caused by mutation of a protein that apparently affects only a subset of the IDAs, expanding our understanding of how dynein arms are positioned during cilia assembly. These findings demonstrate the usefulness of the PSAP algorithm and set a precedent to consider it in the evaluation of other cases of PCD with no obvious structural defects. Identifying the genetic basis of PCD and the functional defects is an important step toward developing personalized treatments for this rare disease.
Human subjects. The individuals included in this study provided informed consents and all protocols involving human studies were approved by the University of North
Carolina Medical School Institutional Review Board.
Human genetic analysis. A cohort of 99 PCD patients with no obvious EM phenotype was assembled for exome sequencing. Exome libraries were prepared using an IDT capture reagent. Genetic variants were discovered and genotyped using a validated analysis pipeline at the Washington University McDonnell Genome Institute as previously described (31). Each case was analyzed using the population sampling probability (PSAP) framework, a published statistical method for identifying pathogenic mutations from n=1 cases of rare disease (24). Segregation analysis was performed on the available DNA from family members (family UNC-1095). The primers used are listed in Table S6.
Airway epithelial cell cultures Human nasal epithelial (HNE) cells from the PCD subject (proband 2-II) and controls
were obtained as described (7). The nasal cells were expanded as conditionally reprogrammed cells (CRC) (8) and cultured as previously described (9).
Human tracheobronchial epithelial cells (hTEC) were obtained from non-smoking donors lacking respiratory pathologies provided by the Cystic Fibrosis Center Tissue
Procurement and Cell Culture Core (59), or were isolated from surgical excess of tracheobronchial segments of lungs donated for transplantation as previously described (11). These unidentified cells are exempt from regulation by HHS regulation 45 CFR Part 46. hTEC cells were expanded in vitro and allowed to differentiate using air-liquid interface (ALI) conditions on supported membranes (Transwell, Corning Inc., Corning, NY), as previously described (11,32,59). These protocols have been approved by the
Analysis of CFAP57 expression. RNA was isolated from cells using an RNeasy Mini
Kit (Qiagen) and RT-PCR was performed using specific primers (Table S6) as previously described (22). For qRT-PCR, RNA was reverse-transcribed using an Applied Bioscience High-Capacity Reverse Transcription Kit (Thermo Fisher Scientific). FluoroPure™ (Life Technologies). Slides were mounted using ProLong Diamond antifade mountant (Thermo Fisher). Images were acquired using a Zeiss -710 microscopy system or a Leica epifluorescent microscope (LAS X, Leica, Buffalo Grove, IL) Images were processed and the fluorescence intensity analyzed using FIJI (10) as previously described (11). Brightness and contrast were adjusted globally using Photoshop (Adobe Systems, San Jose, CA). Isotype matched control antibodies had no detectable staining under the conditions used. The antibodies used are listed in Table S5.
Ciliary isolation, protein extraction and Immunoblot. Ciliary isolation and protein extraction was performed as previously described (35). Protein extraction from tissue or cells and immunoblot analysis was performed as previously described (35,61).
Ciliary beat frequency and waveform analysis. The ciliary beat frequency (CBF) in
HNE cultures (n=6) was measured as previously described (22,63). In hTEC cultures, CBF was measured in at least 5 fields obtained from each preparation. The cultures were maintained at 37 °C using a temperature controller and a stage heater block. Highspeed videos (120 frames/s) were recorded and processed with the Sisson-Ammons Video Analysis system (SAVA, Amons Engineering, Mt Morris, MI) as described (11,64). To analyze the ciliary waveform, cells were lifted off the supportive membranes and imaged directly on slides. High resolution videos of 6 cells in 3 different cultures were recorded as previously described (22). The videos were analyzed by an experienced scientist blinded to the genotype of the cells. Videos were replayed in slow motion. The ciliary length was measured and the waveform of the front and back cilia in 4 ciliated cells was traced manually. Deviation from linearity was determined by measuring the furthest ciliary displacement from the linear axis as defined by the end-effective and end-recovery position, as previously described (65).
Chlamydomonas axoneme isolation and mass spectroscopy. Chlamydomonas cilia were isolated using the dibucaine method (66). Cilia were demembranated with the addition of 1% Nonidet P-40 in HMDS-EGTA (10 mM HEPES, 5 mM MgSO4, 1 Chlamydomonas swimming analysis. Each video were analyzed using ImageJ (69) to create a binary file only displaying the cells. Each pixel had a spatial resolution of 310 x 310 nm and the temporal resolution between 2 consecutive time points was 1 ms.
Cells were tracked using the 2D/3D single-particle tracking tool of the MosaicSuite for Chlamydomonas kinematic analysis of cilium. The uniciliate mutant strain uni1-2 and the double mutants fap57-050; uni1 and fap57-706; uni1 were generated from meiotic crosses, as described by Dutcher (70) . The single mutant uni1 is considered the wild-type reference as previously published (43). Videos were analyzed using a custom-made program written in Matlab R2016a (The Mathworks, Natick, MA, USA) previously published (44). From each video, a sequence of 200 consecutive frames was stored in a 3D matrix of pixel intensity values. Each pixel had a spatial resolution of 169 x 169 nm and the temporal resolution between 2 consecutive time points was 0.5 ms.
Components of the forces exerted by cilia on the fluid are directly calculated from the Cartesian coordinates using parameters previously reported (44).
Statistical analyses. Group variation is described as mean ± standard error (SEM).
Statistical comparisons between groups were made using 1-way analysis of variance (ANOVA) with Tukey post-hoc analysis. Individual group differences were determined using a 2-tailed Student's t-test. A p value of 0.05 was considered to represent a significant difference. Non-parametric data are shown as the median and the 25 th and 75 th intraquartile ranges. Data were analyzed using Prism (GraphPad, La Jolla, CA).
Protein extraction and digestion
The samples were lysed in lysis buffer (8M Urea in 50mM HEPES pH 8.0) and sonicated briefly. Samples were reduced with 10 mM TCEP and alkylated with 25 mM iodoacetamide. Protein concentrations were determined by BCA protein assay (Thermo Scientific). Fifty μg of protein was taken from each sample for digestion. Sequencing grade protease Lys-C was added with 1:250 ratio and sample was digested overnight at room temperature with mixing. A second digestion was performed by diluting the sample with 50 mM HEPES to lower the urea concentration to 1M and trypsin was added with 1:100 ratio for a further 12 hour digestion at room temperature with mixing.
Digests were acidified with formic acid and subjected to Oasis HLB solid phase extraction column (Waters).
Tandem Mass Tag (TMT) Labeling
Digested peptides were labeled according to the TMT 10plex reagent kit instructions.
Briefly, TMT regents were brought to room temperature and dissolved in anhydrous acetonitrile. Peptides were labeled by the addition of each label to its respective digested sample. Labeling reactions were incubated without shaking for 1 h at room temperature. Reactions were terminated with the addition of hydroxylamine.
Subsequent labeled digests were combined into a new 2 mL microfuge tube, acidified with formic acid, subjected to Sep-Pak C18 solid phase extraction and dried down.
High pH Reverse Phase Fractionation
The dried peptide mixture was dissolved in 110 μL of mobile phase A (10 mM ammonium formate, pH 9.0). 100 μL of the sample was injected onto a 2.1 x 150 mm XSelect CSH C18 column (Waters) equilibrated with 3% mobile phase B (10 mM ammonium formate, 90% ACN). Peptides were separated using a gradient (71) Peptides were resolved using 75 μm x 25 cm PepMap C18 column (Thermo Scientific).
Data Analysis
All MS/MS samples were analyzed using Proteome Discoverer 2.1 (Thermo Scientific).
The Sequest HT search engine in the Proteome Discover was set to search (E and F) The localization of CFAP57 to the cilia was confirmed by immunofluorescence in (E) whole ALI culture and in (F) isolated ciliated cells. | 2019-09-26T09:01:40.817Z | 2019-09-24T00:00:00.000 | {
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16296820 | pes2o/s2orc | v3-fos-license | Exploration of risk factors contributing to the presence of influenza A virus in swine at agricultural fairs
Influenza A virus infections occurring in exhibition swine populations at agricultural fairs during 2012 served as a source of H3N2 variant influenza A viruses transmitted to humans resulting in more than 300 documented cases. Prior to the outbreak, this investigation was initiated to identify fair-level risk factors contributing to influenza A virus infections in pigs at agricultural fairs. As part of an ongoing active surveillance program, nasal swabs and associated fair-level metadata were collected from pigs at 40 junior fair market swine shows held in Ohio during the 2012 fair season. Analyses of the data show that the adjusted odds of having influenza A virus-infected pigs at a fair were 1.27 (95% confidential interval (CI): 1.04–1.66) higher for every 20 pig increase in the size of the swine show. Additionally, four of the five fairs that hosted breeding swine shows in addition to their junior fair market swine shows had pigs test positive for influenza A virus. While the current study was limited to 40 fairs within one state, the findings provided insight for veterinary and public health officials developing mitigation strategies to decrease the intra- and inter-species transmission of influenza A virus at fairs.
INTRODUCTION
The comingling of swine from numerous premises with varied management practices and their interaction with large numbers of exhibitors and visitors make agricultural fairs an ideal setting for the intraand inter-species transmission of influenza A viruses (IAVs) between swine and human populations. 1, 2 The frequency with which intraand inter-species IAV transmission occurs in these settings is likely due to a myriad of factors, including but not limited to, management practices, IAV strain, and animal and/or human population immunity. Swine is a host species in which reassortment of IAV genomic segments may lead to emergent novel strains, since they are susceptible to infection from swine, human and avian influenza A viruses. 3,4 For this reason, limiting the bidirectional zoonotic transmission of these viruses at agricultural fairs is important for public and animal health.
The association between human and swine influenza was reported after respiratory disease similar to the human disease was noted in pigs during the 1918 human Spanish flu pandemic. 5 H1N1 IAV subsequently became established in the United States swine population with the relatedness of the swine and human viruses being established in 1931. 6 For nearly 80 years, classical swine influenza H1N1 virus was the dominant endemic IAV strain in the North America swine population. 7 In 1998, triple reassortant H3N2 IAVs containing polymerase basic 1 (PB1), hemagglutinin (HA) and neuraminidase (NA) gene segments from human IAV lineages, polymerase basic 2 (PB2) and polymerase acidic (PA) genes from avian lineages, and nucleoprotein (NP), matrix (M) and non-structural (NS) gene segments from swine lineages, emerged in North American swine. 8 Subsequently, this lineage became established in US and Canadian swine herds and has resulted in an increased rate of genetic and antigenic change among swine-origin IAVs. [9][10][11] Reported cases of humans contracting IAV infections directly or indirectly from pigs have been historically sporadic and these variant IAVs showed limited capability for sustained human-to-human transmission. [12][13][14] However, the emergence of the influenza A(H1N1)pdm09 virus, a strain containing gene segments from North American and European swine lineages, 15 illustrated the pandemic potential of swine lineage IAVs crossing the species barrier to humans. While A(H1N1)pdm09 rapidly spread worldwide and became endemic in the human population, 16 sequencing of this virus has to date failed to elucidate any virulence or adaptation markers that would explain its human-to-human transmission efficiency, highlighting our inability to predict IAVs with pandemic potential. While the origin of the A(H1N1)pdm09 virus remains unknown, the virus was introduced into the North American swine population in 2009 and has since reassorted with other swine-origin IAVs. 17,18 Epidemiological data show that zoonotic transmission of IAV from swine to humans has been documented at unprecedented levels in recent years. More than 320 human cases of infection with variant IAVs were reported to the Centers for Disease Control 2011-2012 19 and likely thousands more H3N2v cases went unreported during those years. 20 These zoonotic IAVs contained seven genes from contemporary North American swine lineage IAV and one gene (M) derived from the H1N1pdm09 virus. 21,22 The majority of the cases were epidemiologically linked to swine exposure occurring at agricultural fairs across several states. 19,[23][24][25] Within Ohio, 107 H3N2v cases documented during 2012 resulted in eleven hospitalizations and one fatality. 26 We recovered IAV from exhibition swine at 10 of 40 (25%) Ohio fairs sampled during 2012. Genomic analyses of H3N2 IAV isolates recovered from pigs at one agricultural fair in the state during 2012 demonstrated .99% nucleotide similarity to H3N2v isolates recovered from concurrent human cases, providing molecular confirmation of zoonotic IAV transmission. 2 This record number of variant influenza A cases created the need for a 'one health' effort to minimize intra-and bidirectional inter-species IAV transmission at swine exhibitions. 27 The reason to prevent IAV infections among swine at fairs is clear; however, the paucity of scientific evidence makes it difficult for veterinary officials to make sound recommendations to protect public and animal health. In the present study, we investigate fair specific risk factors contributing to the emergence of influenza A virus in exhibition swine that could be altered to mitigate the risk of IAV transmission in these settings.
MATERIALS AND METHODS
As part of an ongoing active IAV surveillance project, swine nasal swabs and associated metadata about management practices were collected at 40 Ohio fairs in 2012. Molecular and microbiological assays for IAV were performed on the swabs as previously reported. 28 Sample size was selected to provide a 95% probability of detecting IAV infection if greater than 15% of the pigs at each fair were infected. 29 All pigs sampled in this study were from junior fair market swine shows occurring at agricultural fairs. For the outcome of interest, a fair was considered positive if viable IAV was recovered from one or more pigs at the fair. Data collection focused on fair level variables possibly contributing to the presence or absence of IAV in the pigs at each exhibition. Junior fair shows are limited to local exhibitors approximately nine years of age through 19 years of age participating in 4-H, FFA or another youth organization, whereas open-class shows are generally open to all participants regardless of age, affiliation or residence. Classification of swine included market swine (pigs bred, raised and intended for food purposes) and breeding swine (gilts, sows and/or boars being raised for breeding purposes). Terminal swine shows are those in which all participating livestock are consigned to harvest immediately following the exhibition and partial terminal shows usually require the champion animals to be harvested following the exhibition and other pigs may or may not go to harvest.
To account for the variability of arrival and departure procedures among fairs enrolled in this study, the length of the swine exhibition was defined as the number of days between the required arrival deadline for the pigs and the day the pigs were sampled. Study team members calculated the area per pig (ft 2 /pig) from the recorded size of the pens and the number of pigs per pen. While on the fairgrounds, study team members also documented if there was an easily identifiable and operational hand-wash and/or hand-sanitizer station within close proximity to the swine barn(s). These sanitation stations were used by study team members to determine if they were functional.
Additional variables included the number of pigs at the fair, number of swine exhibitors, fair attendance (number of people) and vaccine requirements, all of which were reported to the study team by the fair organizers. Fair officials also reported if there was a commingling event, such as a pre-fair animal identification or weighing session, during the weeks or months prior to the fair. Exhibition directors also reported if there were other pigs besides the exhibition swine on the fairgrounds (i.e., petting zoo, pig races, educational displays).
The commercial swine inventory was retrieved from United States Department of Agriculture's 2007 Census of Agriculture. 30 The county human population was defined as the value reported in the 2010 US census report. Weather data were collected from the National Weather Service's weather station nearest to each fairground.
Data were analyzed using STATA Version 11.1 (StataCorp, College Station, TX, USA). Fisher exact test was used to assess differences in proportions and the Kruskal-Wallis equality-of-populations rank test was used for continuous variables. Univariate analysis was performed to calculate unadjusted odd ratios to identify factors contributing to the presence of IAV in pigs at fairs. Exact logistic regression was used for multivariable modeling using a forward stepwise model building approach. A cutoff of Pf0.05 was used for inclusion in the model.
RESULTS
Influenza A virus was recovered from pigs at 10 of the 40 fairs included in the investigation. The presence or absence of IAV infection among the pigs at the fairs could not be associated with county population, county swine inventory and number of people attending the fair (Table 1). All IAV-positive fairs and 27 of 30 (90%) negative fairs were mixed sex (barrows and gilts) market swine exhibitions. The average space per pig at the studied fairs was 12.8 ft 2 /pig. Properly functioning hand-wash and/or hand-sanitizer stations were available at 25 of 40 (62.5%) fairs. Average daily mean temperature was almost 4 6 C higher for fairs with IAV-positive pigs (Table 1). Pre-fair tag-in and/or weighin events were rather common with 23 out of 40 (57.5%) of the enrolled fairs holding one of these events. For every increase of 20 pigs at a fair, the odds ratio of IAV infection in the pigs increased by 1.27 times (Table 2).
DISCUSSION
The results of the current study provide the first look at fair-level risk factors associated with IAV infections in swine at agricultural fairs. While it is likely impossible to completely prevent IAV transmission at swine exhibitions, these data can be used to develop and evaluate mitigation strategies to reduce the impact of intra-and inter-species IAV infections at swine exhibitions. Just like all other agriculture biosecurity programs, mitigation strategies which are practical, userfriendly, low cost and do not dramatically alter the fair experience for exhibitors and visitors are most likely to be considered, implemented and maintained.
Not surprisingly, larger pig shows appear to be more likely to have IAV-infected pigs than smaller swine exhibitions ( Table 2). Larger swine exhibitions tend to also have open-class and breeding swine shows in addition to junior market shows. While open-class shows were common among our studied fairs (16 of the 40), only 5 of the 40 (12.5%) fairs in this study had a breeding show; 4 of those 5 (80%) fairs had IAV-infected pigs at the fair. The small number of fairs with breeding shows in this study makes analysis of this risk factor problematic; however, the finding is enough to warrant concern given that breeding swine are intended to leave the exhibition and enter a herd for progeny production. This fair-to-farm movement of pigs is a disease introduction risk for the receiving herd and a potential method to disseminate IAV strains across a larger geographic area.
Previous research has shown that environmental stressors (heat, lack of space, noise) on pigs can affect the course of various diseases in commercial swine operations. 31 The average space per pig at the studied fairs was well above 6-8 ft 2 /finishing pig common throughout the swine industry. 32 The results indicate that heat stress could be a contributing factor to IAV infections in exhibition swine; however, caution must be used when interpreting this result because the vast majority of the fairs with IAV-positive pigs occurred in a 4-week period during the middle of summer. This trend of mid-summer IAV activity in Ohio's exhibition swine was also observed in the previous three years 1 and could be more related to animal and/or people movement between these fairs than the weather. Environmental
Risk factors for influenza A virus at fairs
AS Bowman et al 2 temperature failed to meet the selection criteria for inclusion in the final multivariable model.
While the majority of fairs had hand hygiene stations, their presence at fairs was not linked to the occurrence of IAV among pigs at the respective fairs. The large number of H3N2v infections linked to swine exposure at agricultural fairs during 2012 suggests that hand hygiene stations also had minimal impact on zoonotic transmission of IAV in these settings. However, hand hygiene stations are critically important in protecting human health by controlling zoonotic diseases transmitted via direct contact at these venues. 33 One potential mitigation strategy that has been proposed is to shorten the exhibition period. 34 This would limit the time for IAV to spread among susceptible pigs and decrease the time humans are exposed to IAV-infected pigs. The length of the exhibitions in the current study was similar between IAV-positive and -negative fairs.
Active recruitment of fairs with more diverse management practices is needed to study the impact of a shortened swine exhibition. No matter the length of the exhibition time, the disposition of the pigs following the show must be considered. The majority of fairs in this study (65%) had terminal junior market shows. The practice of having a terminal show was not associated with decreased odds of IAV; however, sending all the pigs to harvest at the end of each fair is expected to help protect subsequent fairs by decreasing the potential for fair-to-fair spread of IAV.
Mandated vaccinations were almost non-existent with only one fair in the current study requiring the pigs be vaccinated for Erysipelothrix rhusiopathiae prior to the fair; no fairs required the pigs to receive IAV vaccination before arrival. Use of IAV vaccine in exhibition swine to decrease the risk of IAV infections in swine and humans has been one of the most debated topics following the H3N2v outbreak of 2012. There are currently several commercially available swine influenza vaccines in the United States, all of which are universally indicated to reduce clinical signs of disease in pigs but appear to have limited efficacy against 2012 H3N2v strains. 35 Although their impact on intraand inter-species transmission dynamics remains unclear, 36 it is expected that IAV vaccines will impart at least partial immunity to circulating strains of IAV, which should decrease viral shedding Risk factors for influenza A virus at fairs AS Bowman et al 3 during a fair. An unintended consequence of IAV vaccine use may be vaccine-associated enhanced respiratory disease, which has been reported in swine vaccinated with swine influenza virus vaccines that are mismatched to circulating strains. 37 Furthermore, decrease of clinical signs may hamper recognition and response to active IAV infections in exhibition pigs.
Some of the major pitfalls of mandated IAV vaccination lie with the practical application of vaccines in this setting. The logistics of vaccine distribution to swine exhibitors prior to the fair becomes difficult because most exhibition swine are raised by youth exhibitors in small, dispersed herds (,10 pigs per herd). Commercial vaccines are usually sold in o50 doses per bottle adding to the cost per vaccinated pig in these small herds. Because youth exhibitors and their family members may not be proficient at administering vaccines, agriculture education advisors often volunteer to assist the youth with this task, a practice that is time-consuming and increases the risk of infectious agents being transmitted farm to farm. Additionally, problematic is that most IAV vaccines labeled for swine require a booster dose given 2-4 weeks later to provide optimal protection, which can be difficult for youth exhibitors and their family members to accomplish. Tagging or weighing events are frequently used as a way for exhibitors to declare ownership of their pig(s) prior to the fair. These pre-fair events could provide an opportunity for mass vaccination of pigs prior to the fair, but the application of such events can facilitate disease spread between animals. Even in properly vaccinated pigs, the immunity stimulated by current IAV vaccines is limited in scope and duration. [38][39][40] The strains used for commercial swine influenza vaccines are irregularly updated and the constant genetic and antigenic change occurring in contemporary swine-origin IAVs makes viral antigens unpredictable and difficult vaccine targets.
The reason for the increase in the number of reported H3N2v cases during 2011-2013 remains unclear, but the strain of IAV is thought to be a major contributing factor. The swine-origin H3N2 IAV isolates recovered from these human cases contains the matrix gene from the A(H1N1)pdm09 virus, 22 a recently emerged genomic constellation that increases replication and transmission in cell culture and animal models. 41 Epidemiological data modeling indicate that children are most susceptible to H3N2v, likely due to lack of strain-specific immunity. 42 Additionally, current seasonal trivalent inactivated IAV vaccine provides little to no protection against H3N2v strains. 43 The limited ability for human-tohuman transmission of H3N2v has minimized the impact of these recent zoonotic transmission events, 44 but the outbreak has illustrated the importance of swine exhibitions in zoonotic IAV transmission.
It is nearly impossible to predict the IAV strain that will infect the swine at fairs because IAV reassortment events and novel strain generation frequently occur in modern swine populations. 7,10,45,46 Agricultural fairs provide a pathway for human exposure to these ever changing viruses; 2,19,47 thus, blanket IAV prevention, without regard for strain, is needed for swine at fairs to decrease zoonotic IAV transmission and protect public health. While IAV can infect pigs at any fair, the data presented here indicate that special attention should be paid to large pig shows where the likelihood of IAV among the pigs is much higher.
The results presented herein are based on one year of data from a limited geographic area of the United States. Additional assessments of swine exhibitions in multiple states across several years are needed to provide more comprehensive evaluations of risk factors contributing to the problem. These data provide a critical first step toward developing effective IAV mitigation strategies in swine populations that benefit fairs, exhibitors, visitors and the swine industry. This information will serve as a baseline for measuring the acceptance and effectiveness as mitigation strategies are developed and implemented.
ACKNOWLEDGMENTS
We thank our collaborators from the participating agricultural fairs. We also thank Wendell Bliss, Gary Bowman and Dimitria Mathys (The Ohio State University, USA) and Tony Forshey (Ohio Department of Agriculture, USA) for their assistance and support with this project. This work has been funded by the Minnesota Center of Excellence for Influenza Research and Surveillance with federal funds from the Centers of Excellence for Influenza Research and Surveillance, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract NO HHSN266200700007C. | 2017-11-08T00:48:04.529Z | 2014-01-01T00:00:00.000 | {
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1317108 | pes2o/s2orc | v3-fos-license | Comparison of clinical features in pathologically confirmed PSP and MSA patients followed at a tertiary center
Background/Objectives: The clinical diagnosis of progressive supranuclear palsy (PSP) and multiple system atrophy (MSA) remains challenging due to heterogeneity of the diseases. AIMS: Here we compared the clinical features of PSP and MSA to gain insight into their diagnosis and prognosis, particularly the prognostic value of down-gaze palsy latency in PSP progression. Methods: We reviewed clinical features of pathologically confirmed 10 PSP and 13 MSA patients, incidentally matched in age-at-onset, gender, and disease duration, followed at Columbia University Medical Center during 1994–2009. Results: The final antemortem diagnosis was incorrect in 30% of PSP (all lacking down-gaze palsy) and 23% of MSA patients. Falls in the first year of the disease, pyramidal involvement and freezing of gait during the course were similar between PSP and MSA. Ataxia and apraxia were in 50% of the PSP patients. Parkinsonism responsive to levodopa treatment was in 30% of the PSP (all with resting tremor) and 50% of the MSA patients. Dysautonomia in MSA could occur as early as 3 years preceding the motor symptoms, with 46% within the first year of the motor onset, but 15% did not have dysautonomia in life. The latency of down-gaze palsy and urogenital dysfunction and MMSE scores at first visit in PSP, and the latency of falls and wheelchair confinement in MSA were all associated with the disease progression. Conclusions: We confirmed most of the previously published characterizations, and identified for the first time the prognostic value of down-gaze palsy latency in PSP progression.
INTRODUCTION
The diagnosis of progressive supranuclear palsy (PSP) and multiple system atrophy (MSA) could be very challenging in the absence of characteristic symptoms and signs or the presence of atypical ones due to the heterogeneity of the diseases. Clinically, the diagnostic hallmarks of PSP are the presence of down-gaze palsy and frequent falls in the first year of the disease onset. [1][2][3] The absence of down-gaze palsy often leads to misdiagnosis of PSP in as high as 60% of the pathologically confirmed patients. [4][5][6] The presence of atypical symptoms in the 'exclusion criteria' of PSP, such as apraxia typically seen in corticobasal degeneration, or dysautonomia and ataxia typically seen in MSA, 2 could make the diagnosis of PSP even more difficult. The presence of dysautonomia is a prerequisite for the diagnosis of MSA, with dominant parkinsonism for MSA-P and cerebellar ataxia for MSA-C. 7 The absence of the dysautonomia could similarly make the diagnosis of MSA very difficult.
Studies on clinical features in pathologically verified PSP cases greatly advanced our knowledge on PSP. [8][9][10][11] PSP was classified into PSP-RS (Richardson-Steele -Olszewski syndrome, 54-63%) and PSP-P (parkinsonism, 26-32%) as the major two types, besides other types including the unclassified one with the features of both PSP-RS and PSP-P, pure akinesia with gait freezing, corticobasal syndrome, and progressive non-fluent aphasia. 8,10,11 An ataxia type was also proposed. 12 PSP-RS is clinically defined as the presence of down-gaze palsy, falls and cognitive dysfunction in the first 2 years of the disease, 8,10 with more rapid progression and shorter disease duration than PSP-P, which has bradykinesia, tremor and some response to parkinsonian medication. 8,10 The pathological basis of these different clinical types is due to the difference in brain areas and severity involved by the tauopathy, with more severe and widespread pathology in PSP-RS than PSP-P and subtle difference in the amount of insoluble tau protein between them as well. 9 The potential prognostic role of the downgaze palsy latency in the clinical progression of the PSP, though very important, has rarely been studied before.
Here we compare the clinical features and the evolution of the clinical hallmarks of down-gaze palsy in PSP and dysautonomia in MSA and other important symptoms, and their relationship with the disease duration in pathologically confirmed patients who happened to be matched in age-at-onset (AAO), gender, and disease duration. They had years of follow-up in a single tertiary movement disorder center with methodological and expertize uniformity. This study would help a better diagnostic and prognostic assessment of PSP and MSA, in particular for the first time, the prognostic value of down-gaze palsy latency in PSP progression.
Pathologically confirmed cases
We reviewed all the charts in patients with PSP or MSA who had been following up during 1994-2009 in the Center for Parkinson's Disease and Other Movement Disorders at Columbia University Medical Center with autopsy confirmed diagnosis. The neuropathological diagnostic criteria for PSP and MSA were as described before by our center. 13,14 In brief, the neuropathological diagnosis of PSP includes (1) the presence of taupositive tufted astrocytes in the cerebral cortex, neostriatum, and amygdaloid nucleus; (2) globose neuronal tangles in at least seven of the nine following sites: cerebral cortex, neostriatum, globus pallidus, subthalamic nucleus, red nucleus, pars compacta of the substantia nigra, pontine nuclei, inferior olivary nucleus, and dentate nucleus of the cerebellum; and (3) scattered tau-positive glial cytoplasmic inclusions. Glial cytoplasmic inclusions, which involve oligodendrocytes, are the molecular hallmark of MSA. They are labeled with antibodies directed against αsynuclein aggregates, or ubiquitinated proteins. MSA currently comprises subtype MSA-C and MSA-P, based on a set of predominant symptoms including autonomic failure, and distinct sites in the brain with relative selective vulnerability. The MSA-C is considered when the chief symptoms reflect cerebellar dysfunction, with the cerebellum and brainstem bearing the brunt of the degenerative process, which used to be referred to as olivopontocerebellar atrophy. The subtype MSA-P is considered when parkinsonism prevails, with the brunt of the changes involving the striatum (caudate nucleus, putamen, and globus pallidus) and the substantia nigra (pars compacta and pars reticulate), which used to be referred to as striatonigral degeneration. However, the overlaps of the symptoms and the main sites or systems of degeneration could be prominent as MSA progresses, despite the predominant involvement of either the cerebellar or the striatonigral system.
All patients had signed an informed consent form approved by the Columbia University Medical Center Internal Review Board. We excluded 4 out of 14 PSP patients due to the coexisting basal ganglia ischemic stroke, Alzheimer's disease and cerebral amyloid angiopathy, and insufficient documentation. We also excluded 2 out of 15 MSA patients with insufficient documentation.
Definitions in symptoms and signs recorded
We only recorded the symptoms and signs documented in 450% of the charts unless specified otherwise. Definitions of the terminologies are as follows. AAO: age at the first reported symptom considered attributable to PSP or MSA. Latency: the period between the onset of the specific symptom and the motor symptom onset. Disease duration: time between the age of onset and death. Falls: any fall related to the disease. Cognitive decline: perceived by the patient, the relatives or the physician, including reports of intellectual or functional decline, or the decline in MMSE (minimental status examination, normal scores cutoff ⩾ 26/30) or mMMSE (modified MMSE, normal score cutoff ⩾ 51/57) score. The total mMMSE score used in minority of the patients here was proportionally converted to commonly used MMSE score for quantitative comparison. Speech disturbance: any alteration in speech quality compared with that before the disease onset. Swallowing difficulty: any reported difficulty, particularly coughing to liquid or choking to solid food, or a formal abnormal swallowing evaluation. Supranuclear gaze palsy: restricted eye movement, with preserved vestibule-ocular reflexes on exam. Down-gaze palsy: restricted downward eye movement, with preserved vestibule-ocular reflexes on exam. Square wave jerk: inappropriate saccades that take the eye off the target, followed by a nearly normal intersaccadic interval (~200 ms), and then a corrective saccade that brings the eye back to the target with same speed. Clinically our cases were judged by a quick sideto-side globe movement of same speed in each direction with visual fixation. Pyramidal involvement: both brisk reflexes and pathologically extensor plantar or Hoffmann response(s). Autonomic dysfunction: either abnormal autonomic function testing or documentation of orthostatic hypotension, urinary incontinence, or any two of the following groups: urinary urgency or frequency; impotence; sweating abnormalities; skin color change; or severe constipation in need of medication on a regular basis. Urogenital dysfunction: urinary incontinence, or at least two of the following symptoms of impotence, urinary urgency or urinary frequency. Ataxia: any two of the following symptoms: ataxic gait (unsteady staggering gait with irregular pace and rhythm and usually broad base as well), dysmetric heel-knee-shin test or finger-to-nose test, broad base, titubation, persistent nystagmus, scanning speech, or unable to tandem walk. Frontal lobe dysfunction: apathy, lack of motivation, perseveration, withdrawal, personality change, or as suggested by neuropsychological tests. Response to levodopa: improvement in parkinsonism from the notes or Unified Parkinson's Disease Rating Scale of 430% coincident with the introduction of levodopa or other medications for parkinsonism.
Statistical analysis
Non-paired Student's t-test was used for demographics or symptoms comparison between PSP and MSA. A P-value of 0.05 was viewed as significant. A linear Pearson correlation was applied to study the relationship between a specific symptom and disease duration.
RESULTS
Demographics, clinical symptoms and signs As presented in the Table 1, 10 PSP and 13 MSA patients (7 MSA-P and 6 MSA-C) were eventually studied, incidentally well matched in AAO, gender, and disease duration between two diseases. The final antemortem clinical diagnosis was correct in 70% of the PSP patients, all with down-gaze palsy, with the remaining 30% of patients being misdiagnosed (due to the lack of down-gaze palsy) as either MSA due to the presence of dysautonomia, ataxia and pyramidal involvement, corticobasal degeneration due to the presence of apraxia, alien hand phenomena, dystonia and abnormal positron emission tomography imaging, or frontotemporal lobe dementia parkinsonism syndrome due to the presence of frontal lobe dysfunction. The final antemortem clinical diagnosis was correct in 77% of the MSA patients. The remaining 23% (3 patients, all MSA-P) of patients were misdiagnosed as atypical or vascular parkinsonism, all presenting with parkinsonism but without ataxia, with no dysautonomia in life in one of them, and delayed dysautonomia at year 3 after disease onset in another two.
Down-gaze palsy and square wave jerks were present in most of the PSP patients (70 and 90%, respectively), but much less common in MSA (8 and 23%, respectively). Dysautonomia was present in at least 80% of the PSP patients during the disease course, but involved only urogenital dysfunction without orthostatic hypotension, and none was present before the onset of the motor symptoms. Dysautonomia was present at motor symptom onset in 38% of the MSA patients, and in 46, 62, and 77% of the patients 1, 3 and, 5 years, respectively, afterwards. Interestingly, 23% of the MSA patients even had dysautonomia as early as 3 years preceding the onset of the motor symptoms, and 15% of the MSA patients never had dysautonomia in life, despite being assessed in the last year of their life.
Dystonia occurred in at least 50% of the patients with PSP, and 46% of MSA, with anterocollis being seen only in MSA patients. Ataxia occurred in at least 50% of the patients with PSP, on average 4.6 years after the motor symptom onset, and in at least 61% (6 MSA-C, 2 MSA-P) of the patients with MSA, with ataxia as the presenting features in all MSA-C. Pyramidal involvement was seen in at least 30% of the patients with PSP and MSA (both MSA-C and MSA-P). Apraxia occurred in at least 50% of the patients with PSP, 20% with alien limb phenomena and 10% with cortical sensory loss. There was no difference in the latency of first fall (1.7 ± 1.8 vs. 2.7 ± 2.3 years, P = 0.278), dysarthria (2.7 ± 2 vs. 2.3 ± 2.5 years, P = 0.724), dysphagia (3.5 ± 1.7 vs. 4.1 ± 2.8 years, P = 0.612), or the percentage of the patients with freezing of gait (50 vs. 46%, P = 0.6217) between PSP and MSA.
Laryngeal stridor was present in at least 30% of the MSA patients (1 MSA-P and 3 MSA-C) on average 4.8 years after motor onset, and all died 2-5 years later, including one who had tracheostomy. Myoclonus was present in at least 38% patients of MSA (3 MSA-P and 2 MSA-C), even in the absence of dopaminergic medications in some patients.
The average MMSE score in PSP was mildly impaired at their first visit of 2.7 years on average after the disease onset. Consistently, frontal lobe dysfunction was documented in 70% of the PSP patients. Cognition was normal to mildly impaired in MSA, as assessed on average of 5 years after motor onset.
Response of the parkinsonism to levodopa treatment Response of the parkinsonism to levodopa treatment was seen in at least 30% of the PSP patients, all of whom had resting tremor, at 450-2000 mg daily in the first 4-7 years of disease; and in at least 54% of the patients with MSA (4 MSA-P, and 3 MSA-C), at 200-1300 mg daily in the first 2-10 years.
Correlation between disease duration and important clinical features
The early manifestation of down-gaze palsy (in seven patients with down-gaze palsy before death, Pearson correlation coefficient r = 0.902, P = 0.005), urogenital dysfunction (r = 0.752, P = 0.031), or poor MMSE score at their first visit (on average the first 2.7 years, r = 0.920, P = 0.009) in PSP, and the early presence of the first fall (r = 0.675, P = 0.016) or wheelchair confinement (r = 0.990, P = 0.01) in MSA were all associated with a short disease duration or rapid progression (Figure 1, all panels except the top right panel). The latency of the down-gaze palsy was still associated with disease duration in PSP even when we included all 10 patients (counting the disease duration as the latency for the three patients without down-gaze palsy before the death, r = 0.8893, P = 0.001) (Figure 1, top right panel). There was no correlation between the disease duration and AAO, and the latency of speech or swallowing dysfunction in PSP and MSA.
Brain imaging Brain magnetic resonance imaging (MRI) was all reported to be normal or to show mild diffuse atrophy in eight PSP patients, one with midbrain atrophy. There was no reported cerebellar atrophy in PSP with cerebellar ataxia patients based on radiologists' report, though the brain MRI was obtained 2 years earlier (2.6 ± 1.1) than the onset of the ataxia (4.6 ± 1.5). Cerebellar atrophy was reported in 90% of the nine MSA patients, with two pontine atrophy (1 MSA-C and 1 MSA-P) and one hot cross bun sign.
DISCUSSION
Our study not only confirmed most of the previously reported clinical features of PSP and MSA, but also for the first time found that down-gaze palsy has a prognostic value in PSP progression (Pearson correlation coefficient r = 0.902, P = 0.005). Our novel finding is supported clinically by the fact that the PSP-RS with early down-gaze palsy (usually in 2 years after the disease onset) has a more rapid disease progression and shorter disease duration (on average 5.9 years) than PSP-P, which has late onset or even no down-gaze palsy with longer disease duration (on average 9.1 years). 8,10 Indeed similarly, our three PSP patients who did not have down-gaze palsy (therefore were all misdiagnosed) had parkinsonism and longer disease duration (9.3 ± 2.5 years) compared with the seven PSP patients with down-gaze palsy (6.5 ± 3.5 years), though not statistically different due to the small sample size, with one having additional unilateral resting tremor and good response to levodopa treatment, one having essential tremor, and the other having corticobasal syndrome. Our novel finding could also be explained pathologically by the fact that most PSP patients started with globus pallidus interna, subthalamic nucleus, and substantia nigra par compacta and gradually extended rostrally to the frontal lobe, parietal lobe, and other regions, and caudally to the brainstem and cerebellar dentate nucleus. 9 Brainstem is suggested to control supranuclear eye movements. 15 Hence, the early occurrence of supranuclear palsy indicates a rapid progression of the disease and short survival duration. Similarly, we could also explain the association of the early occurrence of the cognitive dysfunction with short disease duration, consistent with a previous report, 10 though MMSE may not be the best test to assess cognitive dysfunction in both disorders. Our finding could help predict the PSP progression, which is very important for both family planning and disease management. Urogenital dysfunction is not uncommon in our PSP cases, as reported elsewhere. 6,16,17 Interestingly, the latency of the urogenital dysfunction is associated with the disease duration in our patients. The urogenital dysfunction might well represent frontal lobe dysfunction in PSP, given the lack of orthostatic hypotension and the absence of urogenital dysfunction before the disease onset in our study and another report. 16 Dysautonomia could occur as early as 3 years before the motor onset in our MSA patients. Interestingly, 15% of the MSA patients had no documented dysautonomia, even assessed in their last year of life, consistent with another report, 18 which makes the diagnosis of MSA more challenging. Indeed, 8% of the patients missed the diagnosis partially due to the absence of dysautonomia in our study. Worth noting that one cannot absolutely claim that in some cases MSA patients did not have dysautonomia but rather that this was not clinically reported. It is conceivable that in some cases autonomic dysfunction might be mild and only detected instrumentally. Ataxia used to be listed in the exclusion criteria of PSP. 2 However, it is not surprising that ataxia was also found in at least 50% of the PSP patients, but at a later disease stage of 4.6 years on average after the disease onset, as the dentate nucleus in the cerebellum is involved later on as the disease advances caudally, different from that in MSA-C with ataxia as a presenting feature. There was no reported cerebellar atrophy in PSP patients with cerebellar ataxia based on radiologists' report, which is different from that in MSA, as cerebellar atrophy in brain MRI is commonly seen in our MSA-C and MSA-P patients. The obtaining of brain MRI 2 years earlier than the onset of the ataxia in our patients might contribute to the lack of association between ataxia and imaging change. Ataxia has been reported in another study in PSP. 12 It may be plausible to consider an additional type of PSP, PSP-C (cerebellar type), for those with a prominent feature of ataxia.
Apraxia, another sign that used to be in the exclusion criteria of PSP, 2 was also found in at least 50% of our PSP cases, 20% even with alien hand phenomena, and 10% with cortical sensory loss, which most likely is PSP-corticobasal syndrome, 11 with the morphology of PSP instead of corticobasal degeneration. 19 There was no apraxia in MSA, as expected, which helps to differentiate it from PSP.
The presence of limb dystonia in PSP and anterocollis in MSA cases is also consistent with a previous report. 20 The response of parkinsonism to levodopa was seen only in PSP with resting tremor, consistent with the notion of PSP-P, who has tremor and parkinsonism with good response to dopaminergic medication treatment. 8 The percentage of the patients responsive to dopaminergic medication in PSP and MSA is similar to that reported before, 6,10 indicating that dopaminergic medication is worth trying in these diseases, particularly in PSP with resting tremor. A poor prognosis was found in MSA cases who had early falls and wheelchair confinement, consistent with a previous report. 10 Our study has its unique strength and novelty. First, all the patients had been followed clinically in a single tertiary movement disorder center for several years until autopsy, with good methodological and expertize uniformity. Second, the patients with other concomitant brain diseases were excluded to avoid confounding clinical features. Third, the AAO, gender, and disease duration were all incidentally well matched in PSP and MSA, which make a fair comparison between PSP and MSA possible. Fourth, we studied the diagnostic milestones of PSP (down-gaze palsy) and MSA (dysautonomia), and other important symptoms in temporal evolution for the diagnostic and prognostic value of the symptoms. Most importantly, we for the first time found the novel linear prognostic value of the down-gaze palsy latency in PSP progression, which would have significant clinical implications.
Our study also has its limitations. The sample size is small and information is missing for some patients. We might have a selection bias, as only the atypical cases were sent to our tertiary center, which is probably why our MSA cases had older than usual AAO. The retrospective reviewing of the charts could have less comprehensive information. The assessment of the ataxia in PSP might not be accurate given the presence of postural instability and the complexity of the ataxia in this retrospective study. The MRI results were taken from radiology reports without being read by us, which could have a false negative report. The dysautonomic symptoms could be confounded by the comorbidities of diabetes, enlarged prostate in men or stress incontinence in woman, which may not have been formally diagnosed or well documented.
A further study with large size in a prospective cohort would help to corroborate our results in the future.
In conclusion, our pathologically verified PSP and MSA patients followed for years by movement disorder specialists in a tertiary center provide valuable clinical features for the diagnosis and prognosis of PSP and MSA. We not only verified many previously reported characterizations, but also for the first time found the prognostic value of the down-gaze palsy latency in PSP progression, which would have significant clinical implications. | 2017-11-08T19:18:29.601Z | 2015-05-21T00:00:00.000 | {
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167857813 | pes2o/s2orc | v3-fos-license | Earnings Growth and Value Premium: The Indian Experience
Executive Summary This article addresses two main questions: Does a value premium exist in emerging market like India? If so, how pervasive is it in different market conditions? Value premium is assumed to be the difference in stock returns of undervalued and overvalued firms with a unique industry profile. The sample for the study consisted of 32 companies from the Information Technology (IT) sector, the stocks of which had traded continuously in the National Stock Exchange (NSE) during the period 2000–2010. Prowess and Capitaline constitute the sources for the firm-level financial data, and NSE web sources provide data related to share prices and market capitalization. The study involved a two-step empirical procedure: an exploratory factor analysis and a regression modelling under Ordinary Least Square (OLS) method. Exploratory factor analysis identified earnings growth and Earnings Price (E/P) rate as the prime determinants of stock returns. Expected earnings growth significantly explained E/P rate under OLS regression framework. The study then estimated normal E/P rate for the individual stock and compared the same with the actual E/P. If the actual E/P for a particular stock was greater than its estimated E/P, it was inferred that the stock was undervalued, the reverse being the case for overvaluation. The findings of this research provide empirical validity of use of E/P rate in identifying mispriced stocks in the Indian context. Undervalued stocks can produce better returns compared to overvalued stocks, and their success has been both persistent and impressive. E/P rate or P/E ratio is a valuable analytic device when properly interpreted. The publicly available E/P rate seems to possess information content and warrants an investor’s attention at the time of his portfolio formation or revision. The search process involving E/P rate suggests that the best buy would be the stock whose reported earnings per share is expected to grow most rapidly.
T he question of whether value strategies outperform growth strategies has been a matter of empirical investigation for the last several years. Many academic researches provide conclusive evidence on this issue in different country contexts. As per the financial literature, the difference in return produced by these two investment strategies is the value premium. The fundamental analysts adjust their strategies towards this premium.
Value strategies trace their roots to Graham and Dodd (1934). One of the pioneer studies on the outperformance of growth by value was by Basu (1977). Chan, Hamao, and Lakonishok (1991) observed similar results on value stocks in Japan. Fama andFrench (1992, 1996) and Lakonishok, Shleifer, and Vishny (1994) proved the existence of strong value premium in average returns for the US stocks. By using data on the US, Japan, and European markets, Capaul, Rowley, and Sharpe (1993) found value premium pervasive in international stock returns. Bauman, Conover, and Miller (1998) extended the previous study by Capaul, Rowley, and Sharpe (1993) in terms of time and coverage (21 countries) and confirmed that value outperformed growth, though not necessarily in every country or in every year. Arshanapalli, Coggin, and Doukas (1998) reinforced the relation of such findings with international market further, but suggested that value strategies were not riskier than growth strategies. Abhyankar, Ho, and Zho (2006), by using US data, found strong evidence that value stocks stochastically dominated growth stocks during economic boom periods, but lacked such dominance during recession period. However, in the Indian context, the findings of Saji (2012) provided evidence on the outperformance of growth stocks by value stocks during market downturn. Deb (2012) made similar observation with regard to the existence of value premium in the Indian stock market. While Gulen, Xing, and Zhang (2010) showed time variations of expected returns, Athanassakos (2009) corroborated the findings of earlier studies by using Canadian data.
On reviewing the literature, it is obvious that the academic community has consensus on the outperformance of growth strategies by value strategies, but the underlying reasons for the performance are controversial. Fama and French (1996) attributed the higher returns of value strategies to their increased risk. They argued that the value stocks compensated investors for bearing high fundamental risks. Lakonishok et al. (1994) found cognitive biases in investor behaviour and agency costs as the major causes of higher rewards to value investing. Kothari, Shanken, and Sloan (1995) identified data selection bias as the reasons for superior performance of value investing, but Chan, Jegadeesh, and Lakonishok (1995) repudiated that argument. Dreman and Berry (1995) found analysts' errors having an asymmetrical influence on high and low P/E stocks. Laporta (1996) stated that value stocks generated superior returns due to the behaviour attributes and expectation errors made by investors.
There also exists strong disagreement among academic research as to the choice of reliable indicator of market valuation of firms. Basu (1977) and Campbell and Shiller (1998) found Price to Earnings (P/E) ratio as a good predictor of equity returns. Chan et al. (1991) suggested that Book to Market (B/M) value and Cash flows to Price (C/P) were significant in variation of returns. Fama and French (1992) found firm size and B/M as the factors explaining the cross-sectional variations in returns, while Lakonishok et al. (1994) provided evidence only for B/M effect. Leledakis and Davidson (2001) stood for better predictability of returns by ratios of sales to price and debt to equity. Athanassakos (2009) showed P/E ratio as a better predictor of average equity returns than Price to Book (P/B) ratio.
This article does not offer alternative explanations to value versus growth investment strategies; instead, it aims to provide further evidence on the value premium, which has not been much explored in the Indian context. For the purpose of this study, value premium is assumed as the difference in stock returns of undervalued and overvalued firms with a unique industry profile. Two main questions are examined here: Does a value premium exist in an emerging market like India? If so, how pervasive is it in different market conditions? Several factors make this study important. First, the period of the study, that is, from April 2000 to March 2010, when, in the strict sense, the Indian market had reached its present growth level. This period is significant for India because of other reasons as well. The prominent capital flow episode for the emerging market economies started in 2002and almost ended in 2009(Global Financial Stability Report, 2010. Cardarelli, Elekdag, and Kose (2010) specifically documented the importance of this period for emerging economies like India. To the best of our knowledge, no exhaustive study on value versus growth aspect, under a crosssectional regression framework, has been carried out so far in the Indian context during this period.
Second, this study provides rigorous statistical treatment for analysing the issue under investigation. From factor identification to fixation of style-specific benchmark, the quantitative methodology pursued in the study is expected to proliferate in performance evaluation and attribution analysis.
Third, unlike most prior studies, which focus on the value premium on portfolio basis, this study focuses on it at the individual stock level. This definitely makes the investors more familiar with the firm-specific characteristics, which lead stock valuation in different market conditions. Finally, earning fundamentals and stock returns of firms in the same industry (Information Technology) with identical product mix or product policies can provide comparison that is more meaningful at the firm level analysis.
Data
The sample for the present study consisted of 32 companies from the Information Technology (IT) sector, the stocks of which had been traded continuously (having minimum 15 days of trading in every month of the study period) in NSE (National Stock Exchange) during the period 2000-2010. The focus on IT firms was natural because it was the fastest growing sector in India during the study period. The contribution of IT/ITES industry to the GDP of the country soared up to a share of 7 per cent in 2008 from a mere 1.2 per cent in 1998 (NASSCOM-Deloitte, 2008). Therefore, the theories we aim to test derive asset pricing information from such growth-oriented investment decisions.
Data on the firms under study were collected from three sources: Prowess (Centre for Monitoring of Indian Economy) and Capitaline for data measuring company fundamentals; NSE database for data related to share price and market capitalization; and stock price data adjusted for stock splits and bonus issue.
To determine the potential predictor of stock returns, 12 exploratory financial variables (including beta and stock returns) were drawn from the existing literature and intuitive knowledge of the data. The variables included EPS, earnings growth, Return on Equity (ROE), Return on Capital Employed (ROCE), Debt to Equity ratio, Beta (market risk premium), Earnings Price (E/P) rate or Earnings yield, Book Value per share (BV), Price to Book value ratio (P/B), Market Capitalization (MC), Dividend yield and Average Stock Return (AR). The sum of cash flow received and price change over the year, as a percentage of the price of investment at the beginning, gave the annual return.
Methodology
The methodology pursued in the study involved a two-step empirical procedure: an exploratory factor analysis and regression modeling under Ordinary Least Square (OLS) method. Exploratory factor analysis intended to discover the common factors or latent variables among the variables considered for the study. In this case, factor analysis found its application as a data reduction technique and could identify the variables that were potentially significant in explaining the variations in stock returns. The ultimate aim of this methodology was to select independent variables, which could be included in the regression estimation framework. OLS regression technique can measure and estimate causal relation between the identified fundamentals and stock returns. Further explanations of the methodology elaborate with the results and discussions.
Factor Analysis
Exploratory factor analysis administered in the study investigated the dimension that would have caused correlations among the observed company fundamental variables. The factor extraction method of Principal Component Analysis (PCA) was opted to identify the critical factors. In PCA, the variables must correlate with each other for the factor model to be appropriate. So, a correlation coefficient matrix of selected variables was prepared at first for deciding the feasibility of PCA to the collected financial data and then a test proposed by Kaiser-Meyer-Olkin (KMO) (1974) and Bartlett (1937) was applied to see whether the sample variables were adequate for factor analysis. The correlation matrix and the results of KMO-Bartlett's test are reported in Tables 1 and 2 respectively.
The correlation matrix illustrates the extent to which the selected 12 financial variables are correlated pair-wise in a matrix. Out of 66 cells below the main diagonal, there are only 14 correlation coefficients (the numbers that go from the upper left corner to the lower right) above or equal to 0.25, which are also statistically significant (at 1 % level) and different from zero. The KMO statistic reported in Table 2 represents the ratio of the squared correlation between the variables to the squared partial correlation between those variables. The KMO statistic varies between 0 and 1. Kaiser (1974) recommended values greater than 0.5 as acceptable. For the data used in this study, the value is 0.5469; hence, factor analysis is appropriate for the study.
The Bartlett's measure tests the null hypothesis, that the original correlation matrix is an identity matrix. If the correlation matrix were an identity matrix, all correlation coefficients would be zero. For factor analysis to work, there should be some relationship between the variables and hence the correlation matrix should not be an identity matrix. Therefore, we hope to include some relationships between the variables in the analysis. For the data used in this study, Bartlett's test is highly significant (p < 0.001), and therefore, factor analysis is appropriate for this study.
After testing the adequacy of the data and the explanatory variables, the study undertook factor analysis with the 12 company financial variables. For determining the number of factors, representing the financial data, eigen values were estimated. The eigen values associated with each factor represented the variance explained by that particular linear component. To have better interpretability of factors, the orthogonal factor rotation method Varimax was used. By following Kaiser's (1974) rule of retaining factors with eigen values greater than unity, five factors were extracted from the data. Table 3 shows the proportion (%) of variance explained by each factor, and indicates that the five factors altogether account for 69.48 per cent of the total variance. Since this study intended to verify the existence of value premium in the Indian context, we needed to focus only on the factor that would be loaded to stock returns, along with its probable determinants. Hence, Factor 3, labelled as the valuation factor, was the most relevant for further quantitative treatment as will be observed in the later part of the study. Valuation factor indicates how well the securities are valued in the market, and what determines its valuation and the valuation outcome. Earnings yield (E/P rate), earnings growth, and average return were positively loaded on valuation factor. Positive loading implies that the variables were positively correlated with the factor. Growth in earnings leads to larger increase in Earnings per Share (EPS) of firms relative to the market price of its share (increase in earnings yield/decrease in P/E ratio). An increase in this factor indicates that the company has high earnings per share, but the market price of its share is low which implies that the investment in it has good potential for growth in terms of capital appreciation in future. An increase in this factor over a period shall have a positive effect on the share price, thereby enabling the investors to enjoy increased returns from their investments.
Based on the findings of factor analysis, it was hypothesized that the financial variables included in the valuation factor constituted the major determinants of stock returns in India. To be precise, the variablesearnings growth and E/P rate-were considered as the prime parameters for tracking the price behaviour or returns of stocks in the Indian context. The validity of this hypothesis was further verified under a simple OLS regression framework.
Expected E/P Rate: Benchmark Estimate
The decision on a standard of comparison or an appropriate benchmark must follow the identification of stock return determinants. There are at least three possibilities for the appropriate benchmark. First, the observed determinant could be compared to the temporal benchmark; that is, its average over the previous several years. Here we may take the mean or the median value of that determinant overtime. The other two possibilities could be a cross-sectional benchmark, namely, an industry average and a theoretical benchmark developed under a standard regression framework. Since the earlier part of the analysis in this study found positive relationship between E/P rate and stock returns in India, an OLS regression can estimate a theoretical E/P (hereafterexpected E/P) rate.
One of the first studies to employ regression methodology for estimating PE (reciprocal of E/P rate) ratio is by Whitbeck and Kisor (1963). Their study found that projected earnings growth, expected dividend payout, and growth risk explained the differences in P/E ratios. Bower and Bower (1969) used a similar approach and found almost the same effects of earnings growth and payout on P/E ratio. Malkiel and Cragg (1970) studied the effects of historical growth of earnings, dividend payout ratio, and the stock's rate of return relative to the market in determining P/E. Bernard and Thomas (1989) and Clare and Thomas (1995) used similar methodology to estimate the P/E ratio for a given set of fundamentals. Even though strong disagreements exist among equity researchers as to the relation between company fundamentals and P/E ratios, most of them have consensus as to the existence of causal relationship between expected growth rate and P/E ratios of companies in various markets. This study used the E/P rate rather than the P/E ratio as dependent variable because of many reasons. Some of the studies, for example, Litzenberger and Rao (1971), posited linearity in E/P (not in P/E). The study covered an episodic period of Indian market; hence, during the days of negative earnings, the measurement of P/E ratio in real mathematical terms is not possible. Moreover, the general approach pursued in factor analysis in the earlier section of this study found the co-movement of E/P rate and the growth rate of earnings.
Fundamental analysts generally consider the capitalization rate of the current stock earnings as the outcome of psychological as well as economic factors. The forecast rate of growth in reported earnings is likely to be the single most determinant of the price the investors are willing to pay. The absence of significant correlation between E/P rate and financial variables excluding earnings growth gives sufficient freedom to assume that E/P rate is the sole function of its expected growth rate of earnings. Thus, a simple regression model, which regress the E/P rate of selected firms against their expected earnings growth rate, can validate the fundamental theory. However, this type of analysis may indicate the relative level of E/P rate of selected stocks (compared with the E/P rate for a normalized level), but does not indicate the specific level of E/P rate that is appropriate for a certain stock.
For proceeding further, first we have to fit a single factor regression model explaining the causal relationship between the E/P and the expected earnings growth of a firm: a and u i are constant and error term (which assumed to be zero) respectively Y i = Expected E/P rate X i = Expected earnings growth b i = Impact of expected earnings growth on E/P rate
Expected Growth Rate of Earnings
The expected growth rate in earnings (EPS) is an important factor in equity valuation. If one expects a relatively stable cost and profit structure in the near future, the growth rate in earnings may equate with the projected growth rate in sales. However, as far as a growing service industry is concerned, this will never happen. This is because of the intensity of competition, increasing operating cost especially the employee cost, and the presence of heavy research and development expenditure in its cost structure. It is therefore rational to assume the expected growth rate of earnings in future as the function of the past earnings growth.
There are different approaches followed by analysts in ascertaining past earnings growth of firms. Sometimes they take arithmetic mean of growth rate of earnings of the firms for the past few years and, in some other cases, geometric mean instead of arithmetic average. For many investment problems that deal with rates of return, security analysts are interested in the geometric mean as opposed to the arithmetic mean. This is because arithmetic mean is always greater than the geometric mean. Nevertheless, in those cases when the individual growth rates are constant, the arithmetic mean and geometric mean will be the same (McEnally, 1986). For the purpose of analysis, one should use an average of earnings of 'not less than five years' (Graham & Dodd, 1934).
Previous researches in the area have used arithmetic mean of earnings growth under the assumption of constant average annual growth. The regression model used in this study also holds the same assumption, and hence arithmetic mean of the last five years (including the year of estimation) earnings growth rate was used as the expected growth of earnings in every fifth year. Table 4 displays the results of regression that included earnings growth as independent variable. The expected sign of the relationship between earnings growth and E/P is the same (positive) as in the factor analysis.
E/P Rate and Expected Earnings Growth: Regression Results
The regression results summarize the explanatory power of expected growth rate of earnings in determining the variations in E/P rate. The value of R 2 (i.e., proportion of variance explained) adjusted for degrees of freedom ranges from 5 per cent to 59 per cent. In several years, the expected growth of earnings of the firm could explain more than one-third of the percentage variations in E/P rate. The F statistic, which tests the null hypothesis that all the coefficient are zero (expected earnings growth shall not be able to explain the earning price relationship of a particular year) is found to be significant at 1 per cent level in five out of the six years considered for the study. Only in 2007-2008, the expected earnings growth is insignificant in explaining the E/P rate of that year. This could be due to the independence of stock prices from earning fundamentals during the buoyant market conditions due to the extreme play of investor psychometrics. In all other years, the regression coefficient of expected growth of earnings on E/P rate is found to be significant at 1 per cent level. These results confirm the significance of earnings growth in estimating E/P of firms in the Indian market conditions.
Comparison between Actual and Normal E/P Rates
The practitioners of value strategies often deny the efficient market hypothesis, which proposes the impossibility of earning excess returns in a market, which is efficient in terms of investment information.
The value strategists also assert that P/E ratios, due to exaggerated investor expectations, may be an indicator of future investment performance and the use of which definitely helps investors in assessing the true worth of a stock and provides opportunity for them to take advantage of market disequilibria by acquiring low P/E (high E/P) stocks.
Once the analyst has determined the average relationship between the P/E ratio and the expected earnings growth for all stocks, then he can estimate the P/E ratio (Normal P/E ratio) for the individual stock and compare the same with the actual P/E. If the actual P/E for a particular stock is greater than its estimated P/E, he might conclude that the stock is overpriced, and if actual P/E is smaller than the Normal P/E, he will consider it as underpriced and may purchase with a reasonable expectation that its price will rise. If the actual P/E equals the Normal P/E, his claim is correct pricing of stock at the given market conditions. Since this study has used E/P rate, the conclusions of the comparison shall be just opposite of these investment judgements.
Investors believe in value strategies and ultimately wish to park their funds in undervalued/fairly valued stocks. This is because of their notion that the overpriced stocks have their prices and P/E driven down to appropriate levels by the selling pressure which in turn causes loss to their wealth. So, for the purpose of this study, the stocks were classified as undervalued (including fairly valued) and overvalued based solely on the comparison between actual E/P and expected E/P of each stock included in the sample. Table 5 reports the results of comparison. The analysis shows that 13 stocks were undervalued in 2004-2005, 15 stocks in 2005-2006, another 15 stocks in 2006-2007, and 20 stocks in 2008-2009. Stocks of certain medium-and small-sized companies like GTL, Tata Elxsi, California Softwares (Calsoft), Sterlite Technologies, KLG Systel, and Aftek were found to be undervalued in the market in all the four periods of analysis. It is worth mentioning that the market had overvalued some well-known stocks like Infosys, CMC, Wipro, and HCL Technologies in all years of observation. Similarly, stocks of some small companies (Computech and Cybertech) were successively undervalued by the market in all years except 2008-2009, the crisis year for the Indian economy as well as the Indian stock market. An overvaluation of small stocks during the financial crisis can be attributed to the play of traders/speculators who normally wish to trade in small stocks for capitalizing the opportunities created by the market imperfections.
Return Profile of Undervalued and Overvalued Stocks: A Comparison
Does the security valuation strategy based on E/P rate of fundamentalists really help the investors to produce better returns (value premium) in the subsequent year of market valuation of stocks? For answering this question, it was assumed that an investor should purchase the sample stocks on the first day of the accounting period subsequent to the year in which the valuation is done and hold it until the last trading day of that period. By taking the difference in the market values of the stocks on these two dates, plus any dividend declared and paid during the period, the return that he would have made from it, was computed. Such comparison was done only for four periods-2005-2006, 2006-2007, 2007-2008, and 2009-2010 In 2007-2008, only six stocks included in the sample generated a positive return for their investors, out of which only one was from the overvalued group, while the rest were from the undervalued group of shares. The pattern of return profile of stocks thus, indicates that even during bad market conditions, the undervalued stocks can somewhat protect the investors' interest, though that attribute is not common for all stocks in the group. All stocks (except Smartlink) included in the sample were able to deliver relatively good returns to their investors in 2009-2010. To an extent, we can attribute this fact to the gradual revival of investor confidence after the shock that global economic recession exerted on Indian financial system, and to the reinstatement of the stable political system of the country. Both these factors led to rushed purchase of stocks that in turn made a surge in their market value. However, when we closely look into the pattern of their growth in value during this period, we can see that the growth rate was remarkably high in case of most of the stocks that were under-valued according to Table 5 by the same market in the preceding year. Of the five stocks, which occupied the foremost positions in terms of growth in value among the group, four were from the undervalued group. When six of the undervalued stocks were able to produce returns at a rate, which was 300 per cent or more, only one of the overvalued stocks was able to generate return to its investors at that scale. A close analysis of the results also reveals that the stock investments in small-sized firms earned significantly higher rates of return than the similar investments in large-sized companies. This finding is in conformity with the findings of Banz (1981). Table 7 displays the average returns on an annual basis produced by undervalued and overvalued stocks for its investors during the four assessment periods, along with the return produced by the broad-based Index NSE Nifty (market return). It is implicit from the analysis that during all the periods of observation (except in 2007-2008), undervalued stocks received returns (on average) at a rate which were much higher than the rate of return given by the market index. However, overvalued stocks were able to beat the market in terms of returns only in 2009-2010. In all the other years of observation, the performance of overvalued stocks was poorer than the overall market performance. The t-test checked the statistical significance of the difference in returns of the two groups of stocks. The test procedure demands the checking of the equality of variances of the groups for having inferences that are more useful. The Levene's test of homogeneity of variance based on F statistic was used for this purpose, which rejected the null hypothesis that the variances of the groups under observation were the same. When the variances are not equal, one should use Welch's modification test (Gastwirth, Gel, & Miao, 2009). Therefore, we chose the output of Welch's test for drawing inference as to the significance of difference in stock returns. The Welch's test found the difference in mean returns of undervalued and overvalued stocks statistically significant at 5 per cent level (in 2005-2006 and 2006-2007) and at 10 per cent level (in 2007-2008). In 2009-2010, the analysis has not observed the statistical significance of difference in the performance of two group stocks at any level. The continuity of the superior performance of undervalued stocks relative to the market, as well as overvalued stocks reinforce the validity of the argument of the investment strategists in the use of P/E ratio as the tool for earning excess returns from stock market investments.
CONCLUSION
From the above discussions, it may be right to conclude that value strategies outperform growth strategies in managing stock market investments in India. In other words, existence of value premium is evident in the Indian context. This paper finds earnings growth, E/P rate, and stock returns to be positively correlated. The estimation of E/P rate using earnings growth and the comparison of the same with actual E/P rate can identify mispriced stocks in the Indian context. Undervalued stocks can produce better returns than overvalued stocks, and their success has been both persistent and impressive. In each of the four years following the year in which the stocks were evaluated, the undervalued group had a mean return performance superior to that of the stocks labelled as overvalued in terms of its expected E/P rate. Therefore, the E/P rate or the P/E ratio is a valuable analytic device when properly interpreted. It is of primary importance in assisting investors in evaluating stock purchases and particularly helpful in the difficult analysis of the growth situations. The publicly available E/P rate seems to possess information content and warrant an investor's attention at the time of his portfolio formation or revision. The search process involving E/P rate suggests that the best buy would be the stock whose reported earnings per share is expected to grow most rapidly. On a long-term perspective, investment in fundamentally strong companies, having low P/E ratio or high E/P rate, would definitely deliver better returns than those provided by other strategies. However, one should be more cautious in estimating the growth rate of earnings; otherwise, the outcome may not be as per the expectation. The other company financials do not seem to be of much significance in explaining the variations in stock returns in India. | 2019-05-29T13:14:06.314Z | 2015-12-01T00:00:00.000 | {
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109950719 | pes2o/s2orc | v3-fos-license | COMBATING THE ‘SICK BUILDING SYNDROME’ BY IMPROVING INDOOR AIR QUALITY
The Sick Building Syndrome (SBS) is a term often used to explain symptoms, such as headaches, dizziness, runny noses, itchiness and so on, produced by any harmful environment in buildings, especially in air-conditioned buildings (Royal Australian Institute of Architects, 1991, p.1). As approximately 80 to 90% of people’s time is spent in buildings (EAP, 1998,), concerns about indoor air quality resulting in such symptoms are increasing. There are a number of methods which appear to be able to reduce the SBS symptoms, such as purifying/cleaning indoor air and utilising materials for construction, furniture and finishes that have been proven to reduce adverse effects. O’Brian (1988) and Karnstedt (1991), suggest that ionised air is beneficial to human health and can also reduce the symptoms of SBS. Based on the premise that, if SBS and the potential for harmful conditions within buildings are to be avoided, building professionals require suitable design tools (Ferguson 1987, p.1), this paper describes a method for estimating how much additional negative ions should be added to a room/office to compensate for the losses caused by three major factors namely, video display terminals (VDT), heating ventilation and air-conditioning (HVAC) and building contents (BC).
Sick Building Syndrome (SBS)
The Sick Building Syndrome (SBS) is a term often used to explain symptoms, such as headaches, dizziness, runny noses, itchiness and so on, produced by any harmful environment in buildings, especially in air-conditioned buildings (Royal Australian Institute of Architects, 1991, p.1).As approximately 80 to 90% of people's time is spent in buildings (EAP, 1998,), concerns about indoor air quality resulting in such symptoms are increasing.
There are a number of methods which appear to be able to reduce the SBS symptoms, such as purifying/cleaning indoor air and utilising materials for construction, furniture and finishes that have been proven to reduce adverse effects.O'Brian (1988) and Karnstedt (1991), suggest that ionised air is beneficial to human health and can also reduce the symptoms of SBS.
Based on the premise that, if SBS and the potential for harmful conditions within buildings are to be avoided, building professionals require suitable design tools (Ferguson 1987, p.1), this paper describes a method for estimating how much additional negative ions should be added to a room/office to compensate for the losses caused by three major factors namely, video display terminals (VDT), heating ventilation and air-conditioning (HVAC) and building contents (BC).
Negative Ions
Ions are electrically charged atoms or molecules that can gain or lose an electron (Bionic Products, 1998).An atom gaining electron(s) is called a negative ion and an atom losing electron(s) are called a positive ion.From this formation process of the ions, there seem to be a number of ion effects, positively and negatively in terms of indoor air quality.
With regard to the nature of the movement of electrons, negative ions (atoms with excess electrons) are quicker than positive ions.The positive effects of this phenomenon can be felt near waterfalls, in pine forests and on the seashore where waves are breaking on the shore (Bionic Products, 1998).
Most negative ions are likely to be the negative hydrogen ions (-H) produced by solar continuous spectrum (Massey, 1976, p.673).Massey suggests that the number of negative ions of carbon (-C) and oxygen (-O) produced by ozone reaction are significant.These types of negative ions are therefore also considered for the purposes of the calculations used in this study.
Negative Ion Therapy
There has been a considerable amount of research that has shown positive and beneficial effects from negative ions such as removing air particles and contaminants, and preventing and reducing the SBS (O'Brian, 1988, andKarnstedt, 1991).Some researchers however, are sceptical about the positive effects.They suggest that the reverse may be true and that generating negative ions might emit ozone excess that can irritate eyes and affect the lungs (Nava, 1998).It is not the aim of this study to argue either way but rather to demonstrate a method for estimating the de-ionising effects of key factors in modern buildings.
The following tables show the possible effects of the amount of negative ions in particular areas (Penex Air Ionisers, 1988).Figure 1 shows the effects of the three factors reducing negative ions.
As can be seen from the figure 1, each factor reduces the amount of negative ions in buildings differently.VDT's, appears to decrease negative ions by emitting ionising radiation as low energy xray.HVAC is likely to reduce the negative ion density (ions/cc) in air through its process, especially the process of the airconditioning system.The Building Contents (BC's), which naturally have electrostatics, may be electrically activated by contact, such as sliding and rolling of two insulating materials.The electrical charges generated appear to attract the positive and negative ions in indoor air and stick on the BC's surfaces, and therefore the ions in buildings are likely to be reduced.Quantities And Unit).The negative ions then lose their potential (see also figure 2).
How much
As a consequence of this, the energy of low energy x-ray and the electron binding energy or electron affinity of negative ions (EBE) can be calculated and consequently the amount of negative ions eliminated can also be calculated.
The following equation indicates the relationship of participant parameters for estimating Negative Ion Loss from VDT's (VDT-NIL).
VDT-NIL α LEX, 1/EBE, Rr, and Fn (Equation 1) Where: LEX = Low Energy X-ray = approx.9to 20 keV per a VDT (ARL, 1998 2) shows the loss of negative ions by the low energy x-ray of a VDT.Moreover, it determines by referring to the possible effect of the loss, to the lowest electron binding energy of negative ions.As an example two normal 14" or 15" television and computer screens might eliminate almost all the negative ions (typically 500 neg.ions/cu.cm) in a closed room of 3 x 3 x 2.4 m within 30 mins.
HVAC
The Negative Ion Loss from HVAC (HVAC-NIL) can be calculated from the fact that particles in air are likely to be electrically charged by an electric field intensity (about 30 kV/cm) in any normal condition including air-conditioned buildings (Hendricks, cited in Moore, 1973, p.57).They then attract the opposite charge of ionised air (negative or positive charge), and thus a number of negative ions will stick their electrons on the particle surfaces (see also figure 4).Each negative ion is assumed to have an excess electron of approximate 1.6 x 10^-19 C (Weisstein, 1996) After the air conditioning system filters a number of the atmospheric dust and/or particles, a number of negative ions seem to be also obstructed by filtering.The process is to consider filtering atmospheric particles, and assume the particles as spheres.The electric field intensity can be considered as an average value, which includes the intensity of the air conditioning system and others (Hendricks, cited in Moore, 1973, p.57)The Negative Ion Loss (HVAC-NIL) is estimated as a percentage of the difference between negative ions outside and inside (%NIL).The following equation shows the relationship of the participant parameters for this.%NIL α Qp, rp, Fn, Dneg, 1/Cp, Fh, %Ef, and 1/Dp (Equation 3) Where From the relationship (equation 3), the percentage of negative ions reduced (%NIL) in any area type (or in any particle concentration) can evaluated as shown in figure 5.The parameters assumed are that the flow rate of the air conditioning system (Fh), and negative ions density outside (Dneg) are 200 CFM and 200 to 1000 ions/cc, respectively.For example, in urban air the density (ions/cc) of indoor negative ions is approximately 150 ions/cc.
Building Contents (BC)
This section illustrates the effects of electrostatics on the BC's surfaces such as building materials and furniture.The effect seems to come from normal activities of the occupants in buildings, such as sitting on a chair, documents being put on a table, walking on a floor, and so on (Electro Statics, 1998).Such activities may potentially bring about electrically charged surfaces.The electrically charged ions (negative and positive ions) in the indoor air will be attracted (polarised) by the charged surfaces and eventually be reduced in the air.'When two insulating materials are rubbed together, the surfaces acquire a net electric charge, with one becoming negative and the other positive.'(Cross, 1987, p.17).This is the action of the electrostatic potential on the material surfaces.In addition, the action is likely to be caused by contact between two materials.The unbalancing charges, after that, occur at the material surfaces and become polarising.Effectively, air ions will be attracted by the opposite charged surface and give up their potential (Jowett, 1976, p.56). Figure 6 shows the simply contacting action of two materials during and after contacting.From the effective contacting of two insulators (see also figure 6), the relationship between the negative ions attracted by the positive charges and related parameters can be demonstrated as follows: BC-NIL α σ, %σ,max., and 1/Rσ,m/i (Equation 4) Where: σ = ideal surface charge density of two materials = approx.1.656x 10^10 e/sq.cm(Jowett, 1976, p.116) %σ,max = the maximum percentage of sliding and rolling contacts = approx.8%(Jowett, 1976, p.116) Rσ,m/i = Ratio between the surface charge density of metal and insulate materials = approx.10,000to 26,000 As a consequence of this, while the material surfaces are electrostatically activated and become electrical charge, negative and positive ions seem to be attracted by polarisation of the opposite surface charges of about 5.095 x 10^4 to 1.325 x 10^5 e/sq.cm.Equation ( 5) is a formula to estimating Negative Ion Loss from the Building Contents (BC).
BC-NIL = max.1.325x 10^9 x NoCT x Abc ions (Equation 5) Where: NoCT = Number of effective Contacts Abc = surface Area of Building Contents contacted An example of this is in an office building where the contents, the seat(s) of chairs used, computer keyboard(s) used and so on, may affect the Static Electrification.If we assume their areas to be about 5 sq.m, this room will lose approximately a maximum of 6.63 bil.ions of negative ions.performed in two different areas: a typical air-conditioned office (3m x 3m x 2.4mH), and a room (6m x 3m x 2.4mH), which was used for testing only the effects of an air conditioning system, and the density of negative ions outside the rooms (Dneg)about 1,500 ions/cc.DOF-NIL showed the results in terms of negative ion density (ions/cc) in the rooms, and the rate (ions/hr) of negative ions reduced by VDT's and BC's with +-15% error of VDT and BC effects.The AIC showed the negative-ion density (ions/cc) in each room with 25% accuracy.
NEGATIVE ION LOSS TESTING
The results were as follows: In the air-conditioned office and the control room, the negative ion densities inside were about 1,400 and 1,450 ions/cc with the negative ion decrease rates of about 1x 10^10 and 1 x 10^9 ions/hr, whereas the AIC showed the density of about 600 and 1,250 ions/cc, respectively.
Figure 7 below, shows the rates of additional negative-ions required for the condition of 1,500 ions/cc in the required period of 1 to 10 hours.The additional rate indicated by the AIC drops sharply when longer ion-generation is required.
Whereas according to the DOF-NIL formula the rate remains quite stable over any time period.However the estimated required rate of additional negative-ions for a 3 to 4 hour period (a realistic time period) as calculated using the DOF-NIL formula compared favourably with results obtained by the Air Ion Counter (AIC) -for an air-conditioned office this is approximately 12 billion ions/hr.
CONCLUSIONS
It is proposed that the DOF-NIL formula described provides a method that has a reasonable degree of accuracy for estimating the amount of negative ion loss in air-conditioned buildings for the critical 3 to 4 hour period.The DOF-NIL formula can be used to estimate the loss in terms of negative ion density (ions/cc) by HVAC, and the rate (ions/hr) of negative ions reduced by VDT's and BC's.A weakness with the DOF-NIL formula, is that only considers three factors, whereas a suitable instrument can take account of significantly more factors, for example the effects of air leakages in buildings and the effect of particle size and density.However these factors and the additional factors measured by an instrument appear to be relatively minor in their effect, and the difference in the results obtained are therefore also minor.Currently available instruments require a considerable amount of time to record data -typically up to 10 hours and require a real building to obtain results, whereas applying the formula method can produce data quickly and easily which can be used at the design stage to avoid possibly costly mistakes and/or to introduce measures to counter the adverse effects of deionisation.
Figure 1 :Figure 2 :
Figure 1: Three main factors affecting negative ion decrease in buildings Figure 4: Cross section showing negative ions attracting a particle
Figure 5 :
Figure 5: Relationship between %NIL and Particle Concentrations
Figure 6 :
Figure 6: Electric charge surfaces of two building materials
Figure
Figure 7.1: Typical Office
Figure 7 :
Figure 7: Additional Negative-Ions for 1 to 10 hours required | 2018-12-07T11:33:29.565Z | 2012-11-14T00:00:00.000 | {
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257029493 | pes2o/s2orc | v3-fos-license | Impact of hearing loss on the performance of auditory processing measured by questionnaires in Korean adolescents
Increasing use of personal listening devices has been accompanied by increase in the prevalence of hearing loss (HL) among youth in Korea, as in other countries. Auditory processing disorder (APD) is one of the main factors affecting academic achievement at school. This study aimed to investigate the prevalence of HL in students attending general middle- and high schools and compare the findings with the APD survey results. From June 1 to December 31, 2016, Korean adolescents (n = 2,791) in the first years of middle- and high school underwent audiometric testing and otologic examination and completed questionnaires on APD. The survey was sponsored by the Korean Society of Otolaryngology-Head and Neck Surgery and the Korean Otology Society. The prevalence of speech-frequency hearing loss (SFHL) and high-frequency hearing loss (HFHL) in the poorer ear was 11.6% and 10.3%, respectively, among Korean adolescents. We analysed data from the Scale of Auditory Behaviors, Fisher’s Auditory Problems Checklist, and KNISE-Auditory Behavioral Checklist and compared these with the results of hearing tests. We observed positive correlations among the APD questionnaire results and mean hearing levels. This study suggested that hearing loss, especially bilateral high-frequency hearing loss, may affect central auditory processing.
www.nature.com/scientificreports www.nature.com/scientificreports/ problems 10 . In addition, a 2017 New Zealand study of youth offenders aged 14-17 years reported that 27% suffered from APD and 64% showed language impairment compared to 18% and 10% of adolescents with normal hearing, respectively 11 . Children diagnosed with APD have listening difficulties, and many of those who present at audiology or other paediatric clinics have 'normal' audiograms. Most of these children have other complications such as speech/language or attention impairments and various learning disorders 12 .
Although the central hearing difficulty seems to be a single problem, it may be a cause of language disorder, reading disorder, learning disorder, attention deficit hyperactivity disorder, and other problems 13,14 . Therefore, it must be diagnosed as early as possible. Several test batteries are available to diagnose APDs in patients with normal hearing 15,16 . However, several studies have shown that in patients with cochlear HL of up to approximately 45-50 dB, speech perception is mainly influenced by audibility 17,18 . In patients with more severe HL, poor discrimination of suprathreshold stimuli plays an important role in addition to the loss of audibility.
However, APD was found in children with normal pure tone average or mild high-frequency hearing loss 12,19 . Therefore, having normal pure tone average does not mean that speech perception is normal.
The purpose of this study was to analyse the relationship between different degree of HL and results of screening surveys of the central auditory processing ability of middle-and high-school students in South Korea.
Methods
Study design and participants. We performed a cross-sectional, complex sampling survey of first-year middle-and high-school students, except those attending special schools for disabled children. The sampling frame was the 2015 Yearbook of Educational Statistics prepared by the Korean Educational Statistics Services 20 .
We sought to enrol 3,013 students from 124 middle schools and 124 high schools based on the prevalence of HL in Korean adolescents, the estimated association between noise and HL, and an expected 80% response rate 21,22 . Details of the study design and research methods are presented in another study 2 .
The survey was performed from June 1 to December 31, 2016. After providing written information about the study to students and parents, we selected and evaluated only those children who themselves and whose parents provided written informed consent. This study was approved by the Institutional Review Board of Seoul National University Hospital (No. 1604-086-755). All methods/experiments were performed in accordance with relevant guidelines and regulations (Declaration of Helsinki).
Data collection and measurements. Audiological evaluation. The methods of audiological evaluation are superbly detailed in the previous study 2 . Briefly, four experienced audiologists measured the hearing thresholds of adolescents using an AD229b diagnostic audiometer (Interacoustics, Assens, Denmark) in a soundproof booth inside a mobile vehicle. Prior to audiometry, otological examinations were performed to rule out factors affecting hearing, such as external ear anomalies, ear wax, retraction of the tympanic membrane, cholesteatoma, and middle ear effusion.
The tested frequencies were 0.5, 1, 2, 3, 4, 6, and 8 kHz. We started at 30 dB, adjusting with a 10 dB decrease or a 5 dB increase according to the participant's response, until we decided that his or her frequency-specific hearing threshold was reached. If the hearing threshold was over 25 dB at any frequency, we measured the bone conduction threshold to determine whether conductive hearing loss was present.
We defined HL as a hearing threshold ≥15 dB. The severity of HL was based on the poorer threshold for unilateral HL and better threshold for bilateral HL, as described previously 1 . Speech-frequency hearing loss (SFHL) was the average of the HLs at 0.5, 1, and 2 kHz, while HFHL was the average of the HLs at 3, 4, 6, and 8 kHz.
Questionnaires. The students were asked to complete separate questionnaires to evaluate their everyday difficulties in areas such as listening, learning, and communication.
The KNISE-Auditory Behavioural Checklist (KNISE-ABC) is used for screening children with APDs and other related disorders to provide information necessary for establishing and implementing management and educational plans [22][23][24] . The KNISE-ABC has 36 items rated on a 5-point scale and includes seven test areas, namely, listening, listening in background noise, communication, learning, auditory memory, auditory attention, and related behaviour.
The Scale of Auditory Behaviours (SAB) questionnaire, chosen for this study, has a version that may be used by parents or professionals 25 . The questionnaire is easily administered because it has a small number (12 items rated on a 5-point scale) of questions and answer options that are easily understood. This questionnaire was developed to assess everyday behaviours associated with audition, listening and academic skills, attention, memory, and organisation.
Fisher's Auditory Problems Checklist (FAPC) was developed by Fisher L 26 and comprises a list of 25 items rated on a 2-point scale. To collect information about the perceived auditory processing problems, this checklist includes questions on acuity, attention, attention span, figure ground, discrimination, short-term memory, long-term memory, sequential memory, comprehension, speech and language problems, auditory-visual integration, motivation, and performance.
Statistics. All data in the study are summarised as means and standard deviations for continuous variables and as frequencies and percentages for categorical variables. Comparisons of general characteristics between HL and no HL were conducted using the chi-squared test or Fisher's exact test. Prevalence rates of SFHL and HFHL were estimated with 95% confidence intervals. To evaluate the association between the degree of HL and mean scores of KNISE-ABC, SAB, and FAPC, we adopted Wilcoxon's rank sum test. All statistical analyses were performed using SAS (ver. 9.4; SAS Institute, Cary, NC), and statistical significance was defined as a two-sided p-value <0.05.
Results
In total, 3,013 students from 109 middle-and high schools agreed to complete the questionnaires. Of these, 134 (4.4%) were excluded because of incomplete audiometric data. Additionally, 88 students were excluded because of incomplete survey tests. Finally, a total of 2,791 (90.0%) students participated in this study (Fig. 1).
Demographic characteristics of these 2,791 students are summarised in Table 1. There were no significant differences between the no-HL and any-HL groups in sex ratio, grade level of middle-vs. high school, annual household income, and family history of hearing loss. Among the 2,791 students, 97.2% showed normal shape of the outer ear and 95.8% had normal otoscopic findings. The prevalence of any-HL was significantly higher in participants with an abnormal outer ear or otoscopic findings (P < 0.05).
The KNISE-ABC analysis revealed no significant differences between students with and without hearing loss in the any-HL and unilateral HL groups. However, students with bilateral HL, irrespective of whether they had HFHL or SFHL, showed significantly worse performance scores than those without HL. Moreover, students with = 2,791). The PTA in the most affected ear is used to define any HL, and the PTA in the less affected ear is used to define bilateral HL. Any HL is considered present if bilateral HL is evident at either high or speech frequencies. Participants exhibiting HL either at high frequencies (3, 4, 6, and 8 kHz) or speech frequencies (0.5, 1, and 2 kHz) are classified into these categories. HL, hearing loss; CI, confidence interval; PTA, pure tone average; HFHL, high-frequency hearing loss; SFHL, speech-frequency hearing loss.
Figure 2.
Analysis of the KNISE-Auditory Behavioral Checklist survey results. No significant difference was observed between students with and without hearing loss in the any hearing loss and unilateral hearing loss group. However, students with bilateral hearing loss, irrespective of whether they had high-frequency hearing loss or speech-frequency hearing loss, showed significantly worse performance scores than those without hearing loss. Moreover, students with bilateral high-frequency hearing loss showed relatively larger differences than those with other bilateral hearing loss.
www.nature.com/scientificreports www.nature.com/scientificreports/ bilateral HFHL showed relatively larger differences than those with other bilateral HL (Fig. 2). The SAB analysis revealed no significant difference was observed between students with and without hearing loss in the any hearing loss and unilateral hearing loss groups. However, students with bilateral hearing loss, irrespective of whether they had high-frequency hearing loss or speech-frequency hearing loss, showed significantly worse performance scores than those without hearing loss. Moreover, students with bilateral high-frequency hearing loss showed relatively larger differences than those with other bilateral hearing loss (Fig. 3). FAPC analysis also revealed no significant difference was observed between students with and without hearing loss in the any hearing loss and unilateral hearing loss groups. However, students with bilateral hearing loss, irrespective of whether they had high-frequency hearing loss or speech-frequency hearing loss, showed significantly worse performance scores than those without hearing loss. Moreover, students with bilateral high-frequency hearing loss showed relatively larger differences than those with other bilateral hearing loss (Fig. 4).
Discussion
In this study, the prevalence of SFHL and HFHL in the poorer ear was 11.6% and 10.3%, respectively, among Korean adolescents. There were no significant differences between the no-HL and any-HL groups in sex ratio, grade level of middle-vs. high school, household income, and family history, meaning that environmental factors Figure 3. Analysis of the Scale of Auditory Behaviours survey results. No significant difference was observed between students with and without hearing loss in the any hearing loss and unilateral hearing loss groups. However, students with bilateral hearing loss, irrespective of whether they had high-frequency hearing loss or speech-frequency hearing loss, showed significantly worse performance scores than those without hearing loss. Moreover, students with bilateral high-frequency hearing loss showed relatively larger differences than those with other bilateral hearing loss.
www.nature.com/scientificreports www.nature.com/scientificreports/ such as noise exposure might be more influential on their hearing. In addition, when the three degrees of HL (any, bilateral, and unilateral) and mean scores from KNISE-ABC, SAB, and FAPC were compared, Korean adolescents with bilateral HL showed significantly worse scores on all three screening tests for APD.
Auditory behaviour is defined as behavioural characteristics associated with hearing that appear in relation to the process or outcome of receiving and perceiving auditory information in the peripheral and central auditory systems. Children's hearing behaviour may vary according to the mechanism and function of the central nervous system (CNS). The ability to perceive and manipulate auditory information in the CNS can be used for sound localisation and lateralisation, auditory discrimination, determining the temporal aspects of audition (temporal resolution, temporal masking, temporal integration, and temporal ordering), assessing auditory performance with competing sound signals and acoustic signals, and assessing auditory performance with degraded acoustic signals 27 . One or more deficiencies in this ability are referred to as central APD or APD.
Auditory behavioural characteristics resulting from these central hearing processing problems can be caused by or related to difficulties in higher language, learning, and cognitive function. In addition, they may be associated with language disorders, reading disabilities, learning disabilities, and attention deficit hyperactivity disorder 27 . Children with hearing impairment may have problems with overall auditory behavioural characteristics and may exhibit sluggishness or difficulty in certain areas only. . Analysis of Fisher's Auditory Problems Checklist results. No significant difference was observed between students with and without hearing loss in the any hearing loss and unilateral hearing loss groups. However, students with bilateral hearing loss, irrespective of whether they had high-frequency hearing loss or speech-frequency hearing loss, showed significantly worse performance scores than those without hearing loss. Moreover, students with bilateral high-frequency hearing loss showed relatively larger differences than those with other bilateral hearing loss.
www.nature.com/scientificreports www.nature.com/scientificreports/ However, distinguishing between APD and peripheral HL is sometimes difficult; moreover, both problems may sometimes coexist. Peripheral HL does not have uniform effects on the results of APD tests. Studies have shown that cochlear lesions can be clearly distinguished from cerebral lesions using the dichotic-digit test, dichotic sentence identification test, and frequency-pattern test 9,17 , whereas a less clear distinction can be made using low-pass filtered speech 17 , binaural interaction, and localisation/lateralisation 27 . The usefulness of the dichotic test of binaural integration in children was also doubted 28 . Previous studies have reported these relationships between APD and hearing impairment. Miltenberger et al. 29 reported that auditory processing tests must always be related to the basic audiological assessment. They insisted that if the test results do not correspond with expectations based on the peripheral audiological assessment, the presence of APDs is likely, in addition to peripheral auditory disorders. Moreover, in the case of symmetrical hearing thresholds, auditory processing test results should be symmetrical. If the hearing thresholds are asymmetrical and performance in the better ear is poorer than that in the other ear, then an APD could be present 21 . The problem in APD is a decrease of speech performance in children with normal or mild hearing loss or functional hearing loss 12,30,31 .
In this study, adolescents with bilateral mild hearing loss showed a higher risk of APD. This may be because hearing loss is causally related to functional loss of central auditory processing 32 or because the loss of the cortical auditory function and high frequency hearing loss occurred simultaneously for some reason. The latter case may be considered as having the same mechanism as loss of speech in noise processing and temporal processing from age-related hearing loss, leading to an increase in APD 33 . This idea requires further research.
In this study, we adopted three different survey tools-the KNISE-ABC, SAB, and FAPC-for evaluating the ability of central auditory processing among Korean adolescents. All three test results showed that students with bilateral HL, irrespective of whether they had HFHL or SFHL, had significantly worse performance scores than those without HL. Some authors have concluded that HL has only a slight influence on the dichotic-digit and pattern-recognition tests 34,35 , while Neijenhuis et al. 16 reported that hearing-impaired patients did not show normal scores on these two tests. As with the results of Neijenhuis et al., in addition to the fact that peripheral sensorineural deafness affects the central hearing process, our results for bilateral HL are the most meaningful. We suggest that adolescents with bilateral sensorineural HL are more likely to have learning difficulties than those with normal or unilateral HL; therefore, more active hearing rehabilitation should be considered for such adolescents.
A limitation of this study is that the survey tests conducted to measure the capacity of the central auditory system were originally developed for evaluating children in the elementary school age group. The results of this study showed that only bilateral HL is related to disabilities in central auditory processing and learning; however, in reality, unilateral or various hearing impairments, such as SFHL or HFHL, may be related to learning disabilities in middle-and high-school students. These findings suggest the need for developing a survey to test central auditory processing in students of middle and high school age.
conclusions
Our findings suggest that hearing loss, especially bilateral high-frequency hearing loss, may affect central auditory processing. We should consider providing hearing aid, assistive listening devices, or enforcing educational programs to prevent further noise-induced hearing loss to improve the academic achievement of youth with bilateral hearing loss.
Data availability
All relevant data are within the manuscript and its supporting information files. | 2023-02-20T15:00:29.399Z | 2020-06-22T00:00:00.000 | {
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38411745 | pes2o/s2orc | v3-fos-license | Bacteria on Meat Abattoir Process Surfaces after Sanitation : Characterisation of Survival Properties of Listeria monocytogenes and the Commensal Bacterial Flora
Contamination of food with spoilage bacteria and pathogens from food processing environment remains a challenge for the food industry. Bacteria able to persist in such environments over time must survive several hygienic hurdles. The aim of this study was to identify bacteria surviving practical disinfection and compare their survival abilities with representative isolates of the pathogen Listeria monocytogenes. Bacteria isolated from processing surfaces after cleaning and disinfection in a meat abattoir were identified. Selected isolates of the most frequently isolated bacterial genera along with eight meat associated L. monocytogenes were further characterized with regard to biofilm formation abilities at 12 ̊C and 20 ̊C, tolerance to desiccation (stainless steel at 70% RH at 12 ̊C) and bactericidal effects of recommended in-use-concentrations of four commercial disinfectants on stainless steel surface. The most dominating bacterial genera based on counts on non-selective agar were Aerococcus, Acinetobacter, Pseudomonas, Serratia and Staphylococcus. Isolates of Citrobacter. Enterobacter and Serratia dominated on agar plates selective for Enterobacteriaceae. In general, Gram negative bacteria formed more biofilm than Gram positives, especially at 12 ̊C with the best biofilm formers being Acinetobacter, Citrobacter and Pseudomonas. Listeria monocytogenes were poor biofilm formers. Gram positives survived better air drying than Gram negatives. Strains of L. monocytogenes were more sensitive to desiccation than the other Gram positives; Aerococcus, Kocuria and Staphylococcus. Two disinfectants containing peracetic acid and a disinfectant containing alkylaminoacetate had limited or no antibacterial effect against bacteria dried on stainless steel. A quaternary ammonium compound-based disinfectant provided >2 log reductions of Aerococcus, Acinetobacter and Listeria. Only 0.5 log reductions were obtained against Staphylococcus and no bactericidal effect against Serratia. In this study the dominating flora in a meat abattoir was isolated and identified. Several of these bacteria were better biofilm formers and more resistant to desiccation and disinfection than L. monocytogenes. The disinfectants tested had limited bactericidal activity against surface associated bacteria.
Introduction
The food industry has a strong focus on hygiene in order to produce safe food with high quality.Though cleaning and disinfection are performed daily, few surfaces or equipment are sterile.Bacteria present on surfaces may cross-contaminate the food during processing.In meat production there is a focus on potential faecal pathogens like Salmonella and Escherichia coli, and considerable resources are used to sample the environments for these potential pathogenic bacteria, often with high numbers of negative samples.It has been suggested that survival of E. coli in these environments is not connected to enhance survival properties, but that the bacterium is associated with certain raw materials and niches [1].For L. monocytogenes, it has been proposed that persistence is connected to survival abilities of the bacterium itself [2,3].Several studies have been conducted to describe important properties of L. mononcytogenes associated with survival in food production environments [4][5][6][7][8], but much less is known concerning other commensal bacteria.It may be hypothesized that bacteria surviving cleaning and disinfection is likely to have improved abilities to survive and also to form persistent bacterial populations in food production environments.It is therefore of specific interest to identify bacteria isolated after cleaning and disinfection and characterise their surviving properties.There is limited information about the prevalence of the commensal bacterial flora in meat processing environments.In a study where the bacterial composition was studied on conveyor belts from a lamb boning room, Sphingomonas dominated among non-cultivable bacteria while Pseudomonas, Serratia, Alcaligenes and Microbacterium were identified by culture-dependent techniques [9].In another study Pseudomonas and Staphylococcus dominated on the floor in a ground meat processing facility [10].A comparison of qualitative determination of bacteria from different types of food production environments revealed that Pseudomonas, Staphylococcus, Acinetobacter, Bacillus, lactic acid bacteria and coryneforms commonly dominated [9][10][11][12][13][14][15].The role of the commensal bacterial flora on food safety is not completely understood, but it may be involved in food spoilage, and affect pathogenic bacteria present in the food producing environment [16][17][18][19].Strains of L. monocytogenes may also frequently be isolated after sanitation and still remain the most challenging microbial threat to many parts of the food industry, including meat processing industry.
In the present study, survival characteristics of meat associated L. monocytogenes were compared to isolates of the bacterial genera dominating in a meat abattoir.The flora isolates were collected from processing surfaces after cleaning and disinfection.The bacteria were identified and characterized with regards to factors anticipated as important for growth and persistence in the food production environment.
Isolation and Identification of Bacterial Isolates
A total of 20 surfaces of equipment, floors, doors, knives, saws, handling panels from the slaughter area (from flaying to evisceration, splitting and cooling) in an abattoir for bovine slaughter were swabbed.The swabbing was performed approximately 6 h after cleaning and disinfection and prior to slaughter activities in the abattoir.In each site an area of approx.100 cm 2 was swabbed (3M Swab-Sampler-Letheen Broth; 3 M, St. Paul, USA).In certain sites reduced sampling areas were available for sampling.The samples were cooled and transported to the laboratory for plating within 10 h after sampling.Direct plating of the samples from the separate surfaces was performed by adding 0. [20].The 16S rDNA sequencing were performed using the Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) using the 534r primer [20].Obtained 16S rDNA sequences were compared to known sequences using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) for DNA sequence homology and isolate identification.The L. monocytogenes strains used in this study are described in Table 1.The strains were meat/meat industry related, in addition to the Scott A and EGDe strains which are commonly used as reference strains in scientific studies of L. monocytogenes.
Logging of Temperature and Humidity in the Abattoir
The temperature and relative humidity was monitored in the abattoir for one week using an automatic logging device (EL-USB-2, Lascar electronics Ltd., Salisbury, UK).
Biofilm Formation
Biofilm-forming ability was measured by staining of polystyrene-attached bacteria with crystal violet (CV Total cell mass was measured as absorbance at 600 nm (TitertekMultiskan RC plate reader; Labsystems, Helsinki, Finland).Biofilm formation was quantified according to the following procedure: Bacterial suspensions were pipetted off and the remaining biofilm washed twice with 300 µl distilled water (dH 2 O), using a semiautomatic microtiter plate washer (Wellwash AC, Thermo Electron Corporation, Waltham, Massachusetts, USA).Surface-attached bacteria were dried at 30˚C for 15 min and thereafter stained with 200 µl 0.1% CV for 5 -10 min.After two washes with 300 µl dH 2 O, surface-bound CV was extracted by addition of 200 µl 33% acetic acid and incubation for 5 min.A volume of 100 µl was transferred to a new microtiter plate and absorbance was measured at 600 nm.Absorbance measurements were subtracted the absorbance values from wells containing BHI only.Each strain was tested in three to five independent experiments per cultivation condition.
Tolerance to Drying
Bacterial survival after air-drying on stainless steel was studied in a model system, as described previously [23].
An overnight culture (16 -18 h) in BHI-broth incubated at 30˚C, inoculated from BHI-agar (Oxoid), was diluted ten times in fresh BHI.From the resulting suspension, four drops of 20 µl each was applied to a stainless steel (AISI 304, 2B, NorskStål AS, Nesbru, Norway) coupon of 20 × 20 mm, leading to a start concentration of log 6 -7 cfu per coupon.The coupons were incubated for 1 h at 20˚C in a safety hood before being transferred to a plastic box with lid.A petri dish with a 20 ml saturated solution of lithium acetate was placed in the box, resulting in an atmosphere of approx.70% relative humidity (RH).After 1, 7 and 14 days incubation at 12˚C, a steel coupon was transferred to a tube with 6 ml peptone water.To release cells from the coupon, the tube was sonicated for 15 min in a sonication bath (40˚C, 45 kHz/100 W, Bransonic 3510, Bransonic Ultrasonic B. V., Soest, The Netherlands).The number of surviving bacteria was determined after plating to BHI-agar and incubation at 30˚C.The strains were tested in two to five independent experiments.
Disinfection Test
The effect of disinfectants against bacteria dried on stainless steel was tested by the European surface test EN13697 with a few modifications [24,25].The lowest recommended user-concentration of four commercial disinfectants was used (Table 2).The abattoir where the strains were isolated from used TP-99 for daily disinfection and Topactive DES for disinfection three times a year.50 µl of an overnight culture grown in BHI at 25˚C was applied in one drop to a stainless steel coupon of 20 × 20 mm (AISI 304, 2B, Norskstål, Nesbru, Norway).The coupon was allowed to dry for 1 h in a safety hood at 20˚C.The disinfectant to be tested was diluted and added bovine serum albumin (0.3%) immediately before the test.The bacteria were exposed to the disinfectant for 5 min at 20˚C. 100 µl of the diluted disinfectant was added to the coupon to cover the area where the bacterial suspension was added.After 5 min exposure the coupon was transferred to a tube with 6 ml Dey/Engley Neutralizing broth (Difco Laboratories, Detroit, MI, USA).The bacteria were released from the surface by sonication as de- Copyright © 2013 SciRes.AiM scribed above.The number of surviving bacteria was determined after serial dilution and plating on BHI-agar.As a control, deionized water was used instead of disinfectants.The different strain/disinfectant combinations were tested in three to four independent experiments.
Statistics
Statistical differences between different treatments or genera were calculated using analysis of variance (Anova, Minitab v16, Minitab Ltd, Coventry, UK) and difference between means by Tukeys test (Minitab).Calculations on desiccation and disinfection data were performed on log transformed data.
Temperature and Humidity in the Meat Abattoir
The automatic monitoring of air temperature and humidity was performed in the same slaughter area as swab sampling.During one production week, temperatures were in the range 14˚C -25˚C with temperatures below 20˚C during production and in the weekend.Relative humidity (RH) measurements varied between 35% -90% RH.The highest temperatures and RH were obtained during cleaning and disinfection.The lowest RH was recorded during the weekend with no production while RH during production was in the range 50% -70%.As an example, data from the flaying area is shown in Based on bacterial identification, we selected bacterial isolates for studies on surface survival and biofilm formation (Table 3).Isolates of the genera, Acinetobacter, Aerococcus, Citrobacter, Pseudomonas, Serratia and Staphylococcus were chosen as they dominated on two or more of the sampling sites.In addition, we also included Copyright © 2013 SciRes.AiM two isolates Kocuria based on their reported effect on biofilm formation in bacterial co-cultures [18].
Biofilm Formation
In general Gram negative bacteria formed more biofilm (p < 0.05, all four time/temperature combinations tested) than Gram positive bacteria including L. monocytogenes.The highest biofilm formation was observed for Acinetobacter, Citrobacter and Pseudomonas (Figure 2).Enterobacter formed little biofilm compared to the other Gram negative bacteria.There were no statistical differences in biofilm formation between L. monocytogenes and the other Gram positive bacteria for the four time/ temperature combinations (p = 0.23 − 0.98).The L. monocytogenes isolates 2624 (EGDe) and 3131 formed more (p < 0.05) biofilm than the other L. monocytogenes isolates after 2 d at 20˚C and 7 d at 12˚C.Especially at 12˚C there was very poor biofilm formation among Gram positive bacteria, except for Kocuria isolate 3620.The staphylococci and Kocuria 3621 showed very poor growth at 12˚C, the OD 600nm < 0.2 (measured before removal of culture medium).Also at 20˚C these strains had lower growth (OD 600nm ) than the other strains.To check whether the differences in biofilm formation could be explained by differences in growth, we compared the biofilm/growth ratio between the strains.For the strains 0.0 0.5 with very low growth at 12˚C (Staphylococcus and Kocuria 3621), the denominator in the calculations was very low, which lead to high uncertainty in the ratio, thus we omitted these strains from the comparisons.For the other strains, when comparing biofilm/growth, the highest values were found among the Gram negative bacteria.
Survival Air-Drying on Stainless Steel
Gram positive bacteria were more tolerant to air-drying than Gram negative bacteria, both after 7 and 14 days (p < 0.001, both incubation times; Figure 3).The L. monocytogenes strains were less tolerant (p < 0.001; d7, p < 0.01; d14) than the other Gram positive bacteria, and more tolerant (p < 0.05) than the Gram negative bacteria after 14 d air-drying.There were no differences in tolerance to air-drying (p = 0.44; d7, p = 0.74; d14) between the L. monocytogenes isolates.
Disinfection
The disinfectants TP-99, Topactive DES and Oxydes had only low or no bactericidal effects against the bacteria tested in the surface disinfection tests (Figure 4).The disinfectant Aco Hygiene Ultra DES had a bactericidal effect of 2 -3.5 log reductions against Acinetobacter, Aerococcus and the L. monocytogenes isolates.In general Staphylococcus and Serratia were more tolerant than the other bacteria as no significant (p > 0.05) bactericidal reduction was observed for any of the disinfectants.The most sensitive bacteria were Pseudomonas and Acinetobacter, where significant bacterial reductions (p < 0.05) were observed for all four and three of the four disinfectants, respectively.
Discussion
Control of bacteria in the meat processing industry is a key element to assure meat safety and quality.However, bacterial elimination and control strategies in meat manufacture are difficult tasks.Cleaning and disinfection do not eliminate all bacteria present and bacteria surviving the sanitation process may be a source for crosscontamination and bacterial persistence; hazards potentially affecting both food safety and quality.In the food industry, bacteria will also encounter other stressful conditions, e.g.mechanical shear forces, desiccation, low and high temperature and starvation.It is likely that bacteria dominating after cleaning and disinfection have certain characteristics enabling them to survive in the food processing environments.The bacterial genera dominating in the abattoir after cleaning and disinfection were Aerococcus, Acinetobacter, Pseudomonas, Staphylococcus and Serratia.Several studies have reported the latter four genera to be common in the production environment of many types of food [9][10][11][12][13][14][15].Aerococcus has been found in fish [12] and pastry production environments [10].The semi-quantitative determination of bacterial loads indicated that easy-to-clean sampling sites not in direct product contact had the lowest bacterial counts.Highest bacterial loads were observed on consoles and joysticks for process control which included design not suitable for effective sanitation (rubber with folds).The results indicated lack of a high selective pressure as the diversity was higher than one would expect for example after an efficient disinfection procedure or long periods of drying.However, since a similar microbiota has been reported from production of a range of foods, food processes and countries it is likely that these genera or strains/species within these genera have attributes that enhance survival and growth under conditions common for a range of food processes.Examples of such conditions could be periods with high nutrient availability combined with low temperature, periods of drying and finally cleaning and disinfection.Therefore these scenarios were investigated further.Listeria monocytogenes (mostly meat associated strains) was included in the study as it is regarded among the most troublesome bacteria in several food industries, with numerous reports on their ability to survive and persist in food industry premises [3,26].
The results indicated large differences in the biofilm forming abilities of different genera and also strains of the same genus.The large differences in biofilm formation observed thus indicate that this property alone cannot explain survival of all bacteria isolated.Biofilm formation has been suggested as an important mechanism for L. monocytogenes persistence, but this hypothesis is disputed [3,26].In this study L. monocytogenes was found to be a relatively poor biofilm former compared to Gram negative bacteria.The results are in accordance with other studies showing that L. monocytogenes form less biofilm than Pseudomonas spp.[27,28].The L. monocytogenes isolates producing most biofilms belonged to serovar 1/2a (phylogenetic Division II) while low biofilm producers were serovar 4b isolates (Division I).Differences in L. monocytogenes adherence/biofilm production between persistent and non-persistent strains and between phylogenetic divisions have been reported [4,6,29].The strains used in the present study are not reported as persistent, and testing persistent strains of Listeria or other bacteria could have resulted in different results.Other researchers have reported that the type of growth medium can influence biofilm formation of L. monocytogenes [29][30][31].In addition to BHI, we also tested TSB and LB (with and without NaCl), but the biofilm formation was comparable to that obtained in BHI.In conclusion, the results obtained did not confirm that bacterial strains surviving in the food production environment share a common property of producing high levels of biofilm at low temperature.However, it cannot be excluded that the results would be different in other biofilm models, e.g.models using materials used in food processing environments.
In many food processing facilities the processing equipment is dry for shorter or longer periods, for example in the weekends or between end of the cleaning period and start of production.Indeed, the temperature recordings showed a very large variation of relative humidity during one production day.The Gram positive bacteria were significantly more tolerant to air drying than Gram negative bacteria.This is in accordance with previous findings.It is believed that the Gram positive cell wall has a protective role during desiccation [32].L. monocytogenes is often found in humid areas in a production environment.For control of L. monocytogenesit is recommended to keep the production environment as dry as possible, and allow areas to dry up regularly [33].Interestingly, surfaceassociated L. monocytogenes were less tolerant to desiccation than the other Gram positives, but more tolerant than the Gram negative bacteria.To our knowledge this has not been reported previously, and the mechanisms of lower desiccation tolerance of Listeria than other Gram positives should be subjected to further studies.The presence of a self-produced biofilm matrix, organic matters and food components have been shown to increase the survival of L. monocytogenes and other foodborne bacteria during desiccation [23,34] and the survival during periods of drying may be higher in practice than what was found in this study.
It is important that disinfectants are tested under conditions that resemble the practical usage conditions.It is well known that surface associated bacteria are more tolerant than free living bacteria [25,35,36].Three (including TP-99, the disinfectant used daily in the abattoir) out of four disinfectants tested had very low efficiency in the surface tests (0 -1.3 log reductions).Some manufacturers refer to data from suspension tests when marketing the efficiency of their disinfectant and the disinfectants are tested against laboratory strains.Aco Hygiene Ultra DES was more efficient than the other disinfectants.It is not possible to conclude if the differences in susceptibilities are dependent on too low concentrations used in some disinfectants, or if QAC (Aco Hygiene Ultra DES) is more efficient.Surprisingly, the Gram negative bacte-ria Pseudomonas and Acinetobacter were found to be among the most susceptible to disinfection among the bacteria studied including Gram positive bacteria.This is not in accordance with the majority of previous literature supporting the common opinion that Gram-negative bacteria have higher intrinsic resistance to biocides than Gram positive and among them Pseudomonas being one of the most resistant [37,38].One may speculate that the desiccation process harmed the cells rendering them more sensitive to subsequent stress like disinfection.Another explanation for discrepancies between other studies and the present study is that commercial disinfectants were used.Often commercial disinfectants are containing several active substances including sequestering agents, such as EDTA, that will disrupt the outer protective membrane of Gram negative bacteria [39].It cannot be ruled out that the disinfectants tested in the present study contained such agents, although it was not given in the user information.The differences in the bactericidal effects of disinfectants varied between the type of disinfectant.Further studies should be performed to investigate to what extend bactericidal effects of disinfectants is affected by prior bacterial exposure to meat industry relevant stresses.Serratia and Staphylococcus showed highest tolerance to disinfection.Staphylococcus sp. may harbour plasmid encoding for efflux pumps for QAC, which lead to increased tolerance [40,41] and resistance to several biocides has been linked to mucoid growth [42].Serratia was found to be resistant to the disinfectant containing QAC. Resistance to user-concentrations of amphoteric and cationic tensides has previously been reported for Serratiamarcescens [43,44], and efflux was he proposed resistance mechanism.
As found in the present study and as reported by others, L. monocytogenes is not particularly resistant to environmental stress and not a good biofilm former compared to other bacteria in in vitro tests [27,28].It may be speculated that the background flora may have a role in protecting L. monocytogenes against stress in the food industry.However, other bacteria have been shown both to promote [18,27] and inhibit [17,45] biofilm formation of L. monocytogenes, and further studies are required to reveal the role of background flora on colonisation and persistence of L. monocytogenes in the food industry.
In conclusion, the dominating bacteria isolated from the meat abattoir were similar as reported for many type of food production environments.The bacteria isolated had different properties believed to be important for survival in the food production environment.Gram negative bacteria were better biofilm formers than Gram positive bacteria (including L. monocytogenes), while the opposite was found for tolerance to desiccation, where L. monocytogenes were more susceptible than other Gram positive bacteria.The disinfectant in daily use in the ab-attoir, tested at its recommended user-concentration in a surface test, had a low or limited effect against bacteria from the meat abattoir and L. monocytogenes.Our study showed the necessity of applying proper cleaning and disinfection routines in the meat processing industry.Further investigations are needed to understand the factors and mechanisms affecting desiccation tolerance in bacteria and potential cross-tolerance to other stresses.This could have implications for improved intervention strategies for ensuring microbial food safety and quality.Further, isolation of bacteria surviving processing conditions including sanitation routines may be used for designing improved strategies for elimination and control of bacteria in food industry processes.
Fig- ure 1 .Figure 1 .
Figure 1.Temperature and humidity in flaying department during a five day period.
Figure 2 .
Figure 2. Biofilm formation (OD 600nm ) of bacteria in microtiter plates after incubation for 2 days () and 7 days ().Means f three to five replicates with standard errors in bars are shown.(a) 12˚C; (b) 20˚C.o
Figure 3 .
Figure 3. Bacterial reduction during air drying of bacteria at stainless steel at 70% RH at 12˚C for seven () and 14 days () ncubation.Means of two to four replicates with standard errors in bars are shown.i
Figure 4 .
Figure 4. Effects of disinfectants against bacteria dried on stainless steel.Means and standard errors of three or four independent experiments are shown.Asterisk indicates significant bacterial reduction (p < 0.05).
Table 2 . Disinfectants used in this study.
* quaternary ammonium compound.
disinfection.A semi-quantitative interpretation of total counts after direct plating showed variable bacterial levels in different sites.Highest bacterial loads were on console-joystick surfaces for slaughter process control.Lowest bacterial levels were observed on surfaces not regularly contaminated during the slaughter process (door, curtain for area separation, water hose at splitting saw).Aerococcus was identified as the most dominant bacterial genus, isolated from eight of the swabbed surfaces.Other bacteria identified after PCA plating (15˚C) were Pseudomonas, Serratia, Acinetobacter and Kocuria.All isolates showed 99% -100% identity according to 16S rDNA BLAST sequence homology results.Pseudomonas spp.and Staphylococci were isolated from Pseudomonas agar and Blood agar, respectively.Bacteria of the genera Citrobacter, Enterobacter, Acinetobacter, Pseudomonas and Serratia were isolated on VRBGA from two or more of the sample sites.The presence and survival of presumptive E. coli isolates were tested by plating swab samples on E. coli/coliform agar.No E. coli isolates were found.
Table 3 . Identity and site of isolation for bacteria used in this study.
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198119907 | pes2o/s2orc | v3-fos-license | Consortium of Plant Growth-Promoting Rhizobacteria Strains Suppresses Sweet Pepper Disease by Altering the Rhizosphere Microbiota
Beneficial microorganisms have been extensively used to make plants more resistant to abiotic and biotic stress. We previously identified a consortium of three plant growth-promoting rhizobacteria (PGPR) strains (Bacillus cereus AR156, Bacillus subtilis SM21, and Serratia sp. XY21; hereafter “BBS”) as a promising and environmentally friendly biocontrol agent. In this study, the effect of BBS on a soil-borne disease of sweet pepper was evaluated. Application of BBS significantly reduced the prevalence of phytophthora blight and improved fruit quality and soil properties relative to the control. BBS was able to alter the soil bacterial community: it significantly increased the abundances of Burkholderia, Comamonas, and Ramlibacter, which were negatively associated with disease severity, relative to the control. A redundancy analysis suggested that BBS-treated soil samples were dominated by Burkholderia, Comamonas, Ramlibacter, Sporichthya, Achromobacter, and Pontibacter; abundance of these genera was related to total organic carbon (TOC), total nitrogen (TN), ammonium nitrogen (AN), total potassium (TP), and available phosphorus (AP) contents. This suggests that BBS treatment shifted the microbe community to one that suppressed soil-borne disease and improved the soil chemical properties.
INTRODUCTION
Sweet pepper Capsicum annuum L. var. grossum (Solanaceae) is an annual plant cultivated throughout the world. It is widely valued because of its unique flavor and high nutritional value, especially in terms of its vitamin C content. With the expansion of modern facilities and highefficiency factory farms, sweet pepper cultivation has increased dramatically where cultivated land has expanded (Shu et al., 2016). Global production of chilies and peppers was at 34.5 million tons from 1.9 million ha of crop-growing surface area in 2016; China was the largest contributor, producing 17.45 million tons from 0.75 million ha of land (FAO, 2018).
However, soil-borne diseases such as phytophthora blight have increased through the practice of continuous cropping . Meanwhile, these diseases cause the loss of soil quality which have severely restricted the development of the sweet pepper industry and the income of sweet pepper growers has also been reduced. Farmers have therefore increased the frequency of pesticide application to enhance yields. However, massive application of chemical pesticides has caused many negative impacts, such as the contamination of food, soil, and water by pesticide residues, and loss of biodiversity. Besides, excessive use of chemical pesticides destabilizes the soil microecosystem, and this is an important cause of soil-borne diseases (Avis et al., 2008). The majority of soil-borne pathogens survive in bulk soil; under suitable conditions, they infect host plants to establish parasitic relationships with the plants (Raaijmakers et al., 2009;Liu et al., 2018).
Previous studies have reported that beneficial microbes can be recruited by host plants to counteract pathogen infection (Dudenhöffer et al., 2016). For instance, beneficial microbiomes can induce disease resistance in plants to many plant pathogens such as Ralstonia solanacearum (Aliye et al., 2008;Cao et al., 2018), Phytophthora capsici (Lim and Kim, 2010;Sang et al., 2018), and Botrytis cinerea (Nie et al., 2017;Jiang et al., 2018). Beneficial microbiomes improve soil chemical properties and fruit quality (Song et al., 2015). Furthermore, soil inoculation by microbes alters the resident microbial communities (Trabelsi and Mhamdi, 2013). Zhang et al. (2019) reported that inoculation with Bacillus velezensis NJAU-Z9 to pepper led to a higher rhizosphere bacterial richness and diversity compared to the control without NJAU-Z9 inoculation. In addition, recent work has shown that managing rhizosphere microbial communities contributes to plant disease control (Mazzola, 2010). Wang et al. (2015) revealed that inoculation using bio-organic fertilizer reduced the prevalence of tomato fusarium wilting by altering the soil microbial communities. Liu et al. (2018) found that bio-organic additives (matured chicken manure added with amino acids and PGPR strain Bacillus amyloliquefaciens SQR9) suppressed tomato disease by altering bacterial community composition in the rhizosphere. Xue et al. (2015) reported that B. amyloliquefaciens NJN-6, combined with compost promoted alteration of the rhizosphere bacterial community structure by developing beneficial strains that dominated the microbial community, which contribute to Panama disease control.
Our objective was to evaluate the suppression of sweet pepper disease using a microbial additive ("BBS") that we have previously developed (Wang et al., 2012;Yang et al., 2014). However, the mechanisms by which soil inoculation by microbes alters rhizosphere microflora to control sweet pepper diseases are still not well understood. Therefore, we conducted a 3year field experiment (2014)(2015)(2016) in sweet pepper producing areas in China where soil-borne diseases were prevalent yearround. Specifically, the soil-borne disease pepper blight caused by Phytophthora capsici has posed a serious threat to sweet pepper production in these areas. Using sequencing, we then surveyed the rhizosphere microbiota, to assess the effects of the microbial additive and to examine the relationships between the rhizosphere microbiota and the plant disease.
Bacterial Strains and Culture Conditions
Three PGPR strains (Bacillus subtilis SM21, Bacillus cereus AR156, and Serratia sp. XY21) were cultured at 28 • C for 24 h on Luria-Bertani (LB) agar medium. A single colony from a freshly streaked plate was then selected, inoculated into LB broth, and incubated at 28 • C for 48 h in a shaker at 200 rpm. The broth culture was spun at 6000 × g in a centrifuge for 15 min, and the resulting pellet was resuspended in sterile water and adjusted to a concentration of 10 9 colony forming units per ml (CFU/ml) for further experiments.
Field Experimental Design
The field experimental site was located in Huaian, Jiangsu Province, China (33 • 35 42 N, 119 • 02 11 E), which has a subtropical monsoon climate with an average annual temperature and precipitation 14.2 • C and 940 mm, respectively. The field was continuously utilized for sweet pepper cultivation for several years before our study. Soil-borne diseases pepper blight caused by P. capsici has posed a serious threat to sweet pepper production. The soil was a sandy loam with pH 7.08, 80.76 g/kg total organic carbon (TOC), 139.23 g/kg total organic matter (TOM), 12.51 g/kg total N (TN), 26.24 mg/kg NH4 + -N (AN), 435.10 mg/kg NO3 − -N (NN), 4.95 g/kg total P (TP), 1.15 g/kg available P (AP), and 1.69 g/kg available K. The field experiment was carried out from December 2013 to April 2016. We define "season" as the entire sweet pepper growing season (from early December to mid-April of next year). Plants were cultivated with or without BBS treatment. Each treatment had three randomized independent replications with a single plot of 6 m × 8 m in area. We applied 500 ml of BBS suspension (1:100 dilution) to each seedling at transplanting; the control seedlings were mock inoculated with an equal volume of water. A five-point (each point is 1.2 m × 1.2 m) sampling method was used for random sampling.
Assay of Disease Prevalence and Yield
The prevalence of phytophthora blight was investigated 60 days after transplanting into the field. Five points sampling method was used for random sampling. Sixteen pepper samples were collected at each point 1.2 m × 1.2 m, the diseased plants were counted, and prevalence was calculated as follows: To measure total sweet pepper yield, all mature sweet peppers were harvested and weighed.
Assay of Leaf Chlorophyll Content
Leaf chlorophyll content was measured 60 days after transplanting using a modified version of the method of Jiang et al. (2014). Chlorophyll extraction was conducted using 80% acetone solution (v/v in water); from each sample, 10.0 g plant tissue was cut into 0.5 cm segments and homogenized with acetone solution at −10 • C. The mixture was centrifuged at 12,000 × g for 15 min, and the supernatant was transferred to the flask and covered with aluminum. Absorption of the supernatant was measured at 645 and 663 nm using a HITACHI U-2000 spectrophotometer.
Assay of Fruit Quality
To evaluate sweet pepper quality, the contents of soluble sugar, soluble solids, and vitamin C were assayed at the harvest time. Soluble sugar was determined according to Horowitz and Reynolds (1936). Briefly, 1.0 g fresh fruit was kept in 10 ml of 90% ethanol for 1 h at 60 • C in an incubator. The extract was then transferred into a new flask and the final volume was made up to 25 ml by adding 90% ethanol. 1 ml aliquot was transferred to a test tube and 1.0 ml of 5% phenol was added to it and mixed thoroughly, 5 ml of analytical grade sulphuric acid was then added to it and mixed thoroughly by vertical agitation with a glass rod. For exothermic reaction the test tube was cooled in the air. Absorbance was recorded at 485 nm. Soluble solids were measured using a handheld refractometer at 20 • C ( Barrett et al., 1998). Vitamin C content was detected according to Santos et al. (2016), 2% oxalic acid was used for extraction and the 2,6dichlorophe-nolindophenol dyestuff was added for reduction. Xylene was used for extracting of the excess dyestuff. Absorbance was recorded at 500 nm.
Assay of Soil Properties
Soil total organic carbon (TOC) and total organic matter (TOM) were measured by potassium dichromate (K 2 Cr 2 O 7 ) oxidationreduction titration (Schollenberger, 1931). The content of total nitrogen (TN) was determined using the Kjeldahl method (Page et al., 1982). Soil nitrate nitrogen (NN), ammonium nitrogen (AN) and total potassium (TP) content was quantified using an AutoAnalyzer 3 (Bran and Luebbe GmbH, Germany). To determine soil available phosphorus (AP), we followed the molybdenum-blue method using sodium bicarbonate (Olsen, 1954). Soil total potassium (TK) content was detected by atomic absorption spectrophotometry (ASS), and soil available potassium (AK) was measured in the extract using a flame atomic absorption spectrophotometer (Brown, 1998).
Soil Sampling, DNA Extraction
Both diseased and healthy plants with tightly adherent rhizosphere soil were sampled (Morris et al., 1997). In brief, for each replicate, a five-point sampling method was applied (each point is 1.2 m × 1.2 m). For each point, 16 plants were randomly selected. Eighty plants from five points were collected. After careful removal of 0-5 cm of the topsoil, rhizospheres were excavated, with as much of their associated roots as possible, by digging to a 5-20 cm depth around pepper plants. Soils and Plants were placed into plastic bags and placed on ice for transport to the laboratory for preparations using standard procedures within a few hours. Subsequently, excess bulk soil was removed from the roots by shaking, brushing down firmly adhering soil with sterile brushes, which was defined as rhizosphere soil (Mendes et al., 2017). All of these soils were divided into subsamples: one was frozen at −80 • C for DNA extraction and subsequent molecular analysis, the rest was further air-dried at room temperature and passed through a 0.25 mm sieve for chemical analysis. DNA extraction was performed using FastDNA R SPIN Kit (MP Biomedicals, Solon, OH, United States) following the manufacturer's instructions. The concentration and quality of the DNA samples were evaluated using a NanoDrop 1000, Spectrophotometer (United States). We ensure that adequate amounts of high-quality genomic DNA had been extracted (>90 µg/µl) and no DNA was detected in the negative controls.
MiSeq Illumina Sequencing
The DNA extracted from each soil sample served as the template for 16S rRNA gene fragment amplification. V3-V4 regions of the bacterial 16S rRNA gene were amplified using primers (PAGE purified) 338F (5 -ACTCCTACGGGAGGCAGCA-3 ) and 806R (5 -GGACTACHVGGGTWTCTAAT-3 ) (Dennis et al., 2013). The 16S rRNA V3-V4 amplicon was amplified using KAPA HiFi Hot Start Ready Mix (2×) (TaKaRa Bio Inc., Japan). Reaction was set up as follows: microbial DNA (5 ng/µl) 2 µl; amplicon PCR forward primer (1 µm) 5 µl; amplicon PCR reverse primer (1 µm) 5 µl; 2 × KAPA HiFi Hot Start Ready Mix 13 µl (total 25 µl). The plate was sealed and PCR performed in a thermal instrument (Applied Biosystems 9700, United States) using the following program: one cycle of denaturing at 95 • C for 3 min, followed by 25 cycles of denaturing at 95 • C for 30 s, annealing at 55 • C for 30 s, elongation at 72 • C for 30 s, and a final extension at 72 • C for 5 min. Each sample had three replicates. PCR products were examined on a 2% (w/v) agarose gel, and the band was extracted and purified with the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, United States) according to the manufacturer's instructions and quantified using QuantiFluor TM -ST (Promega, United States). Purified amplicons were pooled in equimolar and paired end sequenced (2 × 300) on an Illumina MiSeq platform according to the standard protocols. The programs of amplification and sequencing were carried on using the Illumina MiSeq platform (United States) at BGI Co., Ltd. (Shenzhen, China). All read sequences were deposited in the Sequence Read Archive (SRA) NCBI database (accession number PRJNA526286).
Bioinformatics Analysis
Raw sequence processing, quality control, and annotation were carried out according to Huang et al. (2015). The representative sequences of bacterial 16S rRNA gene were assigned to taxonomic classifications from genus to phylum at hierarchical levels by RDP Classifier (v2.2) against the Greengene (v201305) with a confidence threshold of 80%. The obtained bacterial OTUs were further modified and only OTUs with >20 counts summed across all samples were retained. The number of sequences per sample ranged from 54 235 to 54 784 (Supplementary Table S1). These were resampled to a depth of 54 235 sequences using the program MOTHUR (v1.31.2), and the resulting new operational taxonomic unit (OTU) table was used for further analyses. Rarefaction analysis was performed by MOTHUR (v1.31.2), and the Observed species, Chao1 and ACE richness estimations, Coverage and the Shannon and Simpson diversities were calculated by MOTHUR (v1.31.2) (Schloss et al., 2009). Principal co-ordinate analysis (PCoA) based on the Bray-Curtis distance metric was carried out using mothur to compare the bacterial microbial community structure among the soil samples . Spearman's rank correlation coefficient was used to assess the correlations between selected rhizosphere soil genera abundance and P. capsici prevalence. Differentially abundant taxa and OTUs between two microhabitats were calculated using moderated t-tests. The resulting p-Values were adjusted for multiple hypotheses testing using the Benjamini-Hochberg correction. Heatmap figures were implemented by R (v3.4.2) packages heatmap. The Mantel test was conducted to reveal the relationships between the selected soil properties and rhizosphere soil microbial genera. In addition, redundancy analysis (RDA) was performed to evaluate the relationships among the soil samples, soil properties and rhizosphere soil microbial genera. In addition, Principal co-ordinates analysis (PCoA) and redundancy analysis (RDA) diagrams were generated using R (v3.4.2) package vegan to demonstrate the clustering of different samples.
Statistical Analysis
Differences between treatment groups were determined statistically at the factorial level by analysis of variance (ANOVA). Differences were considered significant at P < 0.05 or P < 0.01, and differences among treatments were analyzed via Tukey's Studentized Range (HSD) test.
Effect of BBS on Disease Control and Yield
The prevalence of phytophthora blight among BBS-treated plants was significantly lower than among the control plants in all three seasons, at 17.36% (2014), 11.11% (2015), and 6.25% (Figure 1B). Therefore, BBS treatment progressively reduced the prevalence of soil-borne disease and increased sweet pepper crop yields relative to the control.
Effect of BBS on Chlorophyll and Fruit Quality
It is generally known that photosynthesis determines the efficiency with which plants convert incoming sunlight to biomass; therefore, the chlorophyll content in the leaves was measured. Leaf total chlorophyll content was higher in BBStreated plants than in control plants, at 5.88 mg/g in BBS-treated plants, versus 4.93 mg/g in the control (Figure 2A). Furthermore, PGPR agent has been reported to improve crop quality (Song et al., 2015). Thus, we determined the content of soluble sugar, soluble solid and vitamin C of mature sweet pepper at the harvest time. BBS-treated plants had significantly higher soluble sugar content (5.33%) than control plants (4.22%) (Figure 2B). Soluble solids content was higher in BBS-treated plants (5.60%) than in control plants (4.61%) (Figure 2C). BBS treatment increased the vitamin C content of sweet peppers relative to the control ( Figure 2D). Therefore, BBS treatment improved the quality of sweet peppers.
Effect of BBS on the Soil Properties
Previous studies demonstrated that application of PGPR improved the soil nutrient status (Gulnaz et al., 2017). BBS treatment affected the soil properties (Table 1). In the presence of BBS, soil TOM and TOC were significantly higher than in the control soil, at 123.81 g/kg (TOM) and 71.81 g/kg (TOC) in BBS-treated soil, versus 103.51 g/kg (TOM), and 60.04 g/kg (TOC) in the control. AN content was higher in BBS-treated soil (25.84 mg/kg) than in the control (19.02 mg/kg), whereas TN and NN contents did not differ between BBS-treated and control soil. TP and TK levels did not differ between the BBS-treated soil and control soil, whereas AP and AK levels were higher in BBS-treated soil (1.11 g/kg AP and 1.52 g/kg AK) compare than in control soil (0.82 g/kg AP and 1.38 g/kg AK). Therefore, BBS treatment improved soil properties.
Effect of Microbial Community Assemblages by BBS on Disease Control
A previous study has shown that microbe additives change microbial communities (Trabelsi and Mhamdi, 2013). Here, we characterized and identified the complex microbial community by simultaneous DNA amplicon sequencing targeting the 16S rRNA gene in bacteria. In total of 326 683 sequences of 16S rRNA were extracted from treated and control soil samples; the number of high-quality bacterial sequences varied among samples from 54,069 to 54,783 (Supplementary Table S1). Furthermore, 3,615 bacterial OTUs were obtained, with a limited number at the 97% similarity cut-off level. The Good's coverage index revealed 99.00-99.09% of bacteria was obtained in all samples (Supplementary Table S1). The results showed that the probability of gene sequence detection in soil samples was high, and the sequencing results could represent the real situation of soil bacterial community in our experiment. The rarefaction and OTU Rank-Abundance curves of all six samples indicated that there was a smaller variation in the total number of OTUs, and the database of 16S rRNA gene sequences were very abundant which reflected the vast majority of microbial information (Supplementary Figure S1). The dominant phyla across all samples were Proteobacteria, Acidobacteria, Bacteroidates, Chloroflexi, Actinobacteria, Gemmatimonadetes, Planctomycetes, accounting for more than 89% of the bacterial sequences (Supplementary Figure S2a). Among these seven phyla, the relative abundance of Proteobacteria is the largest in the rhizosphere, which is composed of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria (Supplementary Figure S2b).
Principal-coordinate analysis (PCoA) examination of between-sample variation (beta-diversity) based on Bray-Curtis distances revealed that rhizosphere bacterial communities, clustered by treatment, along the second component (Figure 3). Durán et al. (2018) demonstrated that managing microbial communities contributed to plant health. Spearman's rank correlation coefficient revealed a clear positive correlation between phytophthora blight prevalence and the relative abundances of Iamia (P < 0.05), Agromyces (P < 0.01), Kaistia Each treatment was replicated three times, data are presented as means of three replicates ± SD, and error bars represent SD for three replicates. Means with different letters have significant differences (p < 0.05; HSD test). TOM, total organic matter; TOC, total organic carbon; TN, total nitrogen; NN, nitrate nitrogen; AN, ammonia nitrogen; TP, total phosphorus; AP, available phosphorus; TK, total potassium; AK, available potassium.
Effect of Microbial Community Assemblages on Soil Properties
BBS treatment improved the soil chemical properties ( Table 1). The Mantel test revealed striking relationships (r = 0.72, P < 0.05) between soil chemical properties and the abundances of the analyzed microbial genera. Examination of the relationship between the selected soil chemical properties and the abundances of the analyzed microbial genera (redundancy analysis) revealed that the two components explained the 91.41% variance, and BBS treatment was separated from the control treatment ( Figure 5). BBS-treated soil samples were dominated by Sporichthya, Achromobacter, Burkholderia, Comamonas, Ramlibacter, and Pontibacter; bacterial abundance was related to TOC, TN, AN, TP, and AP ( Figure 5). Therefore, BBS modified the microbial community thereby improving the soil properties. In summary, BBS shifted the microbe community to suppress soil-borne disease, increase sweet pepper crop yield and improve soil chemical properties.
DISCUSSION
Application of a consortium of three plant growth-promoting rhizobacteria (PGPR) strains (Bacillus cereus AR156, B. subtilis SM21, and Serratia sp. XY21) significantly reduced plant disease ( Figure 1A); this is consistent with previous findings that PGPR strains can be used as biocontrol agents against plant diseases caused by soil-borne pathogens (Kloeppe et al., 1999;Haas and Défago, 2005). More importantly, BBS treatment improved sweet pepper fruit yield, raised its nutrient contents, and improved soil fertility (Figures 1B, 2A-D). These results confirm previous reports, in which PGPR was shown to act as both a biofertilizer and biocontrol agent (Mohapatra et al., 2015;Prasad et al., 2015). Our finding that rhizosphere bacterial communities differed between the different treatments (Figure 3) is consistent with those of previous studies, which demonstrated that microbial Statistical significance at * P < 0.05 and * * P < 0.01.
Frontiers in Microbiology | www.frontiersin.org FIGURE 4 | Relative abundance of disease-related genus in rhizosphere. Significant differences between different treatments are indicated as different letters on top of the data bars. The statistical analysis was determined by one-side T-test with 5% FDR (+p < 0.1, * p < 0.05, and * * p < 0.01).
FIGURE 5 | Redundancy analysis of soil properties, soil properties and analyzed rhizosphere soil genera bacterial. Soil property: TOC, total organic carbon; TN, total nitrogen; AN, ammonia nitrogen; TP, total phosphorus; AP, available phosphorus.
Frontiers in Microbiology | www.frontiersin.org amendments alter the rhizosphere microbiome (Saravanakumar et al., 2017). Based on Spearman's rank correlation coefficient, the rhizosphere genera Iamia, Agromyces, Kaistia, Rubellimicrobium, Sporosarcina, Aquicella, and Phormidium were strongly and positively associated with sweet pepper disease. In contrast, negative associations between disease prevalence and the relative abundances of Sporichthya, Achromobacter, Burkholderia, Comamonas, Ramlibacter, and Pontibacter were observed ( Table 2). Interestingly, BBS treatment significantly increased the abundance of Burkholderia (moderated t-test, FDR, p < 0.01), Comamonas (moderated t-test, FDR, p < 0.1), and Ramlibacter (moderated t-test, FDR, p < 0.05) (Figure 4), which were negatively associated with sweet pepper disease. Among the genera that occurred in our soil samples, Burkholderia has been used as a biological control agent against plant disease (Parke and Gurian-Sherman, 2001), Comamonas is reported to act against pathogenic fungi (El-Banna, 2007), and Ramlibacter appears to be important in adverse environments (Luca et al., 2011). Hence, we conclude that BBS shaped the rhizosphere microbial community by developing beneficial strains, thereby contributing to the suppression of sweet pepper disease. BBS treatment improved soil chemical properties (Table 1), consistent with previous findings that PGPR can increase soil fertility (Sharma et al., 2017). The findings of our redundancy analysis (to evaluate the relationship between the selected soil chemical properties and the abundance of the analyzed microbial genera) suggest that BBS-treated soil samples were dominated by Sporichthya, Achromobacter, Burkholderia, Comamonas, Ramlibacter, and Pontibacter. Levels of TOC, TN, AN, TP, and AP were correlated with community composition (Figure 5). The abundance of the dominant genera were negatively associated with sweet pepper disease ( Table 2), which suggest that BBS modified the microbial community to suppress soil-borne disease and improve soil chemical properties as well. As a result, BBS improved sweet pepper yield and improved the quality of fruit; this is consistent previous findings that high fertility soil promotes fruit yield and plant quality (Xu et al., 2001;Mahmoud et al., 2009). Liu et al. reported that cucumber roots could sense microbial signals releasing from additive PGPR B. amyloliquefaciens SQR9 and subsequently secrete tryptophan to recruit SQR9 which benefited the cucumber itself and prevented pathogen infection . Wang et al. demonstrated that plant root exudates are involved in B. cereus AR156 biocontrol ability against tomato bacterial wilt caused by R. solanacearum. Furthermore, plant root exudates have been reported to influence the soil bacterial community structure (Szoboszlay et al., 2016). Therefore, it will be interesting to understand the mechanism of the bacteria shaping rhizosphere bacterial communities by regulating plant root exudates.
DATA AVAILABILITY
The datasets generated for this study can be found in the Sequence Read Archive (SRA) NCBI database 162 (accession number PRJNA526286). | 2019-07-23T13:06:44.196Z | 2019-07-23T00:00:00.000 | {
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236358377 | pes2o/s2orc | v3-fos-license | Season of Birth and Cardiovascular Mortality in Atrial Fibrillation: A Population-Based Cohort Study
Background: The fetal origins hypothesis have associated early life exposures with the development of adverse health outcomes in adulthood. Season of birth has been shown to be associated with overall and cardiovascular mortality. Methods: We performed a retrospective database study to explore the association between season of birth and mortality in patients with atrial fibrillation. Results: A total of 8962 patients with AF were identified in the database with 1253 deaths recorded. AF patients born in spring and summer had a higher mortality rate when compared to those born in autumn and winter (hazard ratio (HR) 1.13, 95% confidence interval (CI) 1.01–1.26, p = 0.03). This effect was consistent in the male subgroup (HR 1.25, 95% CI 1.03–1.51, p = 0.02 for males born in spring; HR 1.24, 95% CI 1.03–1.51, p = 0.03 for males born in summer when compared to winter as the reference) but not in females (HR 1.02, 95% CI 0.79–1.31, p = 0.88 for females born in spring; HR 1.11, 95% CI 0.87–1.42, p = 0.39 for females born in summer when compared to winter as the reference). Results persisted after adjustment for baseline characteristics and clinical risk profile. A similar pattern was observed with cardiovascular mortality. Conclusion: Birth in spring or summer is associated with a higher risk of cardiovascular mortality in male AF patients, but not in females. This could be related to the underlying differences in rates of major adverse clinical events between genders. Further studies should aim at clarifying the mechanisms behind this association, which may help us understand the higher level of risk in female patients with AF.
Introduction
The fetal origins hypothesis [1,2] have associated early life exposures with the development of adverse health outcomes in adulthood. Season of birth represent proxies for various complex environmental, familial and socioeconomic factors in prenatal and early postnatal life which could explain the association with disease development in adulthood. Season of birth has been shown in multiple studies from different parts of the world to be significantly associated with overall and cardiovascular disease-related mortality [3][4][5][6][7].
It has been suggested that people born in the autumn in the northern hemisphere live longer than those born during the spring or summer, as they have a decreased risk of cardiovascular disease specific mortality [6]. Not only is there an association with cardiovascular mortality, there has been associations of birth seasons on hypertension [8], one of the main contributory disease to cardiovascular mortality and morbidity worldwide. Most of these reported studies are whole population-based and only a few studies followed populations 2 of 10 longitudinally [9]. Moreover, none of the studies investigated the relationship between season and month of birth and mortality in patients with established cardiac conditions. Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is associated with an increased risk of major adverse cardiovascular events (MACE) [10]. The complex pathophysiology of atrial fibrillation (AF) encompasses many different mechanisms [11,12] and has been associated with modifiable risk factors such as hypertension and non-modifiable risk factors such as genetics and age [13]. Low birth weight has also previously been associated as a risk factor for development of AF, independent of known predictors such as hypertension, body mass index and height [14]. However, it is unknown whether the season of birth is associated with mortality in patients who develop AF.
This complex interaction between environmental and genetic factors, paired with the strong association of season and month of birth with cardiovascular mortality, makes AF an interesting disease to investigate in order to decipher the potential relationship in mortality between AF and the month and season of birth. This study aims to identify the relationship between mortality and season of birth in patients with AF.
Methods
We performed a retrospective database analysis of consecutive patients with AF seen in the cardiology department in our institution between January 2000 and December 2010 were identified through a database [15][16][17][18]. Our hospital covers an area of 4000 km 2 , and a population of 400,000 inhabitants and is the only public institution in the area.
Following identification of eligible patients, basic demographics (age at consultation, gender and season of birth), medical and drug history, biomarkers (left ventricular function, brain natriuretic peptic (BNP) and estimated glomerular filtration rate [19]) and clinical scores including CHA2DS2VASc, HASBLED, Charlson comorbidity index [20], frailty index [21], New York Heart Association (NYHA) and European Heart Rhythm Association (EHRA) functional class were recorded.
During follow-up, deaths from all causes and events of interest were recorded whenever they occurred in our institution. In addition, mortality data were obtained using a search tool from a dedicated website for the Région Centre (http://nrco.lanouvellerepublique. fr/dossiers/necro/index.php). The causes of death occurring in-hospital were collected through computerized hospitalization reports and were investigated to separate between cardiovascular and non-cardiovascular causes. For those that occurred outside our institution, they were collected by telephone from treating physicians, retirement homes or families. Mode of death was adjudicated based on medical reports and autopsy reports or death certificates when available. This information was reviewed by 2 investigators and causes of death were adjudicated after consideration of all the available information, and according to the following prespecified groups: cardiovascular, non-cardiovascular, as well as unknown when the quality of the information could not allow the investigators to appropriately identify cause of death [15]. The relationship between season of birth (autumn, winter, spring and summer) and mortality risk (all-cause and cardiovascular) was assessed using Cox proportional hazard regression models using autumn as the reference and separate analyses were performed for men and women as a gender subgroup.
Further regression analysis was performed using models to adjust for baseline characteristics and clinical risk profile.
The study was approved by the institutional review board of the Pole Coeur Thorax Vaisseaux from the Trousseau University Hospital, on 1 December 2015 and registered as a clinical audit. Ethical review was therefore not required. Patient consent was not sought. The study was conducted retrospectively, patients were not involved in its conduct, and there was no impact on their care.
Within this cohort, a total of 1253 deaths were recorded (1114 in-hospital deaths and 139 out-of-hospital deaths) during the follow-up period and mode of death was identified in 1215 (97%) patients (1080 (97%) for in-hospital deaths and 135 (97%) for out-of-hospital deaths). Of these, (677) 54% was attributed to cardiovascular causes whereas 538 (43%) were non-cardiovascular related causes. The three main causes of death were heart failure (29%), infection (18%) and cancer (12%). There were 715 strokes or thromboembolic events (TE) recorded during follow-up.
Gender Subgroup
When comparing between the 2 genders, the distribution of season of birth was similar as shown in Table 2. There were significant differences between the baseline characteristics of the 2 genders, with females being older and have a lower proportion of with concomitant cardiovascular risk factors and comorbidities (Table 2). Values are n (%) or mean ± SD. AF-Atrial Fibrillation; ACEi-Angiotensin converting enzyme inhibitor; ARB-II-angiotensin 2 receptor blockers; BBB-Bundle branch block; BNP-brain natriuretic peptide; eGFR-estimated glomerular filtration rate; ICD-Implantable cardioverter-defibrillator; LVEF-Left ventricular ejection fraction; SD = standard deviation; VKA-Vitamin K antagonists.
All-Cause Mortality
People born in spring and summer had a higher mortality rate when compared to those born in autumn and winter (hazard ratio (HR) 1.13, 95% confidence interval (CI) 1.01-1.26, p = 0.03) (Figure 1a).
Cardiovascular Mortality
Season of birth was a significant predictor for cardiovascular mortality with mortality rate being highest in the cohort born in spring and summer (HR 1.26, 95% CI 1.08-1.45, p = 0.003 compared to the born in autumn and winter) (Figure 2a). Similarly, the difference in mortality was driven by the higher cardiovascular mortality in males (HR 1.41, 95% CI 1.08-1.83, p = 0.01 for males born in spring; HR 1.39, 95% CI 1.07-1.81, p = 0.02 for males born in summer, HR 0.96, 95% CI 0.72-1.30, p = 0.80 for males born in autumn when compared to winter as the reference) (Figure 2b). This effect was not statistically significant in females (HR 0.92, 95% CI 0.65-1.31, p = 0.63 for females born in spring; HR 1.21, 95% CI 0.88-1.67, p = 0.24 for females born in summer, HR 1.11, 95% CI 0.79-1.56, p = 0.56 for females born in autumn when compared to winter as the reference) (Figure 2c).
Ischemic Stroke and Thromboembolic Events
Season of birth was not a significant predictor for stroke/TE in AF patients born in spring and summer compared to those born in autumn and winter (HR 0.99, 95% CI 0.86-1.15, p = 0.91).
Multivariable Analysis
After adjustment for age, CHA2DS2VASc score, HASBLED score, cardiovascular risk factors, other comorbidities, AF pattern, antithrombotic use and other cardiovascular drugs use, a higher all-cause mortality was still seen in males born in spring or in summer (adjusted HR 1.19, 95% CI 1.02-1.38, p = 0.03 vs those born in autumn or in winter) while this was not seen in females (adjusted HR 1.02, 95% CI 0.83-1.24, p = 0.88 for females born in spring or in summer vs those born in autumn or in winter) Similarly, a higher cardiovascular mortality was seen in males born in spring or in summer (adjusted HR 1.38, 95% CI 1.12-1.70, p = 0.002 vs those born in autumn or in winter) while this was not seen in females (adjusted HR 1.02, 95% CI 0.78-1.34, p = 0.89 for females born in spring or in summer vs those born in autumn or in winter).
There was no significant difference when considering the risk of stroke/TE (adjusted HR 1.11, 95% CI 0.90-1.36, p = 0.34 for males born in spring or in summer vs those born in autumn or in winter; adjusted HR 0.96, 95% CI 0.75-1.22, p = 0.74 for females born in spring or in summer vs those born in autumn or in winter).
There was no statistical interaction when taking into account the early (
Discussion
This study has shown an association between birth season and cardiovascular mortality in patients with AF, which is consistent with prior evidence in large population-based studies in undifferentiated cohort of patients [5]. This lends some support to the fetal origin hypothesis in the AF population. Where our AF cohort differed from an undifferentiated population was that the impact of season of birth on both all-cause and cardiovascular mortality was absent in the female subgroup. Even after adjusting for potential confounding factors, season of birth remains an independent risk factor for mortality only in males with AF. Our results could relate to a small but real seasonal effect of foetal or early life factors in later life, which should be better investigated and possibly addressed in case modifiable risk factors are identified during this specific period regarding the subsequent risk of cardiovascular events.
Our findings conflict with a previous prospective, longitudinal study, which looked at cardiovascular disease mortality in a non-selective cohort of women in the US and has shown an increase in cardiovascular mortality after adjustment of confounders such as age, familial and socioeconomic factors [9]. This may be due to the much smaller sample size and hence it is underpowered to determine the actual effect. However, it could also signify that the presence of AF in females negates the impact that birth season has on cardiovascular mortality.
Sex differences in AF have been previously been known and recognised [22], in particular the higher risk of stroke in females when compared to males [23]. This difference in stroke risk remains present despite treatment with oral anticoagulation (vitamin K antagonists) although it is nullified with the use of non-vitamin K antagonist oral anticoag-ulants [24]. A potential explanation could be differences in the clinical risk profile when female patients develop AF, being at a slightly higher risk of stroke and systemic embolism than their male counterparts, which negates the potentially beneficial or protective effect of being born in the autumn or winter months that can be seen in males. Although low birth weight has been implicated as a potential risk factor for stroke [25], it has not been explored as an outcome when comparing season of birth. As only half the population was on oral anticoagulation and there were no data on the time in therapeutic range, the interpretation of the results on stroke/TE is limited. The absence of significant differences in stroke/TE, a major driver of mortality in non-anticoagulated AF population, renders difficulties in the interpretation of the mortality results. Further studies are needed to examine the mechanisms behind this association.
Season of birth has been shown in previous studies to be significantly associated with cardiovascular disease-related mortality [3][4][5][6][7]. Potential drivers for this observed phenomenon includes dietary and nutritional differences, which could be related to the availability of types of food at different seasons and socioeconomic factors [2,26], inflammation and infective causes [27], environmental and climate differences [28]. The findings from this study may have important clinical implications for the future. Firstly, populations at a higher risk of cardiovascular disease may benefit from closer monitoring to detect early development of cardiovascular disease and initiate early, aggressive primary prevention measures to reduce mortality. Secondly, it may stimulate research into identifying the reasons behind the higher risk. As previously mentioned, the combination of complex environmental, familial and socioeconomic factors likely contributes to this association and to being able to identify and isolate certain modifiable factors which may allow cardiovascular risk prevention to start, even before birth.
Limitations
One of the limitations is the small sample size when compared to other populationbased studies exploring birth season and mortality. This may impact on the effect size and may be underpowered to identify small differences, such as in the female subgroup. Secondly, being a single centre study reduces the generalizability of the results, although the overall conclusion appears to correlate with other published data. Similarly, as the population in the analysis is comprised mainly of pre non-vitamin K antagonists oral anticoagulant (NOAC), the results may not be present in contemporary cohort of patients on NOAC. In the same note, considering the mean age of the patients, the conditions surrounding their birth years and will very unlikely be replicated in the future and hence may not be extrapolated. Third, the lack of data from birth such as birth weight, which could be a confounder, may limit the conclusions drawn. Fourth, the date of diagnosis of AF is unknown which could have an impact on the analysis. Lastly, a major limitation to these findings and that of the fetal origins hypothesis is the inadequate control for some early and later life predictors of mortality which may contribute significantly to variation in lifetime health conditions [1,29]. An example would be that socioeconomic factors could be a confounder as women in more favourable socioeconomic status chose to avoid giving birth in colder months [3,30]. These were not investigated in our study and hence could not be accounted for within the analyses.
Conclusions
Birth in spring or summer is associated with a higher risk of cardiovascular mortality in male AF patients, consistent with other similar studies of undifferentiated cohort. However, this effect was not seen in females which could be related to the underlying differences in rates of major adverse clinical events between genders. | 2021-07-27T00:05:47.872Z | 2021-05-24T00:00:00.000 | {
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88949281 | pes2o/s2orc | v3-fos-license | Effects of Ground and Unground Rice Straw on the Yield and Proximate
A field experiment was carried out to evaluate the influence of ground and unground rice straw application as organic manure yield and proximate composition of Complete Randomized Block Design with four replications. The ground and unground rice straw were in the main plot and differe application rates of unground rice straws at 1,250 and 2,500 kg/ha application rates significantly increased (p < 0.05) yield attributes such as p
Introduction
most popular oil seed crop, cultivated in the tropical and subtropical part of the world (Olayinka and Etejere, 2013).It contains about 45 to 50% oil, 25 to 30% protein, 20% carbohydrate, 5% each of fibre and ash, all wh substantial contribution to human nutrition (Fageria 2010).It is also a rich source of phosphor, potassium, calcium and magnesium, as well as vitamin E, K and B (Savage and Keenan, 1994).Achou consumption as a supplement for the staple carbohydrate food where protein food of animal origin is unaffordable.limited its yield potentials. Inorganic fertilizer application have been considered yield, especially in commercial production; however, its prolong use have been associated with soil degradation, nutrient imbalance and s and practice is gaining attention from researchers, as several studies have been conducted on soil amendment from organic sources ranging from poultry manure vermicompost amongst others, all of which have been repo
Introduction
Groundnut ( most popular oil seed crop, cultivated in the tropical and subtropical part of the world (Olayinka and Etejere, 2013).It contains about 45 to 50% oil, 25 to 30% protein, 20% carbohydrate, 5% each of fibre and ash, all wh substantial contribution to human nutrition (Fageria 2010).It is also a rich source of phosphor, potassium, calcium and magnesium, as well as vitamin E, K and B (Savage and Keenan, 1994).Achou consumption as a supplement for the staple carbohydrate food where protein food of animal origin is unaffordable.
The high nutrient demand of this crop from the soil has limited its yield potentials. Inorganic fertilizer application have been considered yield, especially in commercial production; however, its prolong use have been associated with soil degradation, nutrient imbalance and s and Sami, 2014) practice is gaining attention from researchers, as several studies have been conducted on soil amendment from organic sources ranging from poultry manure vermicompost amongst others, all of which have been repo to enhance yield in groundnut also ( A field experiment was carried out to evaluate the influence of ground and unground rice straw application as organic manure yield and proximate composition of Complete Randomized Block Design with four replications.The ground and unground rice straw were in the main plot and differe application rates of the rice straw (0; 1,250; 2,500; 3,750 and 5,000 kg /ha) were in the subplots.The results revealed that ground and unground rice straws at 1,250 and 2,500 kg/ha application rates significantly increased (p < 0.05) yield attributes such as p a range of 48.33-74.33 pod weight (23.95 38.05 g) as well as some aspect of proximate composition such as ash content, crude protein and crude fats which 2. 79%, 31.47-31.56% and 46.61 unground rice straw applied at the rate of 1,250 and 2,500 kg/ha can serve as an alternative for soil nutrient ame a reasonable yield that is able to meet the nutritional needs of man and livestock.
application rates, groundnut, proximate composition, rice straw, yield attribute Groundnut (Arachis hypogaea most popular oil seed crop, cultivated in the tropical and subtropical part of the world (Olayinka and Etejere, 2013).It contains about 45 to 50% oil, 25 to 30% protein, 20% carbohydrate, 5% each of fibre and ash, all wh substantial contribution to human nutrition (Fageria 2010).It is also a rich source of phosphor, potassium, calcium and magnesium, as well as vitamin E, K and B (Savage and Keenan, 1994).Achou et al. (2005) reported groundnut seed consumption as a supplement for the staple carbohydrate food where protein food of animal origin is unaffordable.
The high nutrient demand of this crop from the soil has limited its yield potentials. Inorganic fertilizer application have been considered as soil amendment to help increase groundnut yield, especially in commercial production; however, its prolong use have been associated with soil degradation, nutrient imbalance and soil acidity (Obi and Ebo, 1995;Sami, 2014).Recently, a better practice is gaining attention from researchers, as several studies have been conducted on soil amendment from organic sources ranging from poultry manure vermicompost amongst others, all of which have been repo to enhance yield in groundnut also (Veluchamy et al., 2013).A field experiment was carried out to evaluate the influence of ground and unground rice straw application as organic manure yield and proximate composition of groundnut seeds, at the University of Ilorin teaching and research farm.The field layout was a split plot Complete Randomized Block Design with four replications.The ground and unground rice straw were in the main plot and differe the rice straw (0; 1,250; 2,500; 3,750 and 5,000 kg /ha) were in the subplots.The results revealed that ground and unground rice straws at 1,250 and 2,500 kg/ha application rates significantly increased (p < 0.05) yield attributes such as p 74.33 pod weight (23.95-42.70),number of seeds (45.67 38.05 g) as well as some aspect of proximate composition such as ash content, crude protein and crude fats which 31.56% and 46.61-47.13%respectively, in order of their mention.The forgoing study revealed that ground and unground rice straw applied at the rate of 1,250 and 2,500 kg/ha can serve as an alternative for soil nutrient ame a reasonable yield that is able to meet the nutritional needs of man and livestock.
Available online:
Not Sci Biol, 2016, 8(2 hypogaea L.) is one of the world`s most popular oil seed crop, cultivated in the tropical and subtropical part of the world (Olayinka and Etejere, 2013).It contains about 45 to 50% oil, 25 to 30% protein, 20% carbohydrate, 5% each of fibre and ash, all wh substantial contribution to human nutrition (Fageria 2010).It is also a rich source of phosphor, potassium, calcium and magnesium, as well as vitamin E, K and B (Savage and . (2005) reported groundnut seed consumption as a supplement for the staple carbohydrate food where protein food of animal origin is unaffordable.
The high nutrient demand of this crop from the soil has limited its yield potentials. Inorganic fertilizer application have as soil amendment to help increase groundnut yield, especially in commercial production; however, its prolong use have been associated with soil degradation, oil acidity (Obi and Ebo, 1995; .Recently, a better soil-friendly amendment practice is gaining attention from researchers, as several studies have been conducted on soil amendment from organic sources ranging from poultry manure, farm yard manure to vermicompost amongst others, all of which have been repo to enhance yield in groundnut also (Veluchamy A field experiment was carried out to evaluate the influence of ground and unground rice straw application as organic manure groundnut seeds, at the University of Ilorin teaching and research farm.The field layout was a split plot Complete Randomized Block Design with four replications.The ground and unground rice straw were in the main plot and differe the rice straw (0; 1,250; 2,500; 3,750 and 5,000 kg /ha) were in the subplots.The results revealed that ground and unground rice straws at 1,250 and 2,500 kg/ha application rates significantly increased (p < 0.05) yield attributes such as p 42.70), number of seeds (45.67 38.05 g) as well as some aspect of proximate composition such as ash content, crude protein and crude fats which 47.13% respectively, in order of their mention.The forgoing study revealed that ground and unground rice straw applied at the rate of 1,250 and 2,500 kg/ha can serve as an alternative for soil nutrient ame a reasonable yield that is able to meet the nutritional needs of man and livestock.L.) is one of the world`s most popular oil seed crop, cultivated in the tropical and subtropical part of the world (Olayinka and Etejere, 2013).It contains about 45 to 50% oil, 25 to 30% protein, 20% carbohydrate, 5% each of fibre and ash, all which makes it a substantial contribution to human nutrition (Fageria 2010).It is also a rich source of phosphor, potassium, calcium and magnesium, as well as vitamin E, K and B (Savage and . (2005) reported groundnut seed consumption as a supplement for the staple carbohydrate food where protein food of animal origin is unaffordable.
The high nutrient demand of this crop from the soil has limited its yield potentials. Inorganic fertilizer application have as soil amendment to help increase groundnut yield, especially in commercial production; however, its prolong use have been associated with soil degradation, oil acidity (Obi and Ebo, 1995; friendly amendment practice is gaining attention from researchers, as several studies have been conducted on soil amendment from organic sources farm yard manure to vermicompost amongst others, all of which have been repo to enhance yield in groundnut also (Veluchamy et al., 2010; .Received in revised form: 16 June 2015.A field experiment was carried out to evaluate the influence of ground and unground rice straw application as organic manure groundnut seeds, at the University of Ilorin teaching and research farm.The field layout was a split plot Complete Randomized Block Design with four replications.The ground and unground rice straw were in the main plot and differe the rice straw (0; 1,250; 2,500; 3,750 and 5,000 kg /ha) were in the subplots.The results revealed that ground and unground rice straws at 1,250 and 2,500 kg/ha application rates significantly increased (p < 0.05) yield attributes such as p 42.70), number of seeds (45.67-77.33),seeds' weight (13.55 38.05 g) as well as some aspect of proximate composition such as ash content, crude protein and crude fats which 47.13% respectively, in order of their mention.The forgoing study revealed that ground and unground rice straw applied at the rate of 1,250 and 2,500 kg/ha can serve as an alternative for soil nutrient ame a reasonable yield that is able to meet the nutritional needs of man and livestock.
application rates, groundnut, proximate composition, rice straw, yield attribute www.notulaebiologicae.ro Print ISSN 2067-3205; Electronic 2067 ):176-180.DOI: 10.15835/ L.) is one of the world`s most popular oil seed crop, cultivated in the tropical and subtropical part of the world (Olayinka and Etejere, 2013).It contains about 45 to 50% oil, 25 to 30% protein, 20% ich makes it a substantial contribution to human nutrition (Fageria et al., 2010).It is also a rich source of phosphor, potassium, calcium and magnesium, as well as vitamin E, K and B (Savage and . (2005) reported groundnut seeds consumption as a supplement for the staple carbohydrate food The high nutrient demand of this crop from the soil has limited its yield potentials. Inorganic fertilizer application have as soil amendment to help increase groundnut yield, especially in commercial production; however, its prolong use have been associated with soil degradation, oil acidity (Obi and Ebo, 1995; Basel friendly amendment practice is gaining attention from researchers, as several studies have been conducted on soil amendment from organic sources farm yard manure to vermicompost amongst others, all of which have been reported ., 2010; The global production of rice straw amounts to approximately 731 million tons annually (Binod Only 20% of the declared amount were used for purposes as ethanol, paper and fertilizer production, as well as fodders for animals consumption (El remaining 80% is left on the field for burning or decomposition.This contributes to atmospheric pollution and soil nutrient lo In order to increase the rice straw percentage in use and minimize the usual burning practice, the present research was conducted to evaluate the effect of ground and unground rice straw on yield and proximate composition of harvested groundnut seeds
Experimental site
The experiment was conducted at the University of Ilorin Botanical Garden, Ilorin.The experimental site was situated between longitude 27.810 of 7.5.
Experimental design and treatment details
The experimental layout was a split plot design with a gross plot dimension of 24.50 × 9.50 m.The net consisting of four ridges with inter net-plot arrangement followed a complete randomized design A field experiment was carried out to evaluate the influence of ground and unground rice straw application as organic manure groundnut seeds, at the University of Ilorin teaching and research farm.The field layout was a split plot Complete Randomized Block Design with four replications.The ground and unground rice straw were in the main plot and differe the rice straw (0; 1,250; 2,500; 3,750 and 5,000 kg /ha) were in the subplots.The results revealed that ground and unground rice straws at 1,250 and 2,500 kg/ha application rates significantly increased (p < 0.05) yield attributes such as p 77.33), seeds' weight (13.55 38.05 g) as well as some aspect of proximate composition such as ash content, crude protein and crude fats which 47.13% respectively, in order of their mention.The forgoing study revealed that ground and unground rice straw applied at the rate of 1,250 and 2,500 kg/ha can serve as an alternative for soil nutrient ame a reasonable yield that is able to meet the nutritional needs of man and livestock.The global production of rice straw amounts to approximately 731 million tons annually (Binod Only 20% of the declared amount were used for purposes as ethanol, paper and fertilizer production, as well as fodders for animals consumption (El remaining 80% is left on the field for burning or decomposition.This contributes to atmospheric pollution and soil nutrient loss.
In order to increase the rice straw percentage in use and minimize the usual burning practice, the present research was conducted to evaluate the effect of ground and unground rice straw on yield and proximate composition of harvested groundnut seeds when applied to the soil as organic manure.
Experimental site
The experiment was conducted at the University of Ilorin Botanical Garden, Ilorin.The experimental site was situated between longitude 27.810 1 N and 8°28.230 of 7.5.
Experimental design and treatment details
The experimental layout was a split plot design with a gross plot dimension of 24.50 × 9.50 m.The net consisting of four ridges with inter plot arrangement followed a complete randomized design A field experiment was carried out to evaluate the influence of ground and unground rice straw application as organic manure groundnut seeds, at the University of Ilorin teaching and research farm.The field layout was a split plot Complete Randomized Block Design with four replications.The ground and unground rice straw were in the main plot and differe the rice straw (0; 1,250; 2,500; 3,750 and 5,000 kg /ha) were in the subplots.The results revealed that ground and unground rice straws at 1,250 and 2,500 kg/ha application rates significantly increased (p < 0.05) yield attributes such as p 77.33), seeds' weight (13.55-25.56g) and hundred seed weight 38.05 g) as well as some aspect of proximate composition such as ash content, crude protein and crude fats which 47.13% respectively, in order of their mention.The forgoing study revealed that ground and unground rice straw applied at the rate of 1,250 and 2,500 kg/ha can serve as an alternative for soil nutrient ame application rates, groundnut, proximate composition, rice straw, yield attribute www.notulaebiologicae.ronsb.8.2.9823 The global production of rice straw amounts to approximately 731 million tons annually (Binod Only 20% of the declared amount were used for purposes as ethanol, paper and fertilizer production, as well as fodders for animals consumption (El-Gammal remaining 80% is left on the field for burning or decomposition.This contributes to atmospheric pollution and In order to increase the rice straw percentage in use and minimize the usual burning practice, the present research was conducted to evaluate the effect of ground and unground rice straw on yield and proximate composition of harvested when applied to the soil as organic manure. ethods
Experimental site
The experiment was conducted at the University of Ilorin Botanical Garden, Ilorin.The experimental site was situated between longitude 4°38.920 1 E and 28.230 1 N.The soil was sandy
Experimental design and treatment details
The experimental layout was a split plot design with a gross plot dimension of 24.50 × 9.50 m.The net consisting of four ridges with inter plot arrangement followed a complete randomized design June 2016.Published online:
Original Article
Effects of Ground and Unground Rice Straw on the Yield and Proximate Arachis hypogeae L.) OLAYINKA, Emmanuel O. ETEJERE eotejere@yahoo.com A field experiment was carried out to evaluate the influence of ground and unground rice straw application as organic manure groundnut seeds, at the University of Ilorin teaching and research farm.The field layout was a split plot Complete Randomized Block Design with four replications.The ground and unground rice straw were in the main plot and differe the rice straw (0; 1,250; 2,500; 3,750 and 5,000 kg /ha) were in the subplots.The results revealed that ground and unground rice straws at 1,250 and 2,500 kg/ha application rates significantly increased (p < 0.05) yield attributes such as pods' number wit 25.56 g) and hundred seed weight 38.05 g) as well as some aspect of proximate composition such as ash content, crude protein and crude fats which ranged between 47.13% respectively, in order of their mention.The forgoing study revealed that ground and unground rice straw applied at the rate of 1,250 and 2,500 kg/ha can serve as an alternative for soil nutrient amendment in groundnut as it The global production of rice straw amounts to approximately 731 million tons annually (Binod Only 20% of the declared amount were used for purposes as ethanol, paper and fertilizer production, as well as fodders for Gammal et al., 2001), while the remaining 80% is left on the field for burning or decomposition.This contributes to atmospheric pollution and In order to increase the rice straw percentage in use and minimize the usual burning practice, the present research was conducted to evaluate the effect of ground and unground rice straw on yield and proximate composition of harvested when applied to the soil as organic manure.
The experiment was conducted at the University of Ilorin Botanical Garden, Ilorin.The experimental site was situated E and 4° 39.971 1 E and latitude The soil was sandy-loam with a pH
Experimental design and treatment details
The experimental layout was a split plot design with a gross plot dimension of 24.50 × 9.50 m.The net-plot was 2.0 × 2.0 m, consisting of four ridges with inter-row spacing of 0.15 m.The plot arrangement followed a complete randomized design Published online: 17 June 2016.
Original Article
Effects of Ground and Unground Rice Straw on the Yield and Proximate
ETEJERE
A field experiment was carried out to evaluate the influence of ground and unground rice straw application as organic manure on the groundnut seeds, at the University of Ilorin teaching and research farm.The field layout was a split plot Complete Randomized Block Design with four replications.The ground and unground rice straw were in the main plot and different the rice straw (0; 1,250; 2,500; 3,750 and 5,000 kg /ha) were in the subplots.The results revealed that ground and ods' number with 25.56 g) and hundred seed weight ranged between 47.13% respectively, in order of their mention.The forgoing study revealed that ground and ndment in groundnut as it The global production of rice straw amounts to approximately 731 million tons annually (Binod et al., 2010).Only 20% of the declared amount were used for purposes such as ethanol, paper and fertilizer production, as well as fodders for 2001), while the remaining 80% is left on the field for burning or decomposition.This contributes to atmospheric pollution and In order to increase the rice straw percentage in use and minimize the usual burning practice, the present research was conducted to evaluate the effect of ground and unground rice straw on yield and proximate composition of harvested when applied to the soil as organic manure.
The experiment was conducted at the University of Ilorin Botanical Garden, Ilorin.The experimental site was situated E and latitude 8 loam with a pH The experimental layout was a split plot design with a gross plot was 2.0 × 2.0 m, row spacing of 0.15 m.The plot arrangement followed a complete randomized design
Original Article
on the groundnut seeds, at the University of Ilorin teaching and research farm.The field layout was a split plot nt the rice straw (0; 1,250; 2,500; 3,750 and 5,000 kg /ha) were in the subplots.The results revealed that ground and h 25.56 g) and hundred seed weight ranged between 47.13% respectively, in order of their mention.The forgoing study revealed that ground and ndment in groundnut as it The global production of rice straw amounts to 2010).such as ethanol, paper and fertilizer production, as well as fodders for 2001), while the remaining 80% is left on the field for burning or decomposition.This contributes to atmospheric pollution and In order to increase the rice straw percentage in use and minimize the usual burning practice, the present research was conducted to evaluate the effect of ground and unground rice straw on yield and proximate composition of harvested The experiment was conducted at the University of Ilorin Botanical Garden, Ilorin.The experimental site was situated 8°l oam with a pH The experimental layout was a split plot design with a gross plot was 2.0 × 2.0 m, row spacing of 0.15 m.The plot arrangement followed a complete randomized design with four replications, each of ground and unground rice straw at different application rates: T1 = 1,250 kg/ha; T2 = 2,500 kg/ha; T3 = 3,750 kg/ha; T4 = 5,000 kg/ha and T0 = 0 kg/ha.
Mineral analysis of rice straw
The rice straw sample used was analysed for both macro and micro nutrients.Total nitrogen was determined by micro-Kjedhal method as adopted by Bremner (1996).Total phosphorus was determined by the ammonium molybdate /vanadate yellow colour method following ternary acidperchloric-nitric-sulphuric acid wet digestion (Anderson and Ingram, 1994), total boron by azomethane-hydrogen method following Ternary acid digestion (Jones, 1991).Total cations: calcium (Ca), magnesium (Mg), copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn) were determined by atomic spectrophotometer.Elements such as sodium (Na) and potassium (K) were analysed using flame photometry following a wet digestion with perchloric-sulphuric ternary acid (Anderson and Ingram, 1994).Sulphur was determined by turbidometry, organic matter and ash content using the method described by Okalebo et al. (2002), while organic carbon was determined using Walkley-Black method as detailed by Nelson and Sommer (1982).
Field planting
The field was mechanically ploughed to loosen the soil after which ground and unground rice straw, at different application rates, were incorporated into the soil at a depth of 0.1 m.Thereafter, ridges were made by the use of hoe.The plots were watered to saturation point daily for a period of two weeks to enable rapid decay of the rice straw.Viable 'MK373' groundnut seeds, collected from College of Agriculture Mokwa, were sown at a depth of 0.03 m and a spacing of 0.1 m following a pretreatment of the seeds with 500 mg/kg dress Force ® 42WS (20% imidacloprid, 20% metalaxyl -M and 2% tebuconazole).Hand weeding was done to remove weeds at an interval of two weeks throughout the period of the experiment.Since the experiment was carried out during the dry season 2,500 L of water were used to irrigate the plots to saturation point at interval of forty eight hours (48 hours) throughout the period of the investigation.
Yield attributes
Number of fully developed pods was manually counted from each of the representative plants and the average was obtained as the number of matured pods per plant.The pod weight was also obtained by the use of a weighing balance after air drying to 12% moisture level.The average was then recorded as the mean pod weight; thereafter, the shells were removed and the seeds were manually counted and recorded; they were weighed and the mean was recorded as average seeds' weight, whereas 100-seeds weight was also determined.
Proximate analysis of the groundnut seeds
Proximate composition of air dried groundnut seeds before planting and at harvest was determined according to the methods of Official Analytical Chemists (AOAC, 2000).Moisture content was determined by drying 2 g of air dried seeds in an oven at 105 °C for 5 hours.Ash content was determined by incinerating 2 g of sample in a muffle furnace at 600 °C for 3 hours, until a greyish ash was formed.Crude fibre was determined by extracting 2.0 g of the ground sample with hexane, in a thimble, for 6 hours, to free the fat sample.
Thereafter, the sample was hydrolysed using 1.25% tetraoxosulphate (vi) acid to remove digestible nutrient in the fat; it was then filtered and the resulting filtrate was incinerated in a muffle furnace at 600 °C for 30 min, cooled in a desiccator and weighed.Crude protein was calculated by multiplying factor (%N × 6.25) following a % nitrogen determination by the Kjedhal method using 2 g of ground seed samples.Crude fat was determined by manually extracting 2 g of samples with 150 ml petroleum ether in a soxhlet extractor at a boiling point of 60-80 °C.Total carbohydrate was determined by subtracting the sum of ash, crude protein, crude fibre, crude fat and moisture from one hundred seeds.
Data analysis
Data were analysed using Analysis of Variance (F test) with Statistical Package for Social Science (SPSS) software version 16.0.Significant test results followed the Duncan multiple range Test (DMRT) at 5% level of probability for identification of important contrasts.
Mineral analysis of rice straw before planting
The proximate composition of rice straw before its application as manure is shown in Table 1.Rice straw contained moderate organic carbon (1.08%), high organic matter (75%), was very rich in phosphorus (1,170.00mg kg -1 ), rich in calcium, magnesium and sulphur (470.30,320.0 and 390.40 mg kg -1 respectively), also had a solid content of iron and zinc (500.25 and 330.50 mg kg -1 ), while other mineral elements such as boron, potassium, manganese, copper and sodium had relatively low values (4.0, 1.5, 115 mg kg -1 ) respectively.The results showed that rice straw was rich in organic matter and other mineral nutrient necessary for plant growth.Similar results were obtained by Olayinka and Etejere (2013).
Yield attributes of groundnut
The effects of ground and unground rice straw at different application rate on yield attributes are shown in Table 2. Ground and unground rice straw at 1,250 kg/ha application rates significantly increased (p < 0.05) the number of pods and pod weight when compared to all other application rates.No significant effect was recorded for pod length at all application rates.Number of seeds per plant, seeds' weight and 100-seeds weight were The increased number of pods per plant and pod weight per plant could be traced to the large amount of calcium and potassium resulting from accelerated mineralization of the rice straw; Jana et al. (1990) has reported increased number of pods per plant, pod weight, pod yield and oil yield in groundnut plant upon increased potassium level of the soil on account of organic manure mineralization.
Enhanced seeds' attributes in 1,250 kg/ha and 2,500 kg/ha application rates over all other treatment was traceable to enhanced rate of decomposition of the applied rice straw (Das et al., 2003).This result was similar that of Muneshwar et al. (2001) regarding the response of wheat on farm yard manure, rice and wheat crop residue.
Proximate composition of groundnut seeds Moisture content
Moisture content of groundnut seeds before planting was 7.80% (Table 3).Regardless of the application rates, groundnut raised under unground rice straw retained more moisture compared to ground rice straw.
Plants grown within 5,000 and 3,750 kg/ha ground and unground rice straw application rates recorded significantly the highest moisture content of seeds compared to all other application rates.Ground and unground rice straw applied at the rate of 1,250 and 2,500 kg/ha had significantly low moisture content of harvested seeds.
The result was a clear indication that seeds harvested from low rice straw treatment rate will have a longer storage life span.Moisture content has been reported to be of importance in seed storage, because of the correlation between the lower the moisture content of food, the higher the storability (Ajayi and Adedire, 2007).
Ash content
The ash content of the seeds before planting was 2.29% (Table 3).Ground and unground rice straw applied at the rate of 2,500 and 1,250 kg/ha significantly increased the ash content of groundnut seeds compared to all other application rates.Rice straw at 3,750 and 5,000 kg/ha application rates had no significant effect on the ash content (Table 3).The increase in ash content of 2,500 and 1,250 kg/ha rates could be due to the increase in soil 4).Superscript with same letters were not significantly different at p < 0.05; T0 = control, T1 = 1,250 kg/ha, T2 = 2,500 kg/ha, T3 = 3,750 kg/ha and T4 = 5,000 kg/ha ).Superscript with same letters were not significantly different at p < 0.05; T0 = control, T1 = 1,250 kg/ha, T2 = 2,500 kg/ha, T3 = 3,750 kg/ha and T4 = 5,000 kg/ha potassium, resulting from the mineralization of the straws at optimum concentration, since the major component of ash is potassium (Shaharadeen and Seran, 2013).This result was in agreement with the ash content reported by Atasie et al. (2009) and Olayinka and Etejere (2013) which were 2.4-3.08% and 2.4 -3.10% respectively.
Crude fibre
The percentage of crude fibre of groundnut seeds before planting was 3.40% (Table 3).In both ground and unground rice straw application forms, 5,000 kg/ha rice straw application rate significantly increased (p < 0.05) the percentage of fibre content.Thus, 3,750 kg/ha application rate also followed the same trend, except in ground rice straw, where the increase was insignificant.
The result depicts a correlation between the percentage of moisture content and the crude fibre.The trend noticed in the content of crude fibre may be probably similar to what was adduced for the percentage moisture content of the seeds.The result which ranged between 3.14-3.79%was at variance with those of Quass (1995), Shahardeen and Seran (2013) or Olayinka and Etejere (2013) who reported a range of 6.00-6.60%,5.95-7.3%and 7.5-9.12%respectively.
Crude protein
Crude protein of groundnut seeds before planting was 30.81% (Table 3).Regardless of the application form, rice straw at 2,500 kg/ha application rate significantly increased (p < 0.05) the percentage of crude protein of the harvested seeds.All other application rates had no significant effect on the crude protein (Table 3).
The increase in crude protein may be traced to enhanced availability of potassium for plant use, resulting from the mineralization of rice straw at optimum concentration.Potassium is required for every major step in protein synthesis, therefore increased protein synthesis with increasing potassium availability and uptake by groundnut plant could form the basis for the increase in seed protein content.
Similar results were obtained by Musa et al. (2010) who reported 31.3%crude proteine, but at variance with Atasie et al. (2009) who reported 38.61%.The differences could be due to genetic variation in the groundnut seeds.
Crude fat
The percentage of crude fat of groundnut seeds before planting was 46.88% (Table 3).Ground and unground rice straw applied at the rate of 1,250 and 2,500 kg/ha significantly increased (p < 0.05) the crude fat of the harvested seeds compared to all other application rates; however, 3,750 kg/ha ground and unground rice straw application rates had no significant effect on the crude fats (Table 3).
The increase in crude fat observed in 1,250 and 2,500 kg/ha application rate could be attributed to the low moisture content in the seeds.The result which ranged between 45.59-46.90%was in agreement with data obtained by Thakur et al. (2005), Atasie et al. (2009), Shawad et al. (2010), Olayinka and Etejere (2013).
Total carbohydrate
Total carbohydrate of groundnut seeds before planting was 8.82% (Table 3).Ground rice straw had no significant effect on seed's total carbohydrate at all application rate considered, with the exception in the case of unground rice straw applied at the rate of 2,500 kg/ha which significantly increased the total carbohydrate content of seeds.
The total carbohydrate ranged between 5.52-6.60%,thus being similar with the results obtained by Olayinka and Etejere (2013), who reported a range between 4.99-10.27%,but at variance with Atasie et al. (2009), who reported 18.1% for carbohydrate content in groundnut.The variation may be due to varietal differences.
Conclusions
In conclusion, the forgoing research had shown that lower application rate of both ground and unground rice straw (1,250 and 2,500 kg/ha) positively influenced the yield attributes and some aspects of proximate composition such as ash, crude protein and crude fats for groundnut seeds.
Veluchamy Mathivanan et al Received: 20 Apr Effects of Ground and Unground Rice Straw on the Yield and Proximate Composition of Groundnut ( Abdul'Aziz AYINLA*, Bolaji U Nigeria;
/
Not Sci Biol, 2016, 8(2 Effects of Ground and Unground Rice Straw on the Yield and Proximate Composition of Groundnut ( Abdul'Aziz AYINLA*, Bolaji U. OLAYINKA, Emmanuel O University of Ilorin, Faculty of Life Science, Department of Plant Biology, PMB 1515, Ilorin, (*corresponding author); Effects of Ground and Unground Rice Straw on the Yield and ProximateComposition of Groundnut ( . Received in revised form: Not SciBiol, 2016, 8(2):176-180Effects of Ground and Unground Rice Straw on the Yield and Proximate Composition of Groundnut (Arachis hypogeae 16 June 2015.Accepted: 1 Effects of Ground and Unground Rice Straw on the Yield and Proximate
Table 1 .
Mineral analysis of rice straw
Table 2 .
Yield attributes of groundnut
Table 3 .
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247782947 | pes2o/s2orc | v3-fos-license | Case Report: Capitalizing on Development and Activity-Dependent Plasticity, an Interaction With Pediatric-Onset Spinal Cord Injury
Background Spinal cord injury (SCI) in infancy halts typical development secondary to paralysis/paresis and the limited ability to engage with the environment. Traditional therapies further restrict a child via bracing, equipment, and medications. In contrast, activity-based restorative therapies (ABRT) promote activation of the neuromuscular system below the level of injury and affords a more typical sensorimotor experience. Case Description A premature male infant exhibiting hypotonia, poor head control, and extremity weakness was diagnosed at age 5 months with a remote incomplete upper cervical SCI based on magnetic resonance imaging (MRI), presumed to have occurred perinatally. From 4 to 15 months of age, he received physical, occupational and speech therapies. Enrolled in an ABRT program at 15 months, he was unable to sit, pull-to-stand, stand, or walk and had upper extremity impairments. Results of the Bayley-III Scales of Infant and Toddler Development revealed gross and fine motor scores consistent with a 4-month-old. Methods Activity-based restorative therapies was provided 5 day/week: 1.5 h of activity-based locomotor training and 1 h of activity-based occupational therapy. Results Activity-based restorative therapies are reported for 177 sessions and are on-going. Improvements are noted in trunk control, standing, walking, grasp, in-hand manipulation, and associated kinematics. Bayley-III fine motor score improved to that of a 16-month-old and gross motor score to that of a 7-month-old. Discussion While the two treatment periods (i.e., 4–15 months old and 15–24 months) were each ∼9 months, the child’s accelerated progress toward typical development during the latter, ABRT period is noteworthy. In comparison to the period of traditional therapies in which paralysis was compounded by a restrictive environment and compensation, ABRT provided a potentially rich sensorimotor experience with an emphasis on active weight-bearing and proper kinematics to activate the neuromuscular system below the lesion in an age-appropriate, task-specific context of activities. Improved physical capacity enabled exploration more typically associated with development at this age expanding the positive impact to other developmental domains.
emphasis on active weight-bearing and proper kinematics to activate the neuromuscular system below the lesion in an age-appropriate, task-specific context of activities. Improved physical capacity enabled exploration more typically associated with development at this age expanding the positive impact to other developmental domains.
INTRODUCTION
Rapid musculoskeletal growth and development characterize the first year of human life. Spinal cord injury (SCI), whether neonatal or in infancy, halts typical development. Paresis and paralysis of trunk and limb muscles results in the inability to move, explore, and learn via interactions with the environment. Traditional physical rehabilitation in young children is typically 1 − 2x/week. With paralysis assumed to be permanent (1,2), therapists apply prone positioning targeting head control, promote the developmental sequence, and compensate for trunk and limb paralysis by focusing on muscles above the injury recommending equipment (e.g., braces, stander) to achieve functional sitting, standing, and mobility. With spasticity resulting from upper motor neuron lesions, physicians may introduce botulinum toxin (Botox) as an antispasticity medication. With SCI and paralysis, the inability to move decreases and alters the sensorimotor experiences of a child essential to development. This is compounded when using braces, standers, and medications, further restricting mobility, and altering the sensorimotor experience.
Activity-based restorative therapies (ABRT), e.g., activitybased locomotor training (AB-LT) (3)(4)(5)(6), neuromuscular electrical stimulation (7,8), transcutaneous spinal stimulation (9,10), in comparison, target activation of the neuromuscular system below the lesion. During ABRT, emphasis is on facilitating task-specific kinematics during repetitive training, 5 days/week. Neither anti-spasticity medications nor trunk or limb braces are used to afford apt sensory input during training in the clinic and home/community. During the delivery of ABRT with children, therapists use age-appropriate play as a driving and meaningful context for the child. Thus, play is an essential tool capitalizing on the child's inherent motivation and intent to move necessary to engage them and achieve therapeutic goals (11)(12)(13). We report the case of an infant with a cervical SCI presumed to have occurred in utero or at the time of birth who received traditional therapies (8 days to 15 months of age), followed by an intensive and on-going course of ABRT (reporting on 15-24 months of age) with the perspective that "experience" guided by activity-dependent plasticity matters to advance and promote a more typical course of development.
Only one other case report has been published to date describing rehabilitation of an infant with an intra-uterine SCI. The child, however, was treated more acutely within the first year following injury with a combination of ABRT and compensation strategies (14). Pape (15) also provides a brief description of rehabilitation of an infant with a cervical SCI. The child's injury, however, occurred during delivery and treatment began at 3 years old with a therapeutic regime focused on neural activation below the injury using neuromuscular electrical stimulation. With neonatal SCI being rare (15), and only one other case study describing rehabilitation beginning in the acute stage, this current study follows the case of a child with a neonatal SCI who received ABRT in the chronic phase. This case thus adds to the limited available literature to guide clinical decision making.
CASE DESCRIPTION
A male child was born at 33 weeks gestation following a pregnancy complicated by premature spontaneous rupture of membranes at 29 weeks and bicornate uterus. Mother was hospitalized on bedrest until spontaneous onset of labor at 33 weeks gestation. Delivery was by non-instrumented vaginal delivery in the cephalic position. He was admitted to the neonatal intensive care unit (NICU) at birth for management of respiratory distress and prematurity. He required positive pressure support for 3 days. He received inpatient occupational therapy for treatment of congenital positional plagiocephaly, torticollis, and mild positional bilateral foot deformities (Figure 1). He was discharged home at 3 weeks feeding orally on demand. He presented to neurology clinic at 4 months old for evaluation of hypotonia and motor delays with persisting torticollis and plagiocephaly. Mild feeding difficulties were reported without signs or symptoms of aspiration. There were no respiratory symptoms. Initial neurological examination was notable for axial hypotonia, poor head control, proximal and distal weakness of all extremities affecting arms more than legs, bilateral thumb-in-palm posture, normal deep tendon reflexes, and upgoing toes. Normal diagnostic studies at age 5 months included brain MRI, chromosomal microarray with limited highresolution chromosomes, serum creatine kinase, and thyroid studies. Initial spine MRI without contrast at age 5 months identified small foci of T2 hypointense signal at the C1 and C2/3 levels as well as possibly at the T11 level consistent with small hemorrhagic foci (Figures 2A,B). Intervening mild volume loss within the spinal cord at the C1/2 level associated mild T2 hyperintensity was also seen ( Figure 2C). Repeat spine MRI with and without contrast at ages 6 months and 1 year showed stable findings. Postcontrast images demonstrated no abnormal enhancement. The possibility of a small vascular malformation was considered and dismissed based on follow up imaging. The presence of chronic microhemorrhage and cord atrophy were most consistent with a remote SCI. In the absence of a known traumatic mechanism, the injury was presumed to occur in utero or around the time of birth. The patient's neurological examination evolved as expected over the first year of life with development of spasticity and hyperreflexia with clonus in extremities. Bowel and bladder function remained within normal limits for age. From 4 to 15 months, he received therapies focused on head control in prone, developmental sequence, fine motor control, and oral motor control. Medical record review provides insight into interventions and positions the patient was placed in during usual occupational and physical therapy care. The developmental sequence was used for positioning during therapeutic activities, as well as an abdominal binder where trunk control was lacking. In summary, the patient was primarily treated in prone, quadruped, supine or facilitated sitting with an abdominal binder. There was no indication of the specific methodology used during intervention but, the focus at that time was reported as bed mobility, weightbearing on arms in a supported quadruped position, and sitting balance/posture with an abdominal binder. Physical therapy goals included: demonstration of midline posture of trunk and head with assist of abdominal binder and minimal assist by provider for 3 mins, demonstrate independent symmetrical load through upper extremities in prone with head upright for 10 s, demonstrate alternating weight shift and upper extremity reach in prone position with head right to midline with minimal assist, independent roll back to tummy with head righting. Occupational therapy goals included: sitting with minimal support while reaching outside of base of support to place items in open container, sit with moderate assist and activate cause and effect toy on 2/3 trials, will imitate play and clap/bang toys together in midline on 2/3 trials, will oppose thumb and fingers to pick up small crackers with minimal assist.
Medical management included recommendations for Botox injections to gastrocnemius muscles, ankle foot orthotics (AFOs), abdominal binder use, wrist splint use and a standing frame. The family consistently used the hand splints for the infant but use of the AFOs was inconsistent due to severely increased plantar flexion posturing. A standing frame was used in the home with trunk, pelvic, lower extremity (LE) and foot supports.
He presented to an outpatient, pediatric ABRT program for evaluation and treatment at 15 months old. His torticollis and feeding concerns had resolved. At initial evaluation, the child was unable to sit (with or without upper extremity support), grasp objects appropriately, complete full shoulder flexion, pull to stand, stand, or walk. His initial Segmental Assessment of Trunk Control (SATCo) (16,17) score was 8/20, meaning that with external support at the inferior angle of the scapula and pelvis, he could maintain trunk alignment above the support in response to trunk perturbations at the sternum. He exhibited an excessive poster pelvic tilt and thoracolumbar kyphosis in shortsitting with support needed to prevent falling. Vertical pelvic alignment could be achieved with manual facilitation.
He demonstrated hyper reflexive patellar deep tendon reflexes and a positive Babinski, bilaterally. Clonus was observed, though specific testing did not elicit the response. With manual support, he could "weight-bear" through LEs exhibiting both excessive genu recurvatum and plantar flexion dominated by extensor tone. With support at the axillae, he initiated steps with plantar flexion posturing (no dorsiflexion) and LE scissoring (i.e., one LE crossing across over the other). Upper extremity (UE) posturing was predominately in wrist and finger flexion with ulnar deviation and avoidance of grasp of objects. Volitional finger extension through partial range was observed, however, the index finger remained relatively flexed bilaterally. He could not flex his shoulder above nipple line and would not weight-bear through an extended wrist. At 15 months of age, the Bayley-III Scales of Infant and Toddler Development (18) revealed gross and fine motor scores consistent with a 4-month-old.
METHODS
The child initiated and is enrolled in an outpatient ABRT program (3,4,6,(19)(20)(21). Therapy is provided 5 day/week: 1.5 h of activity-based locomotor training (ABLT), 1 h on the treadmill and 30 mins of overground and community integration, and 1 h of activity-based occupational therapy (ABOT). During ABLT (5), the treadmill portion consisted of manually-facilitated stepping and standing emphasizing locomotor-and task-specific kinematics with the patient partially unweighted in a body weight support (BWS) system and harness. Treadmill speed was at 1.0 mph or below while BWS was assessed daily and set to lowest amount of support while maintaining kinematics. Manual facilitation and a circumferential strap with the harness were used initially at low ribs to provide support. Ageappropriate play and rhythmic songs were integrated to maintain patient engagement to stepping task while encouraging active participation of stepping and arm swing. Standing activities focused on skill recovery or acquisition in an area most lagging or rate-limiting. Examples include reaching overhead encouraging trunk extension and shoulder extension with appropriate digit extension, squat-to-stands, single leg standing, marching. Treatment off the treadmill (overground), capitalized on the patient's activated neuromuscular system during sitting, standing, age-appropriate transitions (i.e., squat-to-stands, sit-tostands), and walking, encouraging self-initiated movements, and typical task kinematics. Caregiver education targeted integration of principles into the home/community where parental choices of positions and activities supported clinical gains for greater practice and repetition. Progression of recommendations was an evolutionary process and mirrored the patient's habilitation.
Activity-based occupational therapy sessions immediately followed ABLT daily and further focused on appropriate kinematics specifically of trunk and UEs but with a holistic, full body approach during sitting, standing and transitional tasks. Therapeutic activities focused on facilitating wrist and digit kinematics during age-appropriate play, performing "pullto-stands, " active use of arms/hands, employing varied objects to facilitate task-specific kinematics for pinch and age-appropriate grasps, and encouraged weight bearing through an extended wrist. Trunk control was challenged and progressed simultaneous to all UE focused tasks. Formal re-evaluations occurred approximately every 20 sessions in both PT and OT.
RESULTS
Findings are reported through session 177 and 25 months of age as therapy continues. The child rapidly adjusted to the therapy schedule and attended ABRT daily (5x/week) with 95% adherence/attendance rate to scheduled therapy sessions (2% absences, i.e., 4/177 due to illness and 3% absences, i.e., 5/177 due to weather, transportation and medical appointments). There were no adverse events during the period. An orthopedic evaluation was sought due to atypical forefoot positioning (R > L), to confirm development of hip sockets without dysplasia, and to provide a baseline of spine alignment.
Trunk Control Progression
Trunk control improved to SATCo 20/20 (reactive control with no support), sitting independently without UE support for >2 mins, and modified functional reach (22) assessed reaching forward 6.5 , left/right 2.5 . The child now sits with a vertical pelvis, upright extended trunk, and can lift his arms overhead to hold a ball (Figures 3A-C). He can sit in modified ring/long sitting without UE support with posteriorly tilted pelvis and kyphotic trunk posture for 2 mins. While tailor sitting (legs flexed and crossed), he can achieve a vertical pelvis with fully upright trunk. Initially, the patient required harness strapping and overpressure at his trunk to achieve an upright posture in the treadmill environment. Indicative of a change in trunk control, the trunk strap was removed and he was able to maintain his upright posture (Figures 3D-F). In the first 20 sessions, the patient improved to prop-sit 30 s-1 min. At the 160-session evaluation, the patient could independently sit on a bench and play using bilateral UEs for an entire 60-min ABOT session.
Upright Standing and Sit-to-Stand Progression
Timed stand with a pediatric posterior walker improved from unable to stand independently with device to >2 mins. In this position, he demonstrates genu recurvatum and variable foot alignment from foot flat to plantar flexion to supination/pronation. He can stand with his knees in neutral alignment to minimal flexion and feet flat on the ground, though inconsistently. During standing, he can voluntarily initiate knee flexion from a hyperextended position to a more neutral knee position. He can complete a pull-to-stand independently with inappropriate LE kinematics, specifically adduction and internal rotation of the right hip and genu recurvatum bilaterally. From an elevated bench, he can initiate sit-to-stand with weight shift forward over his feet requiring assistance at the LEs to transition fully to stance.
Self-Mobility Progression
Prior to achieving walking, a modified tricycle was introduced, session 90, as a means of community mobility. The patient progressed from being unable to step with an assistive device and requiring trunk assist at axillae, to taking reciprocal steps while in a walker with harness and BWS with assist for walker propulsion, to independent ambulation using a reverse rolling walker with sling seat and pelvic guide for safety. He can navigate obstacles, turns and inclines. At session 177, gait speed was recorded at 0.8 m/s using the 10-m walk test (23) and gait endurance was 42.8 m during the 2-min walk test (24). He continues to demonstrate genu recurvatum, however, with improved foot placement (decreased scissoring) and increased step length (Figure 4).
Home Recommendation Progression
The child's stander was modified to challenge trunk control, decreasing the height of trunk support initially and over time completely removing the trunk strap and having only pelvic, LE and foot straps. A bench was recommended so the patient could practice short sitting during play and snacks. Notably, a tricycle was introduced as a means of independent mobility emphasizing reciprocal LE engagement. Once appropriate, a recommendation was made for walking in a walker with overhead suspension. This progressed to independent use of a posterior rolling walker with pelvic assist.
Upper Limb and Hand Manipulation Progression
The patient improved in-hand manipulation from grasping a 1 block with inappropriate kinematics to grasping the block with appropriate kinematics, specifically of first-and second-digit extension (Figures 5A,B). The patient improved overhead reach in bilateral UEs: able to grasp a half pound weight, fully flex elbow, pronate forearm, flex shoulder overhead with appropriate kinematics except for wrist and fingers in his right UE and complete movement fully with appropriate kinematics on the left (Figures 5C,D). Weight bearing has improved to palmar weight bearing with independent elbow, wrist, and digit extension. At initial evaluation, the patient's mother shared concerns if her son would be able to feed himself as he was only able to "fingerfeed" with inappropriate grasp and lack of coordination. He now consistently utilizes a mature pincer and pad-to-pad grasp for self-feeding and has fed himself an entire meal with a fork. He can independently hold a cup with a straw with sufficient strength to maintain grasp when the cup is full. Initially, the patient reacted to different sensations with tactile defensiveness. Hypersensitivity and avoidance were present throughout messy play experiences such as playdough, finger paint, water play, and food play. The patient now engages and enjoys play throughout sessions utilizing bilateral UEs to engage with various textures throughout tactile play. At the 160th session, the patient demonstrated the ability to perform the following grasps: pad to pad, mature pincer, gross, cylindrical, digital pronated for a writing instrument, and isolated 2nd digit extension. The patient engages in bilateral UE play reaching forward, lateral, and overhead inside and outside base of support with supervision for safety during reaching at the limits of stability. The patient can extend digits to grasp small, medium, and large sized objects with intermittent assist (less than 25% of the time) to correct 2nd digit kinematics for full extension. The patient follows verbal cues to address 2nd digit extension approximately 50% of the time.
Play Assessment
We report on changes in self-selected play at the 20th and 160th session evaluation, adapted from the PACMI (25). Observation of self-initiated play at 20 sessions revealed the patient restricted to two positions for play: (1) manually facilitated long-sitting with trunk kyphosis and bilateral UE support and (2) prone on forearms. At the 160th session, the patient exhibited a wider range of positions for play and freely transitioned from sitting to prone on forearms to side lying. Improved second finger digit extension was noted at the 160th session, with fewer times using inappropriate flexion.
Musculoskeletal Assessment
At 17 months, X-rays revealed no scoliosis, hips in socket, and normal range at hips, knees, and ankles with metatarsus primus varus of the left foot. Orthopedic follow-up at 23 months showed no change except improved forefoot positioning.
Developmental Assessment
Bayley Scales of Infant and Toddler Development (Bayley-lll) (18) was utilized to assess developmental changes across ABRT. Communication (receptive and expressive) and Motor (fine and gross) domains were measured with conversion to both standard score and developmental age. Standard scores across gross motor abilities remained stable between time point 1 (initial evaluation) and 2 (∼session 150). While fine motor abilities significantly improved [SS (standard score) = 55 at time point 1, SS = 75 at time point 2], all standard scores remained well below expected levels. Analysis at the standard score level does not capture the significant changes in development noted in therapy, at home, and on assessment. Across the 9 months of intervention, gross motor abilities improved by 11 months, 10 days. Instead of using the term "going to therapy, " the patient spontaneously now reports, "going to go walking" and "I walk" referring to treadmill stepping during AB-LT sessions. Fine motor abilities improved almost 3 months, nearly doubling his developmental gains in this domain across 9 months of intervention in comparison to only 4 months, 10 days of development across his first 15 months of life. His mother noted on interview, "he insists on getting things for himself now." Of the many developmental changes noted in the patient, the organic spilling of developmental gains across domains was increasingly noted. With his newfound motor skills, the patient demonstrated rapid increases in social and emotional communication. Initially described by his parent as a "reserved" child who "needed to get comfortable with new people", he now would "walk down the hall" initiating interaction with unknown others, saying "hello and bye", turning his head and maintaining gaze until eye contact was reciprocated.
Play and playfulness were reported for the "first time" in the child's life. After 3 months of ABRT his mom reported, "His personality I feel like is developing a little bit more now too, just based on the fact that he can go get the things that he wants, and he can play with my daughter a little bit more. Like they can play together while I'm you know, making dinner or something and she can interact or engage with him a little bit more, because he can move."
DISCUSSION
This is the only known and detailed case report that demonstrates improvements in trunk control, ambulation and upper extremity capacity in a child with a SCI occurring in utero or at birth with standardized ABRT begun after 1 year since injury (i.e., a chronic stage post-SCI). At 1-year post-SCI, there is little expectation of improvements with interventions due to the chronicity of injury. In addition, in the absence of a known traumatic mechanism, the injury in this case was presumed to occur in utero or around the time of birth. Neonatal spinal cord injuries are most often associated with difficult or traumatic deliveries and are rarely reported following uncomplicated delivery in the cephalic position (26,27). Complicated deliveries that result in SCI are also at a high risk for death and if survival occurs, it is with significant physical impairments and paralysis (27). Furthermore, this infant was not diagnosed with a SCI until 5 months of age. The parents initiated further medical examination due to their perception that development was not normal and "something was not right." An MRI was critical to the diagnosis. This case highlights the unique context of rehabilitation with (1) development interrupted and delayed due to neonatal, cervical SCI with delivery in the cephalic position, (2) on-going maturation, and (3) delivery of neuro-therapeutic interventions beginning 1-year post-SCI. The child's subsequent gains in neuromuscular capacity afforded new interactions with his environment and thus has propelled development forward across multiple domains.
Initial standardized assessments at 15 months of age revealed impairments associated with the patient's injury restricted his gross and fine motor development. The Bayley-III, clinical objective findings, and parent report, reveal acceleration of developmental gains across domains during the period of ABRT. These gains included physical goals related to the intervention as well as a broader range of impact within the developmental spectrum. In comparison to traditional rehabilitation in which medication and compensation are the therapeutic strategies to meet immediate functional goals, ABRT provided rich sensorimotor experiences during repetitive and progressive practice based on the concepts of activity-dependent plasticity and the role of the spinal cord in controlling movements (20,28). In this context, "activity drives plasticity and plasticity (circuitry modification) drives the behavior" (21). Acknowledging that development and maturation are not linear, ABRT converged with the typical maturation experience and enabled this patient with delayed motor development to physically explore his world via ambulation, tricycle pedaling, sitting upright independence, and fine motor manipulation providing a rich context for development (12,13). Consistent with development, the repetitive practice in ABRT mimics the extensive number of repetitions children use as they develop new motor skills and learn how to adapt to their environment (29).
Ordinarily, a child would demonstrate a 9-month change in developmental abilities across the initial 4-15 months of life and another 9-month change in abilities during the following 15-24 month time period. When this patient arrived for initial evaluation at 15 months of age, he was unable to complete anticipated developmental milestones such as sitting independently (typically develops between 5 and 8 months of age), cruising (typically develops between 7 and 12 months), standing (typically develops between 9 and 14 months), or walking (typically develops between 11 and 15 months) and already experiencing significant developmental delay. The maturation that took place and the therapies he was receiving during that initial 9-month period did not result in a positive change toward typical development whereby he was able to perform age-appropriate motor tasks. ABRT may facilitate a resolution of neuromuscular incapacity that occurs secondary to SCI as the timing of an injury and initiation of ABRT may have a strong impact on the developmental landscape (30). Further, the improved neuromuscular capacity positively interacted with developmental delay and maturation. Maturation may certainly be a contributing component to change and improvement, but we suggest that maturation requires guidance by experience-dependent therapies. SCI, paralysis, and the associated impairments and inabilities cannot be resolved solely by maturation. For a child with a neonatal-onset SCI, experience and the type of experience likely matters to positively advance habilitation in the context of and interaction with development delayed by injury, physical limitations, and on-going maturation. The relevance of sensorimotor experience and deprivation for development has been detailed in the scientific literature of neglect highlighting the impact of neuromuscular disorders. Disorders of the neuromuscular system resulting in immobility and limiting exploration impede cognitive development (12,13,31,32). Within this setting, ABRT may more effectively align to promote the typical developmental experience (e.g., facilitated, active standing and stepping on a treadmill vs. passive standing in a stander) of a child with SCI, thus, enabling development across multiple domains (12,30).
The patient's gains in motor capacities and control reverberated across developmental domains with self-initiated "classic" and complex skill demonstrations observed and reported by the family across language, social/emotional, adaptive, and play domains. The patient's gains across developmental domains were observed across environments including therapy, home, and within the community. Finally, developmental gains consistent with developmental theory were not coached, practiced, or suggested, rather they were the natural outcroppings of the increased capacity and ability that had been created via the ABRT intervention and its delivery in an age-appropriate context of play. The patient's awareness of his new-found abilities became apparent where he would state, "I do" or "me do." He would become increasingly frustrated when therapists or his parents would attempt to assist him with an activity that he deemed himself independent.
While group comparisons and the randomized clinical trial (RCT) are viewed as the gold standard for scientific evidence, the rarity of cervical SCI whether neonatal or presented with cephalic delivery challenges the possibility of conducting RCTs in this population. Group settings and comparisons are highly unlikely (15) as the incidence of children with SCI either in utero or during cephalic births is extremely rare (27) relative to the already low percentage of pediatriconset SCI (3-5% injured when <15 years of age) occurring within the approximate 10-11,000 injured individuals per year (33). Lesion heterogeneity, presentation variability, and multiple geographical sites of birth makes group comparisons impossible and creates significant risks to the validity of group research. A direct comparison between therapeutic approaches via group comparisons is not feasible. A comparison of sequential findings relative to the timing and introduction of different therapeutic approaches is complicated by developmental delay, as well as ongoing developmental maturation, yet similar to single-subject design with interventions separated by washout periods. The rate of change in this child's neuromuscular capacity relative to the shift in therapeutic approaches and the onset and intensity of ABRT provides at minimum face validity to its positive effect for this individual. Case reports or single subject design studies are thus viewed as appropriate for providing evidence to guide clinical-decision making (15) especially when standardized and well-described therapies (3,5,20,34), and psychometrically-proven pediatric outcome measures (17,18,22,24,35) add to the validity of the intervention, clinical data and potential for future implementation.
While significant gains have been made by this child across the 9-month period of ABRT, therapeutic goals remain to reduce UE support during walking (i.e., progression or even elimination of an assistive device). While functional mobility remains a goal, how this child walks and his neuromuscular capacity to control his body without assistance of a walker or braces using more typical kinematics is paramount to expanding his options for continued exploration for participation as he ages. On-going ABRT will target such gains, as well as refined and consistent patterns of UE movement for successful manipulation with selffeeding, use of utensils, and manipulation of objects for play or writing. Physical and occupational therapists continue to reevaluate progress, adjusting therapeutic goals to maximize the child's neuromuscular capacity and developmental potential via ABRT. For infants injured due to a SCI and paralysis, early delivery of intensive ABRT may be a beneficial intervention to change and accelerate the trajectory of outcomes across domains (e.g., cognitive, motor, perception) during this pivotal period of growth and development.
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.
ETHICS STATEMENT
The studies involving human participants were reviewed and approved by University of Louisville Institutional Review Board. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.
AUTHOR CONTRIBUTIONS
MG-R oversaw the physical therapy clinical data collection and drafted the manuscript. KN and DS oversaw the occupational therapy clinical data collects and provided the manuscript edits. MC oversaw developmental psychology care and provided related input for this manuscript. KB provided input related to qualitative interviews and editing of the final draft. NWD oversaw neurology care prior to ABRT, provided medical diagnostic imaging, and provided the manuscript edits. AB oversaw all clinical and research interventions for this child and drafted and edited the manuscript. All authors contributed to the article and approved the submitted version.
FUNDING
This study was funded by the Kosair Charities Center for Pediatric NeuroRecovery, University of Louisville was provided by Kosair Charities (OGMB141540). The caregiver interviews were supported by a grant from the Craig H. Neilsen Foundation (CCDB190830A). | 2022-03-30T14:00:10.025Z | 2022-03-29T00:00:00.000 | {
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221137061 | pes2o/s2orc | v3-fos-license | A narrative review on trans-nasal pulmonary aerosol delivery
The use of trans-nasal pulmonary aerosol delivery via high-flow nasal cannula (HFNC) has expanded in recent years. However, various factors influencing aerosol delivery in this setting have not been precisely defined, and no consensus has emerged regarding the optimal techniques for aerosol delivery with HFNC. Based on a comprehensive literature search, we reviewed studies that assessed trans-nasal pulmonary aerosol delivery with HFNC by in vitro experiments, and in vivo, by radiolabeled, pharmacokinetic and pharmacodynamic studies. In these investigations, the type of nebulizer employed and its placement, carrier gas, the relationship between gas flow and patient’s inspiratory flow, aerosol delivery strategies (intermittent unit dose vs continuous administration by infusion pump), and open vs closed mouth breathing influenced aerosol delivery. The objective of this review was to provide rational recommendations for optimizing aerosol delivery with HFNC in various clinical settings.
Introduction
In severely hypoxemic patients, supplemental oxygen is routinely administered by high-flow nasal cannula (HFNC). HFNC is superior to conventional oxygen therapy in improving oxygenation and ultimately for avoiding intubation/ reintubation in acutely ill patients [1][2][3][4][5]. Trans-nasal pulmonary aerosol delivery by HFNC combines the benefits of both HFNC and aerosol therapy [6,7]. Since 2008, in vitro and in vivo studies have explored factors influencing delivery of aerosol with HFNC and the clinical effectiveness of this route of administration. In this article, we review the available evidence and provide a scientific basis for optimizing aerosol delivery with HFNC in various clinical settings.
Literature search strategy and results
A search of the published English literature was conducted in PubMed, Medline, and Scopus until February of 2020, using the following keywords: ("high-flow nasal cannul*" OR "high flow cannul*" OR "high flow oxygen therapy" OR "high flow oxygen" OR "high flow therapy" OR "HFNC" OR "trans-nasal") AND ("aerosol" OR "nebuliz*" OR "inhal*"). Publication types included in vitro/ bench studies, scintigraphy studies for animal or healthy volunteers, clinical retrospective and prospective studies, randomized controlled trials, and questionnaire surveys. In total, databases identified 620 records and 42 original studies investigating aerosol delivery with HFNC were finally included. Articles were excluded for the following reasons: duplicates (153), did not investigate aerosol delivery via HFNC (415), conference abstracts (10), review articles (6), and letters (4).
Clinical evidence of trans-nasal aerosol delivery
Trans-nasal aerosol delivery is increasingly employed in the intensive care units (ICUs). A survey of pediatric units in the USA reported that 75% of respondents employed trans-nasal aerosol delivery, while the remainder discontinued HFNC and used more conventional methods for delivering aerosols [8]. While demonstrating the popularity of aerosol delivery via HFNC in children, the survey also revealed concerns about its clinical efficacy. In Table 1, we summarize current clinical evidence regarding aerosol delivery with HFNC.
Adult patients: inhaled albuterol delivery via HFNC
In 2018, Bräunlich and colleagues reported that 26 patients with stable chronic obstructive pulmonary disease (COPD), who inhaled 2.5 mg albuterol and 0.5 mg ipratropium via a small volume jet nebulizer (JN) and mouthpiece or in-line with HFNC (TNI medical AG, Wuerzburg, Germany) at a gas flow of 35 L/min, had similar bronchodilator effect (p = 0.5) [9]. Likewise, Réminiac and colleagues compared delivery of 2.5 mg albuterol with a vibrating mesh nebulizer (VMN) (Aerogen Solo, Aerogen, Ireland) via HFNC (Airvo2, Fisher & Paykel, New Zealand) versus a JN with mask in a cross-over RCT in 25 stable patients with reversible airflow obstruction and reported similar improvements in forced expiratory volume in the first second (FEV 1 ) (p = 0.11) [10]. In a crossover RCT with 12 stable COPD patients, Madney and colleagues compared systemic bioavailability of albuterol administered by JN or VMN in line with HFNC at 5 L/min. Urinary albuterol excretion at 30 min and 24 h [11]. The label dose of albuterol solution in the USA and Europe is 2.5 mg. Li and co-workers performed a doseresponse relationship study in 42 stable asthma and COPD patients with known positive responses to 400 mcg albuterol via metered dose inhaler (MDI) and spacer. The subjects inhaled escalating doubling doses via VMN and HFNC with gas flow of 15-20 L/min. The improvement of FEV 1 at the cumulative dose of 1.5 mg with VMN and HFNC was similar to that with MDI and spacer (p = 0.878) (Fig. 1) [12].
Adult patients: inhaled epoprostenol delivery via HFNC Inhaled epoprostenol, a pulmonary vasodilator, has been used off-label for several decades to treat mechanically ventilated patients with pulmonary hypertension and/or refractory hypoxemia [20,21]. In two small retrospective studies, adult patients with pulmonary hypertension and refractory hypoxemia improved oxygenation after inhaling epoprostenol via HFNC at an average gas flow of 40 L/min [13,14]. Mean pulmonary arterial pressure was reduced more effectively by titrating HFNC gas flow based on individual response to inhaled epoprostenol at the bedside compared with a constant HFNC flow [15].
Future prospective studies with larger sample size are needed to validate these findings.
Pediatric patients: inhaled albuterol delivery via HFNC
In 2015, Morgan and colleagues studied five infants with acute bronchiolitis and respiratory distress unresponsive to three treatments with JN via mask [16]. After inhaling albuterol via VMN and HFNC, infants appeared markedly more comfortable, suggesting that albuterol administration with HFNC was beneficial. An observed increase in heart rate probably reflected delivery of a higher albuterol dose via VMN and HFNC. Likewise, in children receiving albuterol by VMN via HFNC with flow at 2-4 L/min or via face mask, the heart rates increased by 10 beats/min after inhaling albuterol via HFNC (p < 0.001 vs mask) [18]. In a cross-over RCT in 6 infants with bronchiolitis, albuterol delivery via VMN with HFNC (~8 L/min) improved patients' comfort and satisfaction with treatment compared to JN and mask [17].
In a retrospective study of 39 children with status asthmaticus (10 had severe acidosis with pH < 7.30) who failed ≥ 3 treatments with nebulized albuterol via JN, intermittent boluses of albuterol delivered via [12]. In 42 bronchodilator responsive patients with asthma or COPD, FEV 1 improvement after administration of 400 mcg albuterol via MDI and spacer was higher than that after inhalation of 0.5 mg albuterol via VMN with HFNC, but similar to that observed after inhalation of cumulative doses of 1.5 mg or 3.5 mg of albuterol via VMN with HFNC. COPD, chronic obstructive pulmonary disease; MDI, metered dose inhaler; FEV 1 , forced expiratory volume in the first second; VMN, vibrating mesh nebulizer; HFNC, high-flow nasal cannula HFNC at a maximum flow of 1.0 [0.8-1.1] L/kg/min were considered as a contributor in avoiding intubation [19].
Summary
The standard bronchodilator doses delivered via HFNC at 15-35 L/min for adults and 1 L/kg/min for children generated similar clinical responses to those delivered with conventional aerosol devices. Further studies need to quantify aerosol delivery efficiency in critically ill patients.
Aerosol generator: VMN vs JN
When a JN is placed in-line with HFNC, the total gas flow in the HFNC system is greater than 6 L/min, which is the minimal flow to operate the JN. This flow requirement limits the use of JN via HFNC for infants and small children, who require HFNC flow ≤ 6 L/min. Moreover, JN integrated into a HFNC system may be contraindicated in systems that incorporate their own flow generators (e.g., Airvo 2 from Fisher & Paykel) as it alters oxygen, total flow, and pressure. In contrast, VMNs are driven by electricity with no additional gas flow required. Additionally, the residual volume of drug remaining in nebulizers is higher in JN than VMN (45% vs 3%) [41,43]. Consequently, VMN generated 2-3 times higher inhaled dose than JN via HFNC for both pediatric and adult populations (Table 2) [11,33,38,41]. For these reasons, VMNs are preferred over JNs for aerosol delivery with HFNC [8].
Aerosol carrier HFNC gas functions as the "carrier" for aerosol, so that gas flow rate, gas density, and humidity could affect aerosol delivery efficiency.
HFNC gas flow and patient's inspiratory flow
In patients receiving HFNC therapy, the total inhalation flow is a combination of the patient's inspiratory flow and HFNC gas flow. The contribution of each flow influences the efficiency of aerosol delivery. When VMN was utilized to deliver aerosol via HFNC during quiet breathing, aerosol deposition was inversely related to the gas flow (Table 3) [23,26,27,33,38,40,42,44]. Turbulence generated with higher gas flow leads to greater impaction losses of the aerosol particles ≥ 3 μm during their passage through the cannula, prongs, and upper airways, thereby reducing the dose of aerosol delivered to the patient's lower airway. Consequently, one guideline recommends reducing HFNC gas flow to 4 L/min during aerosol delivery to children [45].
In contrast, during simulated adult distressed breathing, two in vitro studies reported that inhaled aerosol dose increased when gas flow decreased from 50 to 30 L/min [26,27] and decreased when gas flow was reduced to 10 L/min [27]. Inhaled doses were higher during distressed breathing than quiet breathing with gas flows of 30 and 50 L/min [26,27], but not at 10 L/min [27]. Subsequently, Li and colleagues utilized 5 different gas flows (5-60 L/min) and 6 different adult breathing patterns in their in vitro study and reported that the ratio of HFNC flow to patient's inspiratory flow was more important than HFNC flow alone [29]. Inhaled drug dose was higher when gas flow was set below the patient's inspiratory flow compared to gas flow exceeding inspiratory flow, and plateaued when HFNC flow was set at~50% of the inspiratory flow [29]. These findings were consistent with a report in infants and children ( Fig. 2) [30] and formed the basis for a RCT to compare albuterol delivery and effective dose at 3 different gas flow settings with a HFNC in patients with COPD or asthma [46].
Currently, no commercial device provides breathby-breath measurement of patient's inspiratory flow during HFNC. However, findings on the gas flow to patient's inspiratory flow ratio [29,30] should remind clinicians to titrate gas flow settings when employing HFNC for aerosol delivery, especially for drugs such NA not available as inhaled epoprostenol that produce immediate clinical responses. In support of this recommendation, a retrospective study in patients with pulmonary hypertension and hypoxemia found that titration of gas flow at the bedside led to a better response to inhaled epoprostenol via HFNC compared with application of a constant gas flow [15].
Gas density: oxygen vs heliox
Heliox (mixture of helium and oxygen) has lower density than oxygen or air and passes through narrow circuits and airways with less turbulent flow than oxygen. Heliox is employed to reduce airway pressures and gas trapping during severe airway obstruction. A meta-analysis reported that heliox provides potential short-term benefits for children with moderate to severe croup [47]. Reducing turbulence with heliox enhances aerosol delivery with HFNC, as previously reported in bench models of mechanical ventilation [48].
In pediatric [23] and adult [27] manikins, aerosol delivery efficiency using heliox showed limited superiority over oxygen only when HFNC gas flow exceeded patient's inspiratory flow [27]. Using heliox as the carrier gas for the sole purpose of increasing aerosol therapy delivery is not cost-effective unless heliox is used to relieve dyspnea in patients with severe airway obstruction [47]. [29,30]. In adult, toddler, and infant in vitro models, as the ratio of HFNC gas flow to patient's inspiratory flow increased, the delivered dose decreased, with a steep decline in aerosol delivery when HFNC gas flow was more than 2-fold higher than the patient's inspiratory flow. Inhaled dose peaked when the HFNC gas flow was 0.1-0.5 of the patient's inspiratory flow. For illustration, data from ratios of 0.1-0. 5, 0.51-1.0, 1.01-2.0, and > 2.0 in the original studies have been combined for this graphic. HFNC, highflow nasal cannula
Dry vs heated humidified gas
In vitro and in vivo studies on mechanically ventilated patients noted that humidification reduced aerosol delivery to the lung [49,50]. Interestingly, during trans-nasal aerosol delivery with flows ≥ 30 L/min, Alcoforado and co-workers found 1-1.5 times higher inhaled dose with dry than humidified gas [42]. Clinically, patient discomfort and potential adverse effects with nasal administration of dry gas at flows greater than 6-10 L/min should be considered. Moreover, turning off the humidifier for 30 min prior to aerosol administration during mechanical ventilation did not improve delivery efficiency [51]. For these reasons, the administration of dry gas in nonhumidified circuits to deliver aerosol for prolonged periods should be discouraged in clinical practice.
Nebulizer placement: close to patient vs at the inlet of humidifier Both pediatric [25,30] and adult [26,39] in vitro studies reported that aerosol deposition with VMN placed at the inlet of humidifier was greater than with the nebulizer placed close to patient. The exception was in infants with extremely low gas flow (≤ 0.25 L/kg/min) where nebulizer placement closer to the patient was more efficient [30]. With the VMN placed farther away from the patient, "carrier" gas flow (including delivery gas flow and patient's inspiratory flow, combined with low tidal volume) was probably insufficient to transport aerosol to the patient before aerosol sedimentation occurred.
Open mouth vs closed mouth breathing
Open mouth breathing reduced inhaled dose compared to closed mouth breathing in the adult manikin during aerosol delivery with HFNC when gas flow was set higher than the patient's inspiratory flow [26]. This observation was consistent with a report by Li and co-workers in a pediatric model [37]. Interestingly, when gas flow was lower than the patient's inspiratory flow, open mouth breathing resulted in a higher inhaled dose than closed mouth breathing [37]. Perhaps aerosol carried with low gas flow collected in the nasal cavity during exhalation via mouth was drawn in with the next inhalation. In contrast, higher gas flows flushed the aerosol from the nasopharynx, thereby reducing the amount of drug available for the next inhalation [37].
Delivery technique Continuous administration using infusion pump vs unit dose
Clinically, aerosol therapy in the acute care setting involves either (1) intermittent unit dose delivery or (2) continuous aerosol delivery. For treatment of severe airway obstruction, administration of larger doses as frequently as every 15 mins over several hours is resource and labor intensive. Initially, "continuous" aerosol delivery was employed to administer high-dose short-acting bronchodilators for prolonged periods, conventionally using a large volume JN with facemask. However, noisy JN operation and cool aerosols produced by them can irritate young patients, causing them to cry during aerosol treatment, which significantly reduces the inhaled dose [52]. In contrast, in-line placement of VMN with active humidification and HFNC provides warm and humidified gas; aerosol generation is silent and significantly improves patients' comfort and tolerance [16,17].
Continuous administration of aerosol using VMN involves a pump feed to control rate and volume of dose emitted. At lower pump feed rates, duration between drops of medication reaching the mesh and producing aerosol is longer. Consequently, "continuous" administration has intermittent bursts of aerosol followed by periods of no aerosol. Li and colleagues reported that inhaled dose with unit dose delivery nebulizing continuously was higher than a similar nominal dose administered via infusion pump at low feed rate during the first 15 minutes of trans-nasal aerosol delivery, independent of gas flow settings [37]. This observation could be due to asynchrony of patient's inhalation with intermittent aerosol production when individual drops reach the mesh during lowrate infusion pump delivery.
High vs low albuterol concentration
In the same study, inhaled dose with albuterol in high concentration was greater than with low concentration whether given by unit dose or infusion pump, with exception of lower delivery with high gas flow (2 L/kg/min) during infusion pump delivery [37].
Aerosol generation: breathing synchronized vs continuous
Continuous generation of aerosol by nebulizers JN or VMN, in-line with HFNC, results in wastage to the atmosphere during the expiratory phase. Synchronized aerosol generation with patient's spontaneous breathing increases inhaled dose during both invasive [53] and noninvasive ventilation [54,55]. With a prototype breath-synchronized VMN, Li and colleagues reported inhaled dose was higher with breath-synchronized versus continuous aerosol generation when placed close to the patient with HFNC gas flow ≥ 10 L/min. However, when placed at the inlet of the humidifier, breath-synchronized VMN generated a higher inhaled dose than continuous operation only when HFNC gas flow was below 50% of patient's inspiratory flow [39]. This finding is likely explained by storage of the aerosol in the HFNC circuit during the exhalation phase. The optimal ratio of HFNC gas flow to patient's inspiratory flow that generates the highest inhaled dose depends on the balance between aerosol storage and gravitational sedimentation in the circuit.
Other aerosol delivery methods during HFNC treatment
Alternatives to placing nebulizers in-line to administer aerosol during HFNC include placing the nebulizer with mouthpiece/mask over the nasal cannula; or discontinuing HFNC to administer aerosol by conventional methods [8].
Nebulizer or MDI+spacer with vs without concurrent HFNC
Administration of conventional aerosol devices (JN, VMN, or MDI with spacer) using mask/mouthpiece during HFNC reduced inhaled dose to a level that was only 6-50% of the inhaled dose with those devices alone without concurrent HFNC [32,34], as high velocity gas from HFNC disperses aerosol away from the upper airway.
Aerosol delivery via HFNC vs conventional aerosol delivery
Aerosol delivery via HFNC at high gas flows (50 L/min for adult and 2 L/kg/min for children) generated similar inhaled dose as a JN and mask [33,37,38], but lower inhaled dose than VMN with mask [32,33]. However, at lower gas flows (0.25-0.5 L/kg/min for pediatrics), the inhaled dose via HFNC was higher than that with VMN and mask [37] and 2-3-fold higher than that with JN and mask (Table 4) [37,38].
Airway model and placement of collecting filter
During in vitro studies, aerosol deposition was lower with collecting filter placed at "trachea" level [26,32] than with filter placed distal to the nasal cannula (Table 5) [27]. This is because the anatomical volumes and structures of the upper airway serve as baffles upon which aerosol impacts, and the "exhalable" fraction of aerosol that stays in the trachea and upper airway at the end of inspiration is exhaled with the filter placed at "trachea." Thus, results from in vitro studies especially with collecting filter placed close to nasal cannula could overestimate the actual aerosol drug delivery in vivo [38].
Breathing profiles
No studies have fully characterized patients' breathing profiles during HFNC treatment. Breathing parameters in the in vitro studies do not truly reflect patients' breathing patterns, which vary breath by breath in individual and also display inter-and intra-patient variability [56].
Safety of trans-nasal aerosol on the nasal epithelium
The potential toxicity or harms of aerosol deposition in the nasopharynx during HFNC are unknown. For example, hypertonic saline, tobramycin solution, and dry air decrease ciliary beat frequency [57]. Elucidation of in vivo nasal toxicity with each drug formulation used with HFNC is necessary because many drugs are
Environmental contamination
During aerosol delivery via HFNC, aerosol leakage from the nasal cannula to the environment combines with aerosol exhaled by patients into the atmosphere. Environmental fugitive emissions decreased as HFNC gas flow increased [44], likely due to turbulence effects of the high velocity gas leading to high impactive losses of aerosol en route. Bedside clinicians should employ personal protection during trans-nasal aerosol delivery, particularly when high-risk medications are administered.
Summary
Among these in vitro studies, the ratio of HFNC gas flow to patient's inspiratory flow was critical; optimal inhaled dose was achieved when HFNC gas flow was set5 0% of inspiratory flow. VMN used with HFNC generated a higher inhaled dose than JN. VMN placed at the inlet of humidifier generated a greater inhaled dose than VMN placed closer to the patient. When using dry gas or heliox as HFNC carrier gas, compared with humidified gas or oxygen, respectively, the inhaled dose was higher only when HFNC gas flow exceeded patient's inspiratory flow. However, patient's inability to tolerate dry gas or the high cost of using heliox particularly for prolonged duration is a deterrent to their routine use. Removing HFNC to use a conventional aerosol device did not improve drug delivery, and placing a conventional aerosol device via mask/mouthpiece concurrent with HFNC reduced drug delivery.
Clinical implications and recommendations: transnasal aerosol delivery strategies for different patients Table 6 provides recommendations on trans-nasal pulmonary aerosol delivery with HFNC, to help optimize aerosol delivery concurrent with HFNC.
Asthma exacerbation
During status asthmaticus, HFNC improves work of breathing and reduces carbon dioxide retention [2,58,59]. The comfort of aerosol delivery via HFNC makes it an ideal option, particularly for young children [8,16,17]. High gas flow setting has some benefits, but it impedes aerosol delivery to the lung. Reduction in HFNC flow from 2.0 to 0.5 L/kg/min resulted in a 21% increase in patient's work of breathing [60]. However, the inhaled dose by trans-nasal aerosol delivery at the lower gas flow increased by 3-to 12-fold [30,37,38,40]. Thus, reducing the HFNC flow improves aerosol delivery at the slight risk of losing breathing support for short periods, while using unit doses could shorten the duration of treatment. Delivery of 1 mL requires 2-4 min; reducing the gas flow for such a short period should not significantly interfere with work of breathing. We caution that when HFNC flow is reduced, the patient should be closely monitored and F I O 2 increased if needed to maintain a target SpO 2 [61].
Patients with severe asthma often require larger than conventional bronchodilator doses. Multiple unit doses are delivered more frequently, requiring intensive use of staff resources. Bronchodilators could be delivered using HFNC with an infusion pump and prepared syringe of albuterol. In this scenario, a higher dose could be delivered to the lung by utilizing a relatively low gas flow and a slightly higher nominal dose compared to conventional bronchodilator aerosol delivery techniques [37].
Stable COPD and COPD exacerbation
HFNC is increasingly utilized for patients with COPD for its effects of washing out dead space and reducing work of breathing [2,62]. Long-term use of HFNC could reduce the frequency and duration of COPD exacerbations and improve patient's quality of life [63,64]. In those studies with stable COPD, HFNC flow was set at 20-25 L/min [63,64] due to the patient's low inspiratory flow demand. HFNC gas flow of 15-20 L/min with a standard dose of 2.5 mg albuterol is sufficient to elicit bronchodilator effects [12].
During exacerbation of COPD, higher than usual patient's inspiratory flow demand requires an increase of flow setting with HFNC. In 12 hypercapnic patients with COPD initially treated with noninvasive ventilation, Rittayamai and colleagues achieved similar work of In vitro pediatric [33,38] In vivo adult [11,41] Discontinue HFNC treatment to deliver conventional aerosol treatment Not recommended.
Use conventional aerosol device with concurrent HFNC Not recommended.
Open mouth breathing during trans-nasal aerosol delivery Adult in vitro [26] Pediatric in vitro [37] Use heliox to deliver aerosol via HFNC
Might be considered for pediatric patients
Adult in vitro study [27] Pediatric in vitro [23] Use dry gas to deliver aerosol via HFNC Not recommended adult in vivo [42] Using frequent unit doses or infusion pump to deliver continuous albuterol for asthma exacerbation If possible, use unit dose to deliver albuterol and decrease gas flow during nebulization; return flow to original setting when nebulization is completed.
Titrate F I O 2 to maintain SpO 2 during the periods of flow reduction.
If infusion pump has to be used, relative low gas flow and a higher nominal dose could be considered.
Pediatric in vitro [37] Stable COPD Standard dose (2.5 mg) of albuterol is sufficient to elicit bronchodilation responses with HFNC gas flow set at 15-20 L/min.
Adult in vivo [9,10,12] COPD exacerbation Standard dose (2.5 mg) of albuterol as a starting dose with HFNC flow set at 20-30 L/min is recommended during trans-nasal aerosol delivery.
Adult in vivo [9,10,12] Pulmonary hypertension without hypoxemia HFNC flow set at 5-10 L/min is recommended Adult in vivo [15] Pulmonary hypertension with refractory hypoxemia Titrating HFNC flow at bedside based on patient's response in order to determine the optimal flow for each individual patient is recommended Adult in vivo [15] HFNC high-flow nasal cannula, VMN vibrating mesh nebulizer, breathing with HFNC flow set at 30 L/min [65]. Discontinuing HFNC to use a facemask significantly increased patients' breathing efforts [66]. Therefore, HFNC should not be interrupted to use mask/mouthpiece with JN when acutely ill patients with COPD require aerosol treatments. In this setting, it is appropriate to deliver aerosol via HFNC at 20-30 L/min flow.
Pulmonary hypertension with/without hypoxemia
In patients with pulmonary hypertension, prolonged continuous inhalation of aerosolized epoprostenol with HFNC is convenient and comfortable for the patient. In the absence of concomitant hypoxemia and with low inspiratory flow demand, low gas flows with HFNC (5-10 L/min) could optimize delivery of inhaled epoprostenol to patients with pulmonary hypertension [15,27,29,42]. When patients with pulmonary hypertension have concomitant hypoxemia, higher gas flow and F I O 2 are required to improve oxygenation by avoiding air entrainment and generating some positive end-expiratory pressure [58,66]. However, high gas flows reduce trans-nasal delivery of inhaled epoprostenol with a potential loss of efficacy because there is a linear correlation of inhaled dose with improvement in oxygenation [20]. A practical solution would be to titrate HFNC gas flow at the bedside to pulmonary arterial pressure and/or oxygenation [15]. This approach could determine optimal setting for individual patient, based on immediate responses to inhaled epoprostenol.
Summary
Patients, who did not require HFNC for administering high F I O 2 and who could tolerate reduced gas flow for short periods, could be benefited by decreasing HFNC gas flow to relatively low settings, such as 15-20 L/min for stable adult patients, 20-30 L/min during COPD/ asthma exacerbation among adults, and 0.25 L/kg/min for children with asthma. Employing unit doses with high drug concentration could shorten the duration for which flow was reduced. After administration of the unit dose, HFNC gas flow should be returned to its previous setting. For patients receiving HFNC therapy mainly for relief of hypoxemia and who simultaneously require inhaled epoprostenol to improve oxygenation, HFNC gas flow should be carefully titrated at the bedside based on patients' response to both inhaled epoprostenol and gas flow.
Future directions
More clinical studies are needed to validate the in vitro findings, such as the impact of gas flow on aerosol delivery via HFNC, and the effective dose at those flow settings [46]. Clinical studies of patient safety are also warranted, particularly the potential for toxicity or harm with off-label use of medication inhaled via HFNC. The use of submicrometer droplets combined with condensational growth technology has shown significant improvements of inhaled dose during trans-nasal aerosol delivery in vitro [67], and its application in clinical practice is awaited.
Conclusion
Trans-nasal pulmonary aerosol delivery via HFNC is a promising method for continuous administration of medication for prolonged periods, especially for children. However, clinicians must consider the features and limitations of the device, and the patient's disease severity. There is increasing evidence to support clinical efficacy and safety of trans-nasal pulmonary aerosol delivery via HFNC. Prospective, well-designed studies in appropriate populations of patients are needed to establish the efficacy of this mode of aerosol administration. We provide practical recommendations for employing trans-nasal pulmonary aerosol delivery via HFNC in acutely ill patients.
Funding
Not applicable.
Availability of data and materials Not applicable.
Ethics approval and consent to participate Not applicable.
Consent for publication
Not applicable. | 2020-08-17T14:27:07.916Z | 2020-08-17T00:00:00.000 | {
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221972414 | pes2o/s2orc | v3-fos-license | Sensitivity Analysis and Impact of the Kappa‐Correction of Residual Ionospheric Biases on Radio Occultation Climatologies
Abstract A new model was recently introduced to correct for higher‐order ionospheric residual biases in radio occultation (RO) data. The model depends on the α 1 and α 2 dual‐frequency bending angle difference squared, and a factor κ, which varies with time, season, solar activity, and height, needing only the F10.7 solar radio flux index as additional background information. To date, this kappa‐correction was analyzed in simulation studies. In this study, we test it on real observed Metop‐A RO data. The goal is to improve the accuracy of monthly mean RO climate records, potentially raising the accuracy of RO data toward higher stratospheric altitudes. We performed a thorough analysis of the kappa‐correction, evaluating its ionospheric sensitivity during the solar cycle for monthly RO climatologies and comparing the kappa‐corrected RO stratospheric climatologies to three other data sets from reanalysis and passive infrared sounding. We find a clear dependence of the kappa‐correction on solar activity, geographic location, and altitude; hence, it reduces systematic errors that vary with the solar cycle. From low to high solar activity conditions, the correction can increase from values of about 0.2 K to more than 2.0 K at altitudes between 40 to 45 km. The correction shifts RO climatologies toward warmer temperatures. With respect to other data sets, however, we found it difficult to draw firm conclusions, because the biases in the other data sets appear to be at similar magnitude as the size of the kappa‐correction. Further validation with more accurate data will be useful.
Introduction
The global navigation satellite system (GNSS) radio occultation (RO) technique (Hajj et al., 2002;Kursinski et al., 1997) has become a valuable source of geophysical data over the past years. A continuous time series exists since the launch of the CHAllenging Minisatellite Payload (CHAMP) satellite mission in the year 2001 (e.g., Ao et al., 2003;Foelsche et al., 2003;Wickert et al., 2001). The data have applications in numerical weather prediction (NWP) (e.g., Cardinali, 2009;Healy & Thépaut, 2006) and also climate research and climate monitoring of the Earth's atmosphere (e.g., Ho et al., 2012;Steiner et al., 2011Steiner et al., , 2013.
RO data have high accuracy over the upper troposphere and lower stratosphere between about 5 to 35 km (e.g., Foelsche et al., 2011;Kursinski et al., 1997). Toward higher altitudes, measurement noise and the influence of the ionosphere become an increasing error source in the RO data. The ionospheric influence is usually corrected to first order by a dual-frequency linear combination of RO bending angles (Ladreiter & Kirchengast, 1996;Vorob'ev & Krasil'nikova, 1994). Nevertheless, residual ionospheric errors (RIE) remain, leading to systematic biases in the data, increasing in relevance at altitudes above about 35 km (Danzer et al., 2013;Liu et al., 2013Liu et al., , 2015Liu et al., , 2018Mannucci et al., 2011;Syndergaard, 2000). Different approaches for higher-order ionospheric corrections exist (e.g., Hoque & Jakowski, 2008;Kedar et al., 2003;Syndergaard, 2002;Vergados & Pagiatakis, 2010, having the disadvantage of the need for additional background information, such as the electron density in the vicinity of the ray path or the geomagnetic field.
second factor depends only on the dual-frequency bending angles α 1 and α 2 , capturing the ionospheric variations from profile to profile.
The aim of this work is to test the kappa-correction for climatological applications. The goal is to improve the accuracy of monthly mean climatologies, potentially raising the current limit in quality of RO data from stratospheric altitudes of about ∼35 km to altitudes of about ∼45 km. To date, the kappa-correction was tested in two simulation studies (Angling et al., 2018;Danzer et al., 2015) using the NeUoG ionosphere model (Leitinger & Kirchengast, 1997) and the NeQuick model (Nava et al., 2008), respectively. The main advantage of the kappa-correction is that it is only based on the two measurable quantities, the α 1 and α 2 bending angles, and the F 10.7 index for the solar activity. Information about the electron density or the geomagnetic field are not needed. For that reason, the kappa-correction is quick and easy to apply at bending angle level to each RO profile.
We combine the kappa-correction with the average-profile-inversion (API), where profiles are averaged in bending angle space, and the average profiles are then passed into the further processing (Danzer et al., 2014(Danzer et al., , 2018Gleisner & Healy, 2013). The API method has the advantage of reducing the influence of background in the data, since the average profiles can be used up to about 80 km. Climatological products of refractivity, density, pressure, and temperature are directly produced in the retrieval process from those average bending angle profiles, instead of obtaining individual RO profiles. In previous studies (Danzer et al., 2015;, it has been suggested to combine the kappa-approach with the API method. The reason is that the kappa-correction does not correct for errors caused by horizontal gradients or by the Earth's geomagnetic field. These errors produce noise for individual profiles, but are assumed to average out in the context of climatologies. This work is structured in the following way: First, we introduce the method and the data sets (section 2). Afterwards, we compute the kappa-correction for each individual profile and discuss filtering and smoothing steps for the RIE (section 3.1). As a next step, we perform a thorough sensitivity study of the kappa-correction through the solar cycle from the solar minimum year 2008 up to the solar maximum year 2015. We analyze the sensitivity of the correction with altitude and latitude for bending angle and temperature RO climatologies (section 3.2). In a final step, we compare RO temperature climatologies with and without the kappa-correction with three other data sets from the European Center for Medium-range Weather Forecast Re-Analysis Interim (ERA-Interim) (Dee et al., 2011), ERA5 (Hersbach et al., 2018), and the Michelson Interferometer for Passive Atmospheric Sounding (MIPAS) (Fischer et al., 2008;Wang et al., 2004) (section 3.3). We conclude with a discussion of the results in section 4.
Method
We combine two different approaches in the production of RO climatologies, with the goal to improve data quality in the stratosphere. The two methods will be introduced briefly here, mainly referring to the original work.
The key research question in this study is the impact of the kappa-correction on RO climatologies. In a first step, we apply the higher-order ionospheric correction on the individual first-order ionosphere corrected Metop-A RO bending angle profiles. The estimate of the neutral atmosphere bending angle (α c ) according to Vorob'ev and Krasil'nikova (1994) is given by where α L1 and α L2 are the L 1 and L 2 bending angles related to the frequencies f 1 and f 2 , given at impact altitude z a . Equation 1 is a correction to the first-order ionospheric effect, neglecting higher-order ionospheric residuals that depend on the GNSS signal frequency, the Earth's magnetic field, and electron number density concentration along the signal propagation (e.g., Danzer et al., 2013;Liu et al., 2013Liu et al., , 2015Liu et al., , 2018. It produces a small negative bending angle error even for a simple spherically symmetric ionosphere, where the geomagnetic term is neglected. Therefore, Healy and Culverwell (2015) propose a modification to the standard ionospheric correction, with an additional positive term to compensate for this error: The latter additional term is the kappa-correction term, consisting of a product of two factors; one expresses the dominant residual ionospheric variation from profile to profile, the other describes the weak altitudinal variation (κ factor). Note the introduction of a temporal dependance t, due to applying a solar cycle dependent correction. The main advantage of the kappa-correction is that the dominant factor depends on two measurable quantities, the difference of the L 1 and L 2 bending angles (α L1 , α L2 ) squared, which is proportional to the total electron content squared (TEC 2 ).
The factor κ(z a ,t) can be approximated by (Angling et al., 2018) κðz a ; tÞ ¼ a þ b · F 10:7 ðtÞ þ c · χðtÞ þ e · z a ; (3) depending on impact altitude z a , the daily solar activity (expressed by the F 10.7 (t) index given in solar flux units sfu, where 1 sfu =10 −22 Wm −2 Hz −1 ), local time, season, and location (comprised by the solar zenith angle χ(t), given in rad). a,b,c,e are scalars found by fitting the model to the data; see Table 1 and Angling et al. (2018). κ(z a ,t) is given in rad −1 . Note that the factor κ is negatively correlated with solar activity; that is, it is low when solar activity is high, and vice versa. In practice, a scalar factor (scal-κ) of κ∼14 rad −1 is a reasonable approximation; see Danzer et al. (2015).
In a further step, we apply the average-profile-inversion (API) to the ionosphere corrected bending angles, analyzing monthly 10°zonal-mean Metop-A climatologies (Danzer et al., 2014(Danzer et al., , 2018Gleisner & Healy, 2013). In the API method, RO climatologies are built on the bending angle level and the mean bending angle profiles are propagated through the RO retrieval. RO data are in general limited in altitude to below about 80 km and show a decreasing signal-to-noise ratio with increasing altitude. For this reason, at the bending angle level, background data (e.g., data from climatological models or from meteorological data) are usually introduced through a statistical optimization (SO) step (e.g., Gobiet & Kirchengast, 2004;Ho et al., 2009Ho et al., , 2012. RO profiles are then individually processed through the retrieval. The problem with the standard method is that different implementations of the SO and structural uncertainties increase through the retrieval chain (Ho et al., 2012;Steiner et al., 2013). The advantage of the API method is that it reduces the influence of background data and circumvents the SO step in the processing, reducing the processing bias in RO climatologies.
Data
For the analysis of the ionospheric kappa-correction, we use example data from the Metop-A satellite mission (e.g., Loiselet et al., 2000;Montenbruck et al., 2008;Von Engeln et al., 2009). The kappa-correction was calculated for each RO profile and afterwards applied to the individual bending angles. For the analysis of the functional kappa (func-κ, Equation 3), the F 10.7 solar flux data were downloaded from Natural Resources Canada (https://www.spaceweather.gc.ca/solarflux/sx-5-en.php, last access: 13 May 2020).
The RO Metop-A data were studied on a monthly basis and binned into 10°zonal-mean climatologies, computed from the solar minimum period (March 2008) up to the solar maximum period (January 2015). We computed three different RO climatology runs for the complete period: • κ equals zero: leading to the standard first-order ionospheric correction; For the validation of the data, we use the three other data sets ERA-Interim, ERA5, and MIPAS. All data sets show high quality at the stratospheric altitudes we are interested in (between about 20 to 45 km). The ERA-Interim reanalysis (Dee et al., 2011) covers the period 1979 to August 2019. It has a horizontal sampling of 79 km (TL255), and 60 vertical levels from the surface up to 10 Pa. This reanalysis uses a four-dimensional variational approach (4D-Var), based on the ECMWF NWP system implemented operationally in 2006 (Cycle 31R2). ERA-Interim generally assimilate operational satellite data, rather than reprocessed data sets so, for example, the change in the operational COSMIC GNSS-RO processing in November 2009 led to a small discontinuity in the stratospheric temperatures. The ERA5 system (Hersbach et al., 2018) is based on the NWP system implemented operationally at ECMWF in 2016 (Cycle 41R2). It has better spatial resolution, with a horizontal sampling of 31 km (TL639) and 137 vertical levels from the surface to 1 Pa. ERA5 also makes greater use of reprocessed data sets, including reprocessed Constellation Observing System for Meteorology, Ionosphere, and Climate (COSMIC) GNSS-RO data. However, both ERA-Interim and ERA5 assimilate the operational Metop-A GRAS data provided by EUMETSAT from May 2008. The MIPAS temperature information is not assimilated in either the ERA5 or ERA-Interim reanalysis.
The consistency of global atmospheric temperature reanalyses-including ERA5 and ERA-Interim-in the lower and middle stratosphere has improved since around 2006 with the assimilation of GNSS-RO Long et al., 2017;Luntama et al., 2008), but ERA-Interim is still around ∼1.5 K warmer than ERA5 globally at 1 hPa. The ERA-Interim and ERA5 data used here are monthly means of daily averages (reanalysis stream = "MODA"), computed at fixed pressure levels.
MIPAS data are available from July 2002 to April 2012 (García-Comas et al., 2012), so comparisons to Metop-A data in the later high solar activity months are not possible. A validation study of MIPAS data relative to WEGC RO OPSv5.6 data has been performed on a profile to profile basis by Innerkofler (2015). The systematic temperature difference is found to be within ±1 K up to about 40 km. Figure 1 shows solar cycle variations, expressed by the F 10.7 index as a function of time. In our analysis, we focus on the period from the minimum year 2008 up to the maximum year 2014. This time period catches the minimum to maximum dependence of RO data on solar activity. We also mark in Figure 1 four test months which catch summer and winter-low and high solar activity conditions, that is, July 2008, January 2009, January 2014, and July 2014. In later analysis, we will sometimes directly compare those test months for illustrating the two solar activity extremes in two opposite seasons.
Characteristics of the Kappa-Correction
In an initial analysis, we discuss the computation and preparation of the kappa-correction. Previous studies computed the kappa-correction using simulated data (Angling et al., 2018;Danzer et al., 2015), but now we focus on real observed RO data. In Figure 2, we plot a map of kappa values for a high and a low solar activity test day. The computed kappa values clearly decrease with increasing altitude, as well as with increasing In a next step, we calculate the complete kappa-correction term using Equation 2. The kappa-correction is correlated with solar activity, increasing with the rising solar cycle, as shown in Figure 4, where we compare a solar high (top row) and solar low (bottom row) example day. The RIE shows large fluctuations, introduced through the α L1 , α L2 bending angle difference squared; see left column of Figure 4. For that reason, an outlier correction combined with a moving average filter was applied. Outliers larger than 0.1 μrad 2 on the α L1 , α L2 difference squared were rejected. Regarding the moving average, a 10-km window on the ðα L1 ðhÞ−α L2 ðhÞÞ difference was applied. Furthermore, the kappa-correction was set to zero below the altitude of 30 km, since from this level and below the neutral bending angle magnitude is greater or equal to about 300 μrad and therefore the impact of the kappa-correction is small. The ionospheric impact on RO data, however, is large at high altitudes and strongly decreases toward the surface. At lower altitudes, the α L1 , α L2 differences show large noise due to neutral atmospheric small-scale dynamical processes, such as from atmospheric wave activity, in particular the wide spectrum of acoustic-gravity waves. Altogether we tested three different cut off heights; 25, 30, and 35 km, resulting in negligible differences on the RO climatologies. Figure 4 shows, for a high and low solar activity test day, the results before and after the filtering and smoothing steps. The outlier correction rejects only a few profiles (7/588-high solar conditions, 5/632-low solar conditions) but is still strong enough to remove the largest fluctuations without a major loss of profiles. After the filtering and smoothing step, we find for the low solar activity day already a rather smooth RIE correction for each profile. For the high solar activity day, still some profiles show larger fluctuations. On the one hand, this simply reflects the nature of the more challenging high solar condition days. On the other hand, it does not introduce a problem when the correction is applied to the bending angle profiles. The bending angles are anyhow binned and averaged for the later average-profile-inversion, which strongly mitigates the impact of the few profiles with larger fluctuations.
Sensitivity Analysis of the Kappa-Correction
In this section, we illustrate the sensitivity of the kappa-correction to the solar cycle. Figure 5 shows the impact of the correction at the bending angle and temperature levels. The kappa-correction corrects at bending angle level only for very small values at the order of about 10 −8 rad. Clearly, the values increase in the high solar activity period, and the values are also largest around the tropics. Of specific interest is the resulting impact at temperature level. First, we detect a distinct increase of the impact of the correction toward higher altitudes. Second, the impact of the correction increases with rising solar activity. And third, maximum values of the correction are found around the tropics, where we present a more detailed illustration in Figure 6. At the 30-to 35-km altitude layer, the impact is for the minimum years around −0.2 K, increasing for the rising cycle and the tropics up to −0.6 to −1.0 K. For the 40-to 45-km altitude layer, the difference can be around −2.0 K in the tropics at solar maximum conditions. The impact of the correction shows a slight but not strong shift toward the northern hemisphere. While on the northern hemisphere, the peak of the correction extends until between about 25°N to 30°N (solid red line), the peak on the southern hemisphere extends usually until 20°S (solid red line). In general, the magnitude of the kappa-correction through the solar cycle is in line with results from our previous simulation study (Danzer et al., 2015). In the simulation study, temperature errors at the tropics increased from −0.2 K up to more than −1.0 K at 35 km. Also, the study of Vergados and Pagiatakis (2011) analyzes the latitudinal, vertical, and solar variation of the second-order residual ionospheric error of RO atmospheric parameters, however, for 40 individual CHAMP profiles. Given the differences in the approach and sample size, these results appear to be reasonably consistent.
In Figure 7, we compare the temperature difference (ΔT = T − T κ ) between a low and a high solar activity month. While for the low solar activity month, the impact of the correction is less than −0.6 K below 50-km altitude, for the high activity month, it increases to about −3.5 K at the same altitude level. In Figures 5 to 7, one can observe the typical northern hemisphere summer and winter conditions, with the peak of the correction for both seasons in the northern hemisphere equatorial region. This is expected due to the combination of, on the one hand, the shift between the geographic and geomagnetic equator and, on the other hand, the equatorial ionization anomaly, leading to higher ionization levels toward the northern equatorial region (e.g., Balan et al., 2018).
Finally, we discuss scal-κ. We investigate the four months July 2008, January 2009, January 2014, and July 2014, catching winter and summer low and high solar activity conditions. Figure 8 shows the daily F 10.7 values of the four respective months. The two low solar activity months show constantly low F 10.7 values, around F 10.7 = 65 sfu. The two high activity months peak around F 10.7 = 220 sfu and show on average an activity of about F 10.7 = 160 sfu. We analyze the difference between RO temperature climatologies computed without kappa-correction, T, and with kappa-correction, T κ , that is, T−T κ = ΔT. Furthermore, we compute the correction using Equation 2; once for scal-κ with κ c ¼ 14rad −1 , and also for func-κ (κ f ) computed For the high solar activity months, the difference between the κ c and κ f corrections during hemispherical summer and within the tropics (±20°) are distinct, and these differences are more pronounced at 50-km altitude. During hemispherical winter, the two kappa-correction techniques present the same results at both altitudes studied here. The analysis of Angling et al. (2018) suggests that a functional modeling of kappa reproduces the kappa found in 25,000 simulations more accurately. It seems that a scalar kappa produces a larger second-order ionospheric correction toward higher altitudes and for northern and southern hemisphere summer season.
10.1029/2019EA000942
Earth and Space Science
Comparison to Other Data Sets
In this section, we compare the ionospheric-corrected RO temperature climatologies against modern reanalyses and observation. We use ERA-Interim, ERA5, and MIPAS monthly 10°zonal-mean climatologies. In general, it was difficult to find comparison data sets which show very high quality at stratospheric altitudes between about 25 to 40 km. We want the accuracy of the comparison data sets to be such that we can assess whether the kappa-correction improves or degrades the GNSS-RO climatologies. The three chosen data sets have reasonably high quality stratospheric data. Figures 10 and 11 show the temperature differences between RO temperature climatologies without kappa-correction (left column), and including the kappa-correction (right column). The results are given at four pressure levels, 50, 10, 5, and 1 hPa, relative to ERA-Interim, ERA5, and MIPAS.
Temperature differences relative to other data in the lower stratosphere at the 50-hPa pressure level are within ±0.2 K for the reanalyses (top panels, Figure 10). The MIPAS differences increase from the midlatitudes toward the poles slightly positive to about 0.5 to 0.7 K (left column, top panels). The larger differences in the northern winter in January 2010 reflected in all three data sets are due to a sudden stratospheric warming (SSW) (e.g., Dörnbrack et al., 2012). In general, the different high-altitude initializations of RO data have the tendency to somewhat smooth strong anomalies from about 35 km upwards (Angerer et al., 2017;Schwarz et al., 2017); that is, their magnitude can be underestimated for features such as strong SSWs. On the other hand, the reanalysis and MIPAS data sets have their own biases (García-Comas et al., 2012;Simmons et al., 2019) and may slightly overestimate anomalies. At this pressure level, the kappa-correction does not have a large impact on the climatologies (right column, top
10.1029/2019EA000942
Earth and Space Science panels). However, we still can see that negative differences slightly decrease when applying the kappacorrection; see, for example, time series at the latitude band 30°N.
At the 10-hPa pressure level (bottom panels, Figure 10), the differences increase with respect to other data sets. Negative differences can amount up to −0.5 K, positive differences relative to MIPAS data partly increase to between about 0.7 to 1.0 K. The impact of the correction on the climatologies becomes more evident (right column). Negative differences clearly decrease, but positive differences increase (see differences relative to MIPAS). This indicates that RO temperature data move toward warmer temperatures when the kappa-correction is applied. Interestingly, in the high solar activity years (starting around the year 2011), the sign changes in the differences relative to ERA-Interim (from positive to negative, left column). For ERA5, temperature differences are always negative, but slightly increase in the high solar activity years. After applying the kappa-correction, temperature differences are dominantly in the range of ±0.2 to ±0.5 K (bottom panels, right column).
The general pattern stays the same in the upper stratosphere. At the 5-hPa pressure level (top panels, Figure 11), negative differences increase relative to ERA-Interim and ERA5 to about −1 to −1.5 K. In addition, temperature biases slightly increase with rising solar cycle. In general, agreement is again better when including the higher-order ionospheric correction, moving to between about −0.5 to −1.0 K (right column). Relative to MIPAS agreement is also quite good, however; the bias shows in general a positive tendency, while ERA-Interim and ERA5 biases tend to be negative. The northern midlatitudes show, to MIPAS data, good agreement around 0.2 K. Between about 10°N to about 50°S, the bias is around 0.2 to 1.0 K.
Finally, we study the 1-hPa pressure level (bottom panels, Figure 11). The temperature differences are now strongly pronounced, showing also varying differences during the rising solar cycle. RO temperature data including the kappa-correction show again the shift toward warmer temperatures (bottom panels, right column). In the comparison with ERA-Interim, this has the consequence that obviously negative differences decrease (bottom panels, top row). However, in the comparisons to ERA5 and MIPAS, the sign of the biases flips, and positive biases become more enhanced after including the kappa-correction. The biases increase even more strongly in the high solar activity years.
Overall, the largest impact of the kappa-correction was found over the tropical belt and in geographical regions near 30°N, as expected from Figures 5 and 6. This is due to the equatorial ionization anomaly (EIA) which leads to larger ionization "bulks" northward and southward of the equator (Balan et al., 2018). In general, the EIA strongly depends on local time, having an ionization maximum in the afternoon and evening. Nevertheless, the EIA is also clearly seen in the analysis of climatologies. The impact of the EIA on the residual ionospheric error for individual RO profiles was also investigated by Liu et al. (2013Liu et al. ( , 2015, showing also the strong increase in the residual bias. With respect to the intercomparison analysis, the kappa-correction led to a warming of the RO data, resulting in a general decrease of negative differences and an increase of positive differences relative to other data sets.
Conclusions
RO data have high quality from the upper troposphere to the middle stratosphere (e.g., Steiner et al., 2019). Toward higher altitudes, the impact of errors resulting from measurement noise and ionospheric residuals increases. Usually, ionospheric errors are corrected to first order at bending angle level, leaving residual ionospheric errors of about 6-7% of the neutral value at 60 km for solar maximum conditions (e.g., . Noise and higher-order ionospheric effects are then reduced at the processing centers through different high-altitude initializations, using different background data. This study aimed to improve the quality of stratospheric climatologies. To achieve this, we combined the average-profile-inversion technique with the ionospheric kappa-correction. The study provided for the first time a thorough sensitivity analysis for the kappa-correction through the solar cycle from the solar minimum year 2008 up to the solar maximum until 2015, using Metop-A RO data.
Results show that the kappa-correction warms the climatology by about 0.2 up to 1.0 K in an middle stratosphere altitude range from 30 to 35 km, with the largest warming over the tropical belt. In the upper stratosphere altitude range from 40 to 45 km, the ionospheric correction implies to a warming of about 2.0 K under solar maximum conditions.
Earth and Space Science
Interesting in that respect is how RO data compare relative to other data sets. We used three different data sets for our analysis: ERA-Interim, ERA5, and MIPAS. One of the key challenges was to find data sets with errors sufficiently small in the stratosphere, so that we can assess the impact of the kappa-correction in the evaluation. Summarizing the intercomparison results, we found the following behavior: First, temperature biases relative to other data sets increase with increasing altitude. Second, temperature biases relative to other data sets vary also with the rising solar cycle. Third, ERA-Interim and ERA5 temperature data tend to be warmer than RO temperature data (T RO −T ERA <0). Fourth, MIPAS temperature data tend to be colder than RO temperature data (T RO −T MIPAS >0). Finally, after applying the kappa-correction to RO data, ERA-Interim and ERA5 in general tend to agree better, while agreement to MIPAS data tends to decrease.
These mixed results illustrate how challenging it can be to validate potential improvements of RO data quality in the stratosphere. This is likely to be the case for other proposed changes to GNSS-RO processing in the future. Other data sets have their own biases in the stratosphere, and higher-order ionospheric corrections are relatively small. In this context, it will be interesting to compare different approaches which correct for higher-order ionospheric residual biases. More sophisticated approaches, which need additional background information, such as, for example, information about the geomagnetic field, are especially interesting in a profile-to-profile comparison study (Liu et al., 2019). In a climatological context, we expect that differences mostly average out between more advanced approaches and the rather simple kappa-correction; at least in global or zonal-mean large-scale averages. The impact of the geomagnetic term as well as the inbound and outbound electron density asymmetries along the RO ray paths will be analyzed in follow-up investigations.
Finally, we conclude that the ionospheric kappa-correction is a very simple and operationally fast applicable approach to correct RO profiles on bending level for ionospheric residuals. The main ionospheric variation is captured by using only RO observational data itself and the daily F 10.7 index, leading to a solar activity-, local time-, geographic location-, season-, and altitude-dependent correction. We emphasize the importance of the kappa-correction method in operational applications, postprocessing climatological analyses, and the potential extension of the radio occultation profiles to higher altitudes above the middle stratosphere. We hope that the improvements reported in this study will aid the quality of future reprocessings of stratospheric climate data records from RO, for the benefit of atmospheric and climate science. | 2020-06-18T09:10:21.569Z | 2020-07-01T00:00:00.000 | {
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237922262 | pes2o/s2orc | v3-fos-license | Metal phthalocyanines: thin-film formation, microstructure, and physical properties
Metal phthalocyanines (MPcs) are an abundant class of small molecules comprising of a highly conjugated cyclic structure with a central chelated metal ion. Due to their remarkable chemical, mechanical, and thermal stability MPcs have become popular for a multitude of applications since their discovery in 1907. The potential for peripheral and axial functionalization affords structural tailoring to create bespoke MPc complexes for various next generation applications. Specifically, thin-films of MPcs have found promising utility in medical and electronic applications where the need to understand the relationship between chemical structure and the resulting thin-film properties is an important ongoing field. This review aims to compile the fundamental principles of small molecule thin-film formation by physical vapour deposition and solution processing focusing on the nucleation and growth of crystallites, thermodynamic and kinetic considerations, and effects of deposition parameters on MPc thin-films. Additionally, the structure-property relationship of MPc thin-films is examined by film microstructure, morphology and physical properties. The topics discussed in this work will elucidate the foundations of MPc thin-films and emphasize the critical need for not only molecular design of new MPcs but the role of their processing in the formation of thin-films and how this ultimately governs the performance of the resulting application.
Introduction
In the simplest form, MPcs (C 32 H 18 N 8 M) consist of four isoindole groups connected by nitrogen atoms forming an 18 pelectron ring structure, with two covalent bonds and two coordination bonds chelating a metal or metalloid center (Fig. 1). 1,2 Additionally, trivalent and tetravalent metal cations allow for the introduction of axial substituents providing an additional handle for tuning material properties. The choice of metal and the inclusion of peripheral, bay, or axial functionalization groups can strongly inuence the physical and chemical properties of MPcs facilitating specic material tailoring. The extensive delocalization of the p-electron system and the exceptional stability of MPcs has resulted in their use for a myriad of applications since their discovery in 1907 and the rst patent in 1929. 1,2 Historically, due to their vibrant blue, purple, or green colour, MPcs have been, and are still, used as commercial colourants in paints, plastics, textiles, printing inks, dyes, and even some food colouring. 3 Noncolourant applications have included catalysts, lubricants, indicators, and semiconductors, with recent interest focusing on more advanced applications. 3 The ability of MPcs to form highly ordered thin-lms coupled with their chemical and mechanical stability has led to their use as the active layer in a number of electrochemical and photo-electrochemical sensors for drug analysis and the detection of pharmaceutical products, 4 gas sensing including the detection of alcohol vapours, 5-8 cannabinoid sensing, 9 and gamma radiation sensing. 10 MPc thin-lms are also a vastly growing area of research for emerging organic electronic devices having found promising success in organic photovoltaics, 11,12 thin-lm transistors, 13,14 and light emitting diodes. 15 In this review, we focus on the formation of MPc thin-lms and their physical properties. The rst section considers how thin-lms of MPcs are formed from solid material by physical vapour deposition (PVD), highlighting the general principals of the nucleation and growth of organic small molecules, kinetic and thermodynamic considerations, and effects of deposition parameters. The second section focuses on MPc thin-lms formed from solution, with a discussion on the relevant nucleation principles and a comparison of solution deposition methods. The third section illustrates the general microstructure of MPc thin-lms with an examination of the commonly seen packing motifs, polymorphs, and lm morphologies. The fourth section focuses on specic physical properties of MPc thin-lms, mainly the optical absorption and vibrational properties which are most relevant to emerging photophysical MPc applications. Lastly, the nal section reviews some of the most relevant and promising synchrotron based X-ray techniques which can be used to characterize and study MPc thin-lms.
Physical vapour deposition
Small molecule thin-lms are commonly fabricated by PVD, where under high vacuum (10 À6 to 10 À8 torr) the solid deposit material is heated above its sublimation temperature creating a vapour which then condenses on a target substrate. Numerous PVD techniques exist that employ different heating sources/ mechanisms or different processing conditions but in all cases no vapour phase chemical reaction occurs such that thin-lms are produced strictly through physical means. As the vapour reaches the substrate, thin-lm formation proceeds through the nucleation and growth of molecules of the deposited material. [16][17][18][19] While on the substrate, the free energy of the deposited molecules is reduced from that of the vapour phase, creating a low-density distribution, randomly diffusing among surface lattice sites. [16][17][18][19] Molecules in this distribution may then diffuse across the substrate until they are lost by one of several processes (Fig. 2). The molecules may re-evaporate back into the vapour phase (desorption), nucleate to form 2D or 3D growth, aggregate into existing nucleation clusters, get captured at defect sites, or diffuse into the substrate (interdiffusion). [18][19][20][21] For perfectly at surfaces molecule capture at defect sites and interdiffusion are excluded from these possibilities, however in practice due to imperfections on the substrate these process oen occur. 16,20,21 Aer the initial formation of nucleation clusters, rearrangement to more thermodynamically stable forms can also occur. This can include mixing of different species, and shape changes caused by surface diffusion or coalescence brought on by post deposition treatments such as annealing. Thus, diffusion processes occur at several stages of thin-lm formation, including the formation, mobility, and rearrangement of nucleation clusters. 16,[19][20][21]
Thermodynamics and kinetics
Nucleation occurs in the beginning stages of phase change when a new phase forms from a prior parent phase oen as a result from a change in temperature that triggers vapourphase condensation, solidication, or solid-state phase transformations. [17][18][19] In thin-lm formation the initial nucleation stage oen dictates the resulting grain structure, lm morphology, and thin-lm properties. The principal theories of inorganic thin-lm growth can largely be used to model the nucleation behaviour of organic small molecules, however some fundamental differences do exist. Most notably, inorganic atoms are assumed to be isotropic in shape such that the orientation of the atom relative to the substrate is irrelevant, whereas many organic small molecules are highly anisotropic and thus the strength of the molecule-molecule and moleculesubstrate interaction will depend on their orientation to the substrate. 20,22,23 Additionally, inorganic lm growth relies on strong covalent or ionic bonds, whereas organic materials rely on van der Waals interactions. 22,23 For the vapour deposition of thin-lms the thermodynamic driving force for nucleation is the difference between the chemical potential of organic molecules in the vapour phase (m v ) and crystalline phase (m c ). 16,17,20,24 The Gibbs free energy change (DG) needed to form a nite-sized crystal composed of a number, j, of molecules can be described by: where the rst term (ÀjDm) describes the thermodynamic driving force, dened as the difference in chemical potentials Dm ¼ m c À m v , and the second term describes the energy associated with creating or adding to a new surface. 16,17,20,24 The term g i corresponds to the surface energy associated with surface i with an area A i . 16,17,20,24 Eqn (1) gives the macroscopic relationship in terms of free energy, between crystal size and surface energies and is a reasonable approximation of nucleation behaviour. In general, the barrier to nucleation where the surface energy effects are greatest (DG*) can be determined by setting the derivation of eqn (1) with respect to the number of molecules (j) to zero, this represents the point at which thermodynamic equilibrium is achieved.
However, due to the anisotropic nature of organic molecules, nucleation is oen governed by kinetic processes rather than thermodynamic ones. 20,22,23 Therefore, thin-lm growth is better described as a non-equilibrium kinetic process resulting in a macroscopic state that is dependent on the respective rates of the different physical processes illustrated in Fig. 2. 20,21 Atomistic theories of nucleation describe the role of individual atoms, or molecules, during the initial stages of thin-lm formation. 17,18,20,21 An important advantage of the atomistic models is that nucleation can be expressed in terms of measurable parameters such as deposition rate and substrate temperature, instead of quantities such as DG* and g i , whose values cannot be known with certainty or easily estimated. 17,20,21 By this approach, the most simplied kinetic rate equation relating the time dependent change in monomer cluster density to surface processes is given by the following: where N 1 is the monomer density, _ R is the deposition rate, s s is the length of time atoms remain on the substrate before desorption, N i is the critical concentration of clusters per unit area of size i, and K 1 is a second-order rate constant. 17 Eqn (2) states that the monomer density change with time is given by the deposition rate, minus the desorption rate, minus the rate at which two monomers combine to form a dimer, minus the loss in monomer population due to their capture by larger clusters. 17,18,20,21 This equation can be generalized further to dene the rate equation for clusters of i size: where the rst term expresses the increase in rate caused by the attachment of monomers to smaller i À 1 sized clusters, and the second term describes the decrease in rate due to formation of larger i + 1 sized clusters. 17,19 While eqn (2) and (3) are valuable in understanding the basic kinetics of nucleation, the inclusion of surface diffusion terms, coalescence, and transient and steady-state solutions offer a much more complete account of nucleation events, however increases the mathematical and physical complexity of these models greatly. More rigorous kinetic models can be found in other works. 18,20,21,23,[25][26][27]
Nucleation density
For vapour deposited materials the rate of heterogeneous nucleation, dened as the number of stable clusters that form per unit volume per unit time, is a function of the deposition rate, substrate temperature, substrate surface properties, intermolecular interactions, and molecule-surface interactions. 16,17,20,24 Greater nucleation rates typically result in ned grained thin-lm morphologies due to the large number of crystallites that nucleate on the substrate in a short period of time. 16,17 Conversely, if the nucleation rate is low large crystal growth is favoured. 16,17 In terms of the energetic contributions, the energetic barrier to diffusion (E diff ), energetic barrier to desorption (E des ), and thermodynamic barrier (DG*) are critical to heterogeneous nucleation and thin-lm growth. 16,17,20,24 Considering these energetic terms, the nucleation density (N D ) of stable clusters is given by eqn (4): where a is a constant related to the critical cluster size, k is Boltzmann's constant, T s is the substrate temperature, and E i is the crystal disintegration energy dened as the energy required to disintegrate a critical cluster containing i molecules into i separate molecules. 16,17,20,24 For systems with a low crystallization driving force, E i is approximately equal to negative the crystal formation energy which can be approximated by E i ¼ (ÀE des + E diff + DG*) for the vapour deposition of most organic small molecules. 16,17,20,24 Thus the three energetic barriers (diffusion barrier, desorption barrier and thermodynamic barrier) directly impact the nucleation density. The relationship between the energetic terms of eqn (4) and surface interactions of the substrate show that the processes illustrated in Fig. 2 (diffusion, desorption, and nucleation) are therefore a function of the interaction between the substrate and deposit material. 16,17,24
Growth modes
Thin-lm formation is generally characterized by three basic growth modes: island (Volmer-Weber), layer-by-layer (Frank-Vander Merwe), and Stranski-Krastanov (SK) growth. Island growth occurs when molecules of the deposited material are more strongly attracted to each other than to the substrate, resulting in 3D growth (Fig. 3i). [16][17][18]20,24 Layer-by-layer growth exhibits the opposite characteristics as the molecules are more strongly attracted to the substrate resulting in planar 2D sheet formation oen referred to as epitaxial growth (Fig. 3ii). [16][17][18]20,24 SK growth describes the formation of one or more complete monolayers where subsequent 2D growth is unfavourable and 3D island growth continues (Fig. 3iii). [16][17][18]20,24 Typically, organic thin-lms, such as those composed of MPcs, experience SK growth. The relationship between growth mode, surface energy of the deposited material, and the substrate can be related by eqn (5): where g s is the surface energy of the substrate, g* is the interfacial surface energy between the deposited material and substrate, g d is the surface energy of the deposited material, and q is the contact angle (Fig. 3iv). [16][17][18]20 In the case of layer-bylayer growth the deposited material wets the substrate and q z 0, therefore, g s $ g* + g d . [16][17][18]20 However, for island growth the opposite is true and q > 0, therefore, g s < g* + g d . [16][17][18]20 SK growth combines features of both island and layer-by-layer growth where initially g s > g* + g d until island formation occurs. [16][17][18]20 2.5 Effect of deposition parameters The formation of thin-lms by PVD is a complex process that can be inuenced by many factors such as material properties, deposition parameters, and environmental constraints resulting in lm microstructure ranging from the formation of single crystal, polycrystalline, to amorphous lms. From eqn (4) nucleation density is largely reliant on substrate temperature and deposition rate. Due to the Arrhenius nature of eqn (4), at elevated substrate temperatures the overall barrier to heterogeneous nucleation is reduced. 16,17,20,24 At high substrate temperatures, molecules have increased kinetic energy and are able to easily migrate to lower energy sites creating nucleation points, resulting in polycrystalline structures with large crystallites and fewer grain boundaries. 24,28,29 This phenomenon has been well documented in MPcs 28-36 which at room temperature exhibit ne grained morphologies, whereas large rod-like bers occur at increasing substrate temperatures, as exhibited by scanning electron microscopy (SEM) and atomic force microscopy (AFM) images of copper phthalocyanine (CuPc) in Fig. 4. 28,30 For fabrication of MPc thin-lms by PVD, substrate temperatures between 30-120 C are commonly used leading to morphologies with large regular crystals and minimal grain separation, which tends to be preferable for various applications. 28,31-33 At greater substrate temperatures (>200 C) the sticking coefficient of the deposited material is reduced and nucleation is limited, resulting in a sparse network of very large crystallites separated by wide gaps (Fig. 4i). 30,31,[34][35][36] At very low temperatures (<0 C) the surface mobility and diffusion are decreased such that molecules lack the energy required to nd favourable nucleation cites, and amorphous lms are formed, as illustrated by low temperature depositions of pentacene. 23,37,38 In addition to substrate temperature, deposition rate effects the nucleation density and subsequently thin-lm formation by determining the density of molecules diffusing on the surface. Increasing the deposition rate increases the rate of nucleation, as more molecules can interact to form a stable cluster in a dened area per unit time, oen leading to smaller and denser crystallites. 23,[39][40][41] Conversely, decreasing the deposition rate decreases nucleation density as this allows more time for incoming molecules to migrate to a favourable orientation prior to the arrival of additional molecules. 23,39-41 Low deposition rates typically lead to large crystallites, and fewer grain boundaries. 28,29,39,40 Similar to substrate temperature, the effects of deposition rate on the fabrication of a variety of MPc thin-lms has been extensively studied 28,39-41 with Fig. 5 displaying the effects on CuPc lms. 40 If the deposition rate is very high, growth becomes kinetically dominated and typically low crystallinity, polycrystalline, or amorphous lm formation is observed. 28 For the fabrication of MPc thin-lms deposition rates of 0.01-5Å s À1 are commonly used as they generally result in morphologies with large crystallites, favourable p-p stacking, connected grain boundaries, and greater crystallinity, which are typically desired for many solid state applications. 28,[39][40][41] Physical surface roughness and surface chemistry of the substrate can have a signicant impact on nucleation and thin-lm formation. Areas of high surface roughness decrease the barrier for heterogeneous nucleation by decreasing the diffusion distance of molecules. 16,[42][43][44][45][46][47] This results in small grain formation, enhanced defects, and oen a different molecular orientation relative to the substrate as exhibited by thin-lms of CuPc (Fig. 6i). [42][43][44] Altering the surface chemistry of the substrate through self-assembled monolayers (SAMs) is a common strategy to inuence the morphology and crystallinity of small molecule thin-lms. SAMs are highly ordered 2D structures consisting of a head group, terminal group, and linker. The head group typically has a specic affinity for a substrate which facilitates spontaneous monolayer formation. 48 The most common SAMs used in thin-lm engineering are thiols on gold, silanes on silicon oxide (SiO 2 ), and phosphonic acids on metal oxides. 48 Using SAMs to modify the substrate can affect the uniformity, morphology, packing structure, and molecular orientation of the resulting thin-lm, as seen by the growth of CuPc on bare SiO 2 versus SiO 2 treated with trichloro (octyl)silane (OTS) (Fig. 6ii). 45,48,49 As discussed, the surface energy of the substrate will greatly inuence the initial nucleation behaviour of the deposited material and determine the nal growth mode. 17,20,45,48 By using SAMs to selectively tune surface energy, island, layer-by-layer, and SK growth can be achieved using the same deposited material and fabrication conditions. 50,51 Overall PVD is an effective fabrication method, already employed in industry and used for the engineering of thin-lms with tunable molecular structures.
Solution deposition
Solution deposition of organic small molecules involves the dissolution of the deposit material into an organic solvent where it can then be deposited onto a substrate by one of four main methods: drop casting (also referred to as dispensing), spin coating, printing, and meniscus-guided coating (Fig. 7). As the solvent evaporates the solution becomes supersaturated, driving nucleation and crystal growth, to form a thin-lm. Compared to PVD, the nucleation and growth of solution deposited materials is more complex due to added solventvapour, solvent-substrate, solute-solvent, and solute-substrate interactions. 52 Additionally, control over the formation of thin-lms by solution processes is limited due to the rapid progression of nucleation, crystallization, and growth stages that can occur in a matter of seconds. 53 Drop casting and spin coating are common lab scale techniques used to deposit material on small area substrates. Drop casting involves depositing solution droplets onto a stationary substrate with controlled droplet size and momentum, where the solvent is le to slowly evaporate, leading to the formation of a thin-lm. 52 As no outside forces are applied, nucleation begins along the edge of the droplet with crystal growth occurring in the direction of the contact line recession. Drop casting can oen lead to non-uniform deposition since the recession of the contact line is typically irregular. Spin coating is a more consistent fabrication method used to create uniform thin-lms by dropping solution onto a rotating substrate which simultaneously spreads the solution by rotational forces while quickly evaporating the solvent. 52 Printing is a broad denition of different deposition techniques, however it typically refers to large area solution processing methods that do not primarily rely on directional shearinduced alignment such as meniscus-guided coating. 52 Inkjet printing is one of the most common and popular printing methods where a jet of solution is ejected from a chamber by a piezoelectric or thermal actuator and is deposited onto a substrate. 52 Similar to inkjet printing, spray coating ejects solution from a nozzle where small droplets are formed by aerosolization with an inert gas and are deposited onto the substrate. 52 Inkjet printing and spray coating are particularly useful as their compatibility with roll-to-roll manufacturing facilitates effective high throughput fabrication.
Meniscus-guided coating methods are scalable large area techniques that use the linear movement of either the substrate, or coating tool, to fabricate thin-lms with uniformly aligned crystal growth. 52,54 Dip coating, involving the vertical withdrawal of a substrate from a solution bath, blade coating, involving the use of a at rectangular edge to spread solution across a substrate, and slot die coating, involving the ow of solution through an orice and shaping device onto a horizontally moving substrate are common examples of meniscus-guided coating methods. 52,54 The alignment and size of the growing crystallites relies on the shear force directing solution ow and is largely inuenced by the speed at which movement occurs.
Thermodynamics and kinetics
When a solution is introduced onto a substrate surface, solvent evaporates, increasing the concentration of the solution until it becomes supersaturated and the dissolved molecules begin to precipitates to form a thin-lm. The formation of precisely controlled thin-lms with specic grain structures and morphologies remains a challenge for solution processing due to the rapid nucleation and growth steps. The same thermodynamic principals that describe PVD apply to solution deposition such that the thermodynamic driving force for nucleation is the difference between the chemical potential of organic molecules in the liquid phase (m l ) and crystalline phase (m c ). 55 In the case of solution deposition, Dm corresponds to the difference between the concentration of the solution at equilibrium (C N ) and the concentration during growth (C), which can be expressed as a function of the substrate temperature (T s ): 55 Thermodynamically, C and T s are the two thermodynamic parameters that determine the nucleation and growth of crystallites during solution deposition, however, similar to PVD, solution deposition methods are largely governed by kinetic processes and rates of crystallization. 56 In the case of solution deposition, the kinetic driving force for nucleation is the rate of solvent evaporation which directly determines the rate of crystallization, and is thus key to the fabrication of consistent small molecule thin-lms. 52,57 Due to variations in the respective solution processing methods the governing principals for the rate of solvent evaporation will be method specic.
Drop casting and printing techniques use the release, impact, and spreading of one or more solution droplets that may form a continuous thin-lm before drying or may dry individually to create a thin-lm composed of many islands. Controlling the rate of solvent evaporation, and thus the nucleation and growth stages, depends solely on the solvent and substrate properties as no external rotational or shear forces are applied. [58][59][60] The solution and substrate surface properties can inuence the deposition by causing splashing, spreading, receding, and/or rebounding. [61][62][63] Additionally, temperature and concentration gradients within solution droplets can lead to coffee ring and Marangoni effects, leading to poorly controlled lm formation. 59,60 Thin-lm formation by spin coating can be accurately represented when the evaporation rate of the solvent, the viscosity increase resulting from the increase in solute concentration, the surrounding vapour phase above the substrate, and the solvent's properties are taken into account. 64,65 The simplest and earliest models describing liquid ow on a rotating surface are formulated with three main assumptions: (i) the gas and liquid phases are Newtonian uids; (ii) the uid ow is axially symmetric and laminar; and (iii) the rotating surface is at and extends innitely. 64,65 It is widely accepted that the early stages of spin coating are ow dominated while late stages are dominated by the rate of solvent evaporation. At the transition point, when evaporation and ow become equal, the evaporation rate (n e ) depends on the rotational speed (u), yielding: 64-68 This simple relationship has been observed experimentally using polymer thin-lms with only small reported variations in the exponent value. 64,65,[68][69][70][71][72] However, as solvent evaporates the physical properties of the solution change, inducing non-Newtonian behavior. More rigorous models describing the spin coating process take into account heat and momentum transfer by including the effects of solution viscosity and solvent volatility. 65,70,71 The two stage ow dominated and evaporation dominated process of spin coating has been corroborated with experimental data from spin coated small molecule thin-lms by in situ grazing-incidence wide-angle X-ray scattering (GIWAXS). 73,74 These experiments show how the rapid ow dominated crystallization stage, which occurs over a sub-second time scale, is independent of the rotational speed, and the slower more gradual evaporation dominated stage is rotation speed dependant. 73,74 Therefore, the rate of solvent evaporation during spin coating can be described by eqn (7). Meniscus-guided coating methods depend mainly on solvent properties and coating speed. Solvent evaporation is dominate in the meniscus region leading to supersaturation, precipitation, and ultimately to nucleation. However, most meniscusguided methods use an external shear force to enhance thin-lm uniformity and crystallite alignment. The intensity of this force, determined by the coating speed (n c ), can be divided into two categories: fast coating speeds (n c z 1 mm s À1 ) and slow coating speeds (n c z 1-100 mm s À1 ). Fast coating speeds where solution is spread out by shear forces and solvent evaporation is separated from the meniscus region is known as the Landau-Levich-Derjaguin (LLD) deposition regime where solvent evaporation is a function of n c . [75][76][77] At slow coating speeds deposition corresponds to the evaporation regime where n c is approximately equal to n e of a pinned drop of solution that is receding primarily due to evaporative mass loss. 77 Thus, in contrast to the LLD regime where solvent evaporation is separate from thin-lm deposition, the evaporation regime is complicated by the interactions between solvent evaporation, uid ow, and lm formation. 75-77 A number of models have been purposed to describe n e most of which take on the general form of eqn (8). 54,77,78 Here V m is the molar volume of the liquid solvent, DS vap is the entropy of vapourization of the solvent, T b is the boiling point of the solvent at atmospheric pressure, and A is a single tting parameter combining all temperature independent variables. 77,78
Effect of deposition parameters
Solution deposition processes can produce wide variations in thin-lm microstructure depending on solution concentration, solvent type, substrate temperature, and substrate surface chemistry. Solution concentration inuences thin-lm coverage, such that at low concentrations low coverage submonolayer formation is observed, whereas at increasing concentrations the coverage and interconnectivity increase with the formation of mesh layers and multilayers. This phenomena has been documented in spin coated and dip coated CuPc thin-lms where, at low solution concentration, CuPc molecules form a sub-monolayer of interconnected ribbons typically 20-50 nm wide, approximately 100 nm in length, and 1 nm thick (Fig. 8). [79][80][81] As the concentration of CuPc in the deposited solution increases, multiplayer formation is observed, however complete coverage for a single layer is never achieved due to the anisotropic nature of CuPc which effects surface diffusion and subsequent nucleation. [79][80][81] Solvent choice plays an important role in the formation of thin-lms by solution deposition. As discussed, the rate of solvent evaporation directly determines the crystallization rate, dictating the nal thin-lm morphology and microstructure. Solvents with a faster rate of evaporation generally leads to lms with a greater surface roughness due to the occurrence of well separated clusters. Solvents with high evaporation rates, such as chloroform, can lead to the formation of these clusters since the rapidly evaporating solvent leaves little time for surface mobility or diffusion of the molecules on the substrate. This oen results in lower aggregation and lms with a non-coalesced morphology. Solvents with low evaporation rates, such as dimethylformamide, facilitate greater molecular mobility on the surface due to the longer evaporation time and oen results in a more highly packed and ordered lm. This has been demonstrate with tetrakis-(isopropoxy-carbonyl)-copper phthalocyanine (TIP-CuPc) 82 and a number of other semiconducting small molecules. 58,[83][84][85] The choice of solution processing method will have signicant inuence on thin-lm microstructure. A recent study by Gojzewski et al., exhibited the differences in CuPc lm formation by drop casting, spin coating, dip coating, and spray coating (Fig. 9). 79 The authors used CuPc dissolved in tri-uoroacetic acid (TFA) that immediately spreads to cover the hydrophilic surface of SiO 2 to form a liquid lm. Upon drop casting, outward capillary ow from the center of the drop brings dissolved CuPc molecules to the edge, creating the morphology shown in Fig. 9i, a. Spin coating using the same solution produced a multi-layer formation of nanoribbons similar to that of drop casting (Fig. 9i, b), however the added rotational force increases the rate of solvent evaporation creating a rougher lm surface (Fig. 9ii). 79 Dip coating yielded similar lm characteristics (roughness, coverage and lm volume) to drop casted lms, however exhibited a unique morphology consisting of a sub-monolayer mesh-like lm made of long, asymmetrically curved and interconnected nanoribbons approximately 600 nm wide where the CuPc molecules were orientated in-plane to the substrate (Fig. 9i, c). 79 Spray coated lms displayed a similar morphology and comparable surface roughness, coverage, and lm volume to spin coated lms with large rod-like CuPc aggregates (Fig. 9i, d). 79 Due to the added rotational force during spin coating, noticeable differences in lm morphology between the two fabrication methods are expected. However, as discussed, morphology is dependent on the rate of solvent evaporation. The specic fabrication parameters used for spray coating and spin coating in this case allows for sufficient TFA evaporation to create lms of large rodlike CuPc aggregates. 79 This further corroborates the relationship between thin-lm microstructure and solvent evaporation as the driving force for the nucleation and growth of solution deposited thin-lms.
Packing motifs
The growth mode and packing structure of inorganic thin-lms is well understood by reason of the strong covalent bonds, and the inherent isotropic shape of inorganic atoms. In contrast, due to the anisotropic geometry and weak van der Waals forces of organic molecules more variable crystallite growth, molecular packing structures, thin-lm textures, and morphologies are observed. 86,87 Molecular packing can not only impact the solid state properties of organic molecules but it can also effect the thermodynamic, kinetic, mechanical, electrical, and optical properties of the nal thin-lm. 88 The identication and clas-sication of different packing structures is therefore crucial for applications in various industries including pharmaceuticals, 89 organic semiconductors, 90 pigments, 91 and explosives. 92 Conjugated aromatic small molecules have been known to form two main crystal packing structures: herringbone and p-stacked (Fig. 10). 93 The herringbone structure exhibits altering face-toedge and face-to-face molecular packing, and mainly occurs in planar MPcs such as CuPc, silicon phthalocyanine (SiPc) and zinc phthalocyanine (ZnPc), whereas the p-stacked conguration exhibits face-to-face packing and is adopted by non-planar MPcs such as titanyl phthalocyanine (TiOPc), chloro-aluminum phthalocyanine (AlClPc), and lead phthalocyanine (PbPc). 93 Polymorphism refers to the ability of molecules to form multiple distinct crystal structures. Controlling polymorphism in organic thin-lms is challenging since p-conjugated molecules typically have similar cohesive energies and a low kinetic barrier to solid-solid transformations, making polymorphs difficult to isolate and stabilize. 88 Common methods of obtaining different polymorphs in thin-lms is through varying lm thickness, temperature, surface chemistry and post deposition processes such as thermal and solvent annealing. 88 The identication of polymorphs and the differences in morphological, structural, and spectroscopic properties have been documented through electrical conductivity measurements, 94,95 optical absorption spectra, 96-99 electron paramagnetic resonance spectroscopy (EPR), 94 The polymorphic character of MPcs was rst reported by Hamm and Norman in 1948 for CuPc 102 and has since been extensively studied in a number of MPcs. 33,101,[103][104][105][106][107][108][109] MPcs are known to exists in various polymorphic forms identied as a, b, g, d, 3, and x-phases with the metastable a-phase and stable bphase being the most common and commercially signicant. 94,100,101 The phase transition from a to b occurs in most MPc thin-lms through exposure to temperature (200-300 C) 97,99,100,104 or organic solvents, 110,111 and is characterized by a change in tilt angle between planes and the degree of pelectron overlap (Fig. 11i and ii). 96,112 The stable b-phase is monoclinic in structure and forms long crystallite needles, 113 whereas the metastable a-phase has been reported to be tetragonal, 114 orthorhombic, 115 or monoclinic 94 in structure, and generally forms into spherical crystallites. As an example, Fig. 11 highlights some of the differences between aand bphase CuPc polymorphs. For both polymorphs the CuPc molecules align in the herringbone packing structure with a 65 angle between molecules and the b axis for a-phase CuPc and a 45 angle for b-phase CuPc. 96 The larger angle of a-phase CuPc results in increased p-electron overlap and is likely the reason for the higher conductivity displayed by this polymorph. 94,95 The XRD pattern of aand b-phase CuPc (Fig. 11iii) shows the distinct crystallographic differences between polymorphs. a-Phase CuPc exhibits a primarily polycrystalline structure with crystallites preferentially oriented with their (001) planes (approximately 2q ¼ 7 ) parallel to the substrate. 97,98,100 In the case of b-phase CuPc alignment in the (20À1) direction is preferred as seen by the high intensity peak at approximately 2q ¼ 9 . 97,98,100 Through Raman spectroscopy differences in the vibrational frequencies of aand b-phase CuPc are shown in Fig. 11iv. Vibrational shis between polymorphs can be observed in ve Raman bands with the largest differences exhibited by the n 52 vibration (Cu-N deformation), n 14 vibration (C-H bending of the benzene ring), and the n 28 vibration (stretching of the phthalocyanine macrocycle). 91 Differences in CuPc packing structure determine solid state properties such as conductivity, optical absorbance, and even colour which are critical for determining appropriate use in some applications. aand b-phase CuPc are oen used as organic semiconductors in electronic devices with particular interest in a-phase CuPc due to the high carrier mobility and high-frequency capacitance and conductance demonstrated by a-phase CuPc OTFTs, 116 and aphase CuPc-Si hetero-structures. 117 Additionally, in the ink industry the most widely used blue pigments are CuPc based, with a-(purple), b-(green-blue), and 3-phase (red) CuPc being the most popular in printing inks, paints, plastics, and textiles. 91,118 4.2 Thin-lm morphologies MPcs will form different surface morphologies depending on the molecular structure and corresponding packing. Fig. 12 displays AFM images of a number of MPc thin-lms deposited by PVD onto heated substrates, including planar and nonplanar structures and divalent, trivalent, and tetravalent metal inclusions. The planar divalent MPcs, such as ZnPc, CuPc, cobalt phthalocyanine (CoPc), iron phthalocyanine (FePc), and magnesium phthalocyanine (MgPc) exhibit comparable morphologies with ribbon-like grains of similar structure and shape with only small variations in grain size. 119 Typically, ribbon-like grain morphologies are observed for lms deposited on heated substrates whereas smaller more cylindrical shapes are observed at lower substrate surface temperatures. 32,33,120,121 The non-planar trivalent and tetravalent MPcs, such as AlClPc, TiOPc, PbPc, and vanadyl phthalocyanine (VOPc) have much larger rectangular plate-like features owing to their different pstacked packing structure. 119,[122][123][124][125][126] Additionally, these four MPc thin-lms have a greater surface roughness and lower surface area to volume ratio compared to the planar divalent MPc thin-lms. 119,[122][123][124][125] Unlike metal center, uorination of the outside ring (F x MPc, x ¼ 4, 8, 16) yields little effect on the morphology of MPc thin-lms as studied in peruorinated CuPc and ZnPc. 9,[127][128][129] In general the addition of uoro molecules to the outside ring slightly alters grain size however, the packing structure and grain shape remain analogous to non-uorinated MPcs. 9,[127][128][129] The packing and resulting thin-lm morphology of MPcs can also be greatly altered through the inclusion of axial substituents as demonstrated by the AFM images of R 2 -SiPc presented in Fig. 13. R 2 -SiPc thin-lms with phenoxy and uorophenoxy groups fabricated by PVD (Fig. 13ii and iii) show two distinct morphologies either consisting of small regular circular grains or more elongated rectangular grains depending on the structure of the phenoxy substituent. 130,131 Additionally, R 2 -SiPc molecules with alkyl axial substituents fabricated by solution processing (Fig. 13iv) highlight how the alkyl chain length, branching position and symmetry affect thin-lm morphology; creating lms with either small dense cylindrical grains, large interconnected grains, or very large plate-like features. 132 By changing only the axial substituent, wide variations in thin-lm morphologies are observed, where in general, it is hypothesized that large substituents alter molecular packing resulting in morphologies with sizeable features as demonstrated in R 2 -SiPcs and other conjugated small molecules. [132][133][134] In electronic devices, morphology has been shown to impact the charge carrier mobility of transistors, 24,135,136 the power conversion efficiency of solar cells, [137][138][139] and the performance of light emitting diodes. 140 Additionally, the mechanical stability of thin-lms, including the exibility and sensitivity to stress and strain, will affect the degree of reorganization in lm morphology with mechanical deformation. [141][142][143] In particular, lms with large grains and broad boundaries are more susceptible to mechanical deformation as the formation of wide interconnected cracks are more prevalent compared to lms with smaller grains and a smoother surface morphology. 141,143,144 5. Physical properties of metal phthalocyanines
Absorption properties
The unique ultraviolet-visible (UV-vis) absorption spectra of MPcs are a result of their extensively conjugated p-electron systems and the overlapping orbitals of the central metal. UV-vis spectroscopy is oen performed on liquid samples which display sharp, well dened peaks. However, solvent coordination and aggregation can result in peak shis uncharacteristic to the MPc itself. 145 Additionally, solid state UV-vis absorption includes effects of thin-lm molecular packing and crystal structures that are not visible in solution. MPcs typically display two strong absorption bands, one in the UV region of 280-350 nm known as the B (Soret) band, and the stronger, oen better resolved, band in the visible region between 550-750 nm known as the Q band (Fig. 14i). [146][147][148] For most planar MPcs, the B band displays three peaks and two shoulders as exhibited by CuPc in Fig. 14i, 147 whereas non-planar MPcs display one to two broad peaks as seen in chloro-gallium phthalocyanine (GaClPc) and VOPc lms (Fig. 14iii). 149,150 In the low energy region of the B band (around 288 nm) changes to the absorption spectra between MPcs is thought to be due to orbital overlap of the phthalocyanine ring and the central metal. 146,147 The high intensity peak in the low energy B band region exhibited in CuPc, CoPc, FePc, and nickel phthalocyanine (NiPc) suggests dband association with the central metal, arising to p-d transitions as a result of the partially occupied d-orbitals of these metals. 146,147 Changes in the higher energy region of the B band (210-275 nm) are thought to be a result of d-p* transitions. 146,147 For all MPcs, the Q band region displays a single peak with characteristic Davydov splitting. 99,100,146,[151][152][153] In contrast, metal free phthalocyanine (H 2 Pc) can exhibit a split Q band due to asymmetry of the isoindole nitrogen atoms. 148,154 The Q band has been interpreted in terms of p-p* excitation between bonding and anti-bonding molecular orbitals. 99,146,147,155 The high energy peak of the Q-band has been assigned to the rst pp* transition on the MPc macrocycle with the low energy peak explained as either a second p-p* transition, 155 an excitation peak, 156 a vibrational internal interval, 157 or a surface state. 157 The extent of Davydov splitting observed in the Q band is related to the degree of available molecules able to participate in electronic transitions, in particular interactions between the transition dipole moments from adjacent molecules. 99 Davydov splitting is common in all MPcs, however is more prominent in lms which adopt a herringbone packing structure as seen by the spectra of CuPc and GaClPc in Fig. 14. 151,152 This is also evident by Q band shis and intensity changes of aand b-MPcs UV spectra (Fig. 14ii), demonstrating how packing angle and therefore the degree of p-electron overlap alters Q band absorption. 153 Several factors can inuence Q and B band absorption, mainly the metal center and the inclusion of substituent groups. MPcs with different metal centers can lead to a Q band shi of around 100 nm as a function of metal size, coordination, and oxidation state. MPcs with closed-shell metals (lithium, magnesium, and zinc) typically exhibit a red shied (bathochromic shi) maximum Q band peak, while open-shelled metals (iron, cobalt, and ruthenium) display a more blue shifted (hypsochromic shi) maximum peak due to stronger interactions with the phthalocyanine macrocycle. 148,151 The chemical tunability of MPcs facilitates the functionalization in the peripheral, bay, and axial positions providing control over the physical, optical, and electronic properties. Functional groups can generally be categorized as electron withdrawing, such as sulfonyl, carboxyl, and uoro groups, and electron donating, such as amino, alkoxy, and alkyl groups. Peripheral substituents with electron withdrawing character typically result in a red shied Q band, whereas electron donating groups have little effect on the Q band absorption in solution samples. 148,151 However, the addition of substituent groups may impact the molecular packing in thin-lms and thus result in changes to the absorption spectra of solid samples. Additionally, functionalization at the bay position tends to result in a greater change in the absorption spectra of MPcs compared to similar groups in the peripheral position. 148,151 The addition of axial substituents to MPcs will similarly affect the absorption spectra by altering the molecular packing resulting in shied peaks of different intensity exhibited by the thin-lm UV-vis spectra of axially substituted R 2 -SiPc shown in Fig. 14iv. 132,158 The general trends relating MPc functionalization and absorption properties may not always hold true since the effects of added substituent groups will depend on the individual nature, number, and position of the group.
Vibrational properties
The vibrational properties of MPc thin-lms can elucidate changes to the conguration of the MPc macrocycle as a result of substituent groups or large central metals, and insight into the orientation and packing structure of MPc molecules relative to the substrate. The vibrational modes of MPc thin-lms assessed by both Raman and IR spectroscopy exhibit very similar spectra with the same structural trends and characteristic vibrational bands observed in powder samples and calculated data. 101,[159][160][161][162][163] Raman spectroscopy of MPc thin-lms exhibit a distinctive band pattern with vibrations under 600 cm À1 attributed to the deformation of the macrocycle ring, N-M stretching, and the deformation of isoindoles. [164][165][166] The 600-900 cm À1 vibrations are generally due to the deformation of the benzene and isoindole rings, with 1330-1445 cm À1 assigned to isoindole stretching and vibrations of the N-M and C-H bonds. [164][165][166] The most intense vibrational band observed in MPcs is around 1500 cm À1 which exhibits a clear sensitivity to changes in the central metal with a denite trend correlating metal size to shis in vibration. [159][160][161]163,164 Bands in this region correspond to the displacement of the C a -N b -C a bridges between isoindole groups in the MPc macrocycle (Fig. 15i). [159][160][161]163,164 The change in wavenumber of this band observed in different MPcs correlates to the cavity size (N a -M-N a distance) of the phthalocyanine macrocycle. 159,160 MPc cavity size varies widely depending on the central metal with four possibilities: (i) the metal is smaller than the cavity size, (ii) the metal is approximately equal to the cavity size, (iii) the metal is larger than the cavity size, and (iv) the metal is much larger than the cavity size. 159 These four scenarios result in either ring contraction, equilibrium ring geometry, ring expansion, or nonplanar geometry and ring doming. 159 Consequently, due to its high intensity this band allows for the identication of the central metal ion and orientation of MPc molecules in thin-lms. 101,162,167 Using the integral intensity obtained from polarized Raman spectra the angle between the MPc molecule and the substrate can be determined and used to ascertain the effects of fabrication parameters such as substrate temperature, deposition method, and lm thickness, and identify polymorphic phases and lm order. 101,162,167 For MPcs with a cavity diameter similar to that of H 2 Pc (3.93Å) such as CoPc, FePc, CuPc, and manganese phthalocyanine (MnPc) a planar equilibrium geometry is adopted. 159 With a cavity diameter of 3.66Å, NiPc is an example of an MPc with a metal inclusion that is smaller than the cavity of the phthalocyanine ring such that the four isoindole groups are pulled towards the metal center resulting in ring contraction. 159 Conversely, ZnPc with a cavity diameter of 3.96Å is an example where the metal is larger than the cavity of the phthalocyanine ring causing ring expansion but not large enough to result in a non-planar geometry. 159 Lastly, PbPc and tin(II) phthalocyanine (SnPc) have much larger metal centers and are pushed out of the MPc ring resulting in a nonplanar geometry and ring doming. 159 The effects of metal ion size on the MPc macrocycle are observed by shis in the vibrational band corresponding to the C a -N b -C a bridge bonds, with the wavelength noticeably decreasing with an increase in metal size. 159,160 NiPc has the most shied position at 1545 cm À1 (Fig. 15i), with all other MPcs ordered according to metal size ( Fig. 15i and ii). 159 Although ZnPc, PbPc, and VOPc have similar located bands in the lower wavenumber region, PbPc and VOPc display signicant ring doming and a non-planar geometry, suggesting that the packing structure has less of an impact on the vibration properties compared to metal ion size. 159,160 Additionally, this trend holds for uorinated MPcs as seen in Fig. 15ii, with a more dramatic shi observed in F 16 MPcs compared to their non-uorinated analogs as the addition of uoro substituents has a noticeable impact on the N a -M-N a distance. 160 Other than the metal dependant band around 1500 cm À1 , the spectra region from 1350-1500 cm À1 , known as the nger print region, changes depending on the individual MPc and can display up to six unique bands. 159,160 This region has been known to change depending on the metal center, degree of uorination, and the inclusion of substituent groups. 159,160,164 A change in metal ion band intensity in MPc lms is attributed to changes in the molecular packing and lm organization whereas band location is a result of metal ion size. 101,162 Polarized Raman spectroscopy using parallel and cross polarization allows Raman surface mapping to determine the angle distribution of MPc molecules in thin-lms and the identication of polymorphic forms (Fig. 15iii and iv). The change in MPc orientation can be observed by an increase or decrease in band intensity with different polarizations, indicating a change in angle between MPc molecules and the substrate. Szybowicz et al., demonstrated this through the polymorphic forms of various MPcs studied by polarized thin-lm Raman spectroscopy. 101,162,167 Fig. 15iii shows the average angle between MPc molecules and the substrate determined by the C a -N b -C a bridge vibration before and aer thermal annealing to induce a polymorphic phase transition. 101 For the MPcs studied an increase in angle was observed between the substrate and MPc, with a smaller increase exhibited by MPcs with a large cavity diameter (ZnPc) compared to MPcs with a cavity size similar to that of H 2 Pc (CuPc and MgPc). 101 The Raman surface map reveals additional information on the angle and orientation of MPc molecules in lms. Using MgPc as an example Fig. 15iv shows the angle distribution of molecules estimated by polarized Raman surface mapping before and aer thermal annealing. 101 Before annealing, the lm consists of the metastable aphase with molecules aligned 26-36 to the substrate while aer annealing Raman mapping shows the transition to the more stable b-phase with molecules aligned 39-46 to the substrate. 101 Through Raman and IR spectroscopy the vibrational properties of MPc thin-lms can be used to determine fundamental thin-lm characteristics such as molecular alignment and lm homogeneity, and identify MPc lms by their metal ion and polymorphic forms.
Synchrotron techniques for thinfilm characterization
High performing organic thin-lm devices rely on the specic interfacial orientation and alignment of molecules to achieve optimum opto-electric properties and thus the characterization of these molecular interfaces is critical to the development of advanced devices. The variable nature of organic thin-lms can lead to an imbalance in property optimization where oen the ability to ne tune molecular structure to optimize nano-scale properties, such as intermolecular charge transfer, negatively impacts large-scale thin-lm formation properties. From molecular packing to crystallite formation, analysis of the thin-lms must be performed at various size scales in order to fully characterize the lms. Fig. 16 illustrates the relevant size scales and corresponding structural characteristics important to organic thin-lms and the synchrotron based X-ray techniques which can be used to provide information at each scale. 168 X-ray techniques using synchrotron light sources provide additional information not possible with other methods like optical microscopy, scanning probe techniques, or transmission electron microscopy (TEM). 168 The ability to select specic wavelengths and vary the incident and collection angles facilitates the resolution of nano-scale features such as bond lengths, molecular packing, and phase segregation through the entire lm, rather than strictly at the surface. Additionally, unlike lab scale X-ray methods, synchrotron X-ray techniques can be used to study weakly scattering samples due to the greater ux, brilliance, and collimation of synchrotron light sources, making them ideal for investigating organic thin-lms. 168 X-ray scattering techniques employ the distribution of incident X-rays through a sample where a fraction of the waves are diffracted and collected creating distinct diffraction patterns with high intensity peaks characteristic to the specic lm properties. The angle of the diffracted peaks provides information on the spacing between molecular planes in the lm, whereas the direction of the peaks correspond to the orientation of the planes. Grazing-incidence X-ray scattering (GIXS) is a common X-ray scattering technique where the scattering vector is directed along the sample plane and the diffracting planes are perpendicular to the sample plane. 168 GIXS can be used to analyze the bulk or surface lm properties depending on the chosen incident angle and detection method, for example signal can be collected by a point detector for high accuracy or more commonly using a 2D detector for rapid data collection over a large area with minimal sample damage (Fig. 17i). 168 Grazing-incidence wide-angle X-ray scattering (GIWAXS) and grazing-incidence small-angle X-ray scattering (GISAXS) are two of the most commonly used synchrotron techniques to investigate organic thin-lms with the ability to resolve features in the range of approximately 1Å -100 nm for GIWAXS and 1-100 nm for GISAXS. 168 2D GIWAXS patterns can be used to determine crystal packing through the size and symmetry of the unit cell by analysing peak position and intensity, crystallite size and disorder by analysing peak width, and the degree of crystallinity by analysing the integrated diffraction intensity. 168 Additionally, the molecular orientation and alignment can be determined by performing an azimuthal scan where a diffraction peak is selected and the intensity recorded while the sample is rotated about the substrate normal to determine the orientation distribution. 168 GIWAXS has been demonstrated to be useful for determining how fabrication parameters, such as annealing temperature, effect the molecular orientation of small molecules, 56,73,132 and how molecular structure effects orientation as demonstrated by R 2 -SiPc thin-lms (Fig. 17iii). 130,132 GISAXS is used to analyze the nanoscale surface morphology of polymer and multi-component thin-lms with some use in quantifying domain size in singlecomponent small molecule lms as demonstrated in Fig. 17iv which characterizes CuPc thin-lm formation using different annealing temperatures on different surfaces. [168][169][170] However, GISAXS is predominantly used to study the phase segregation and morphology in polymer and small molecule-polymer blends and is typically used in conjugation with GIWAXS in order to obtain a more complete analysis. 168,169 Scanning X-ray microscopy techniques combine X-ray scattering or spectroscopy methods with a spatially resolved rendering of an image using rasters through a focused X-ray beam (Fig. 17ii). 168,171 Scanning transmission X-ray microscopy (STXM), oen called near-edge X-ray absorption ne structure spectroscopy (NEXAFS) microscopy, is a common method which combines high resolution images with NEXAFS spectra to obtain composition and orientation maps of single and multicomponent thin-lms. 168,171 Typically STXM is used for largescale features (10 nm to 5 mm) and similar to GISAXS has found the most utility in lms consisting of polymer and small molecule-polymer blends. [171][172][173] Orientation and order mapping of single-component thin-lms is achieved by polarized STXM measurements. Different molecular orientations with respect to the polarization axis can be determined by tuning the photon energy to a specic dichroic NEXAFS resonance while measurements with a linearly or elliptically polarized X-ray beam provide contrast between molecules. 168,171 Thus, by rotating the sample about the surface normal and collecting multiple images in the same region with different in-plane polarizations, areas of varying molecular orientation can be mapped and information such as packing structure, tilt angle, and domain size can be acquired for the bulk lm. 168,171 Therefore making STXM a useful tool for large area visualization of organic thin-lms with recent use demonstrated in analyzing the composition of bis(tri-n-propylsilyl oxide) SiPc/ poly-(3-hexithiophene) blends in thin-lms. 173
Conclusion
For over 90 years MPcs have demonstrated their utility as colourants, catalysts, and semiconductors, with particular interest as thin-lm active layers in a myriad of electrical devices. With nearly endless molecular structure possibilities, the ongoing research into the physical, chemical, mechanical, electrical, and optical properties of MPc thin-lms is an evolving discipline. Understanding the building blocks in the formation of MPc thin-lms from deposition, to nucleation and lm growth, helps recognize the inuences of chemical structure and fabrication conditions on lm microstructure, morphology, and properties. Herein the fundamentals of small molecule nucleation and growth in the context of MPc thin-lms fabrication by PVD and solution processing have been discussed with focus on the thermodynamic and kinetic considerations, and how various fabrication parameters and methods effect lm formation. The structure-property relationship of MPc thin-lms was considered in terms on lm microstructure, surface morphology, and optical and vibrational absorption properties. This review provides a valuable resource for the design and application of MPc based thin-lms.
Conflicts of interest
There are no conicts to declare. | 2021-08-27T17:10:57.014Z | 2021-06-15T00:00:00.000 | {
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253784172 | pes2o/s2orc | v3-fos-license | A Deep Learning Approach to Detect Anomalies in an Electric Power Steering System
As anomaly detection for electrical power steering (EPS) systems has been centralized using model- and knowledge-based approaches, EPS system have become complex and more sophisticated, thereby requiring enhanced reliability and safety. Since most current detection methods rely on prior knowledge, it is difficult to identify new or previously unknown anomalies. In this paper, we propose a deep learning approach that consists of a two-stage process using an autoencoder and long short-term memory (LSTM) to detect anomalies in EPS sensor data. First, we train our model on EPS data by employing an autoencoder to extract features and compress them into a latent representation. The compressed features are fed into the LSTM network to capture any correlated dependencies between features, which are then reconstructed as output. An anomaly score is used to detect anomalies based on the reconstruction loss of the output. The effectiveness of our proposed approach is demonstrated by collecting sample data from an experiment using an EPS test jig. The comparison results indicate that our proposed model performs better in detecting anomalies, with an accuracy of 0.99 and a higher area under the receiver operating characteristic curve than other methods providing a valuable tool for anomaly detection in EPS.
Introduction
Vehicle control systems have been significantly influenced by electrical power steering (EPS) due to the increased demand for environmental friendliness. In contrast to hydraulic power steering systems, in EPS systems, power is consumed only when assistance is required. This results in power efficiency since the EPS receives current from the alternator without direct connection with the vehicle engine (when the engine is powered).
EPS is typically preferred over hydraulic power steering for various reasons, including its ability to address leaking hydraulic hoses, its lower weight, and its potential for higher fuel savings [1,2]. With increasing use, EPS is becoming more sophisticated and widely adopted in the automobile industry. This necessitates enhanced safety, reliability, and performance [1,3] which can be achieved through implementing effective system design during the production process, enhancing anomaly detection, and implementing prognostics and health management of the EPS system.
An EPS system comprises various mechanical and electrical components, such as a torque sensor, handwheel angle sensor, speed sensor, and electronic control unit. These elements work together dynamically. Possible EPS system failures include anomalies or failures of components such as sensors and actuators, as well as impending failures such as insulation failure of the stator coil, damaged or defective bearings that impact friction [3]. Early detection of component failures is considered the most important type of failure detection, particularly of the incorrect steering caused by sensor malfunction. This type of failure can lead to catastrophic events due to driver panic [4]. However, the safety design for EPS has not been thoroughly investigated to avoid critical issues concerning the reliability, performance, and safety of EPS systems.
Anomaly detection, also known as "outlier" detection, can be defined as a technique to identify unexpected deviations of data points from typical patterns in a dataset. These deviations mainly occur due to gradual or sudden failures. The three main approaches to detecting this outlier are model-based, knowledge-based, and data-driven [5]. In recent years, the data-driven approach has demonstrated high potential with the development of deep learning models. The advent of Industry 4.0 has benefited industries by generating numerous datasets that can be utilized to monitor and improve production efficiency. This type of data, known as time-series data, has been widely adopted in forecasting and anomaly detection. However, obtaining data with an anomalous event is costly and difficult, which limits the use of the traditional data-driven method. The use of a deep learning algorithm for anomaly detection provides a solution to this challenge through an unsupervised or semi-supervised learning algorithm. The semi-supervised method is based on the assumption that all data used during training are normal data. In the testing stage, any data that deviates from the normal data are considered anomalies. In unsupervised learning, the state of the dataset to be used is undefined [6].
In this paper, we introduce a method for detecting anomalies of the EPS component by using deep learning with long short-term memory (LSTM) on raw sensor data. LSTM is a type of recurrent neural network (RNN) that mitigates the vanishing gradient problem by taking advantage of the temporal dependency of each time step [7,8]. The encoder compresses multidimensional data into a latent space while the decoder utilizes data in the latent space to reconstruct the representation of the input from the encoder. The reconstruction loss from training the normal dataset is used as a threshold for the anomaly score; that is, any value greater than the threshold during testing is classified as an anomaly. To evaluate the performance of our proposed method, we designed an EPS test jig and collected torque sensor data. Common sensor anomalies in EPS are caused by torque sensor and include difficulty turning the steering wheel, uneven left-right power steering assist, and a reduce amount of assistive torque when driving [9]. The main contributions of this paper as follows: • Most existing methods for detecting anomalies in EPS components are physics-based modeling. In this paper, we propose a deep learning (data-driven) approach. • We propose a two-stage approach for detecting anomalous scenarios in sample EPS data. Training is conducted using normal data and anomaly detection based on the reconstruction error. • We utilized a dataset obtained experimentally from a test jig of an EPS system and compared the performance analysis to other methods used to detect anomalies.
The remainder of this paper is organized as follows:-Section 2 presents the related work used for detecting anomalies, while Section 3 describes our proposed method. Section 4 presents the experimental results, including the dataset used, model evaluation, and performance. Section 5 concludes the paper and describes future work.
Related Works
Recent advancements in sensors have increased the amount of data that can be captured and analyzed during the production process. These data can be used to improve decision-making for quality assurance and more effectively observe the manufacturing process [10]. Sensor data have enabled data-driven methods such as machine learning, which can be used for anomaly detection [11,12], intrusion detection [13], and quality prediction [14]. Data-driven defect detection is often associated with anomaly detection and is considered one of the most difficult tasks in machine learning. There are three methods of implementing data-driven defect detection: supervised, semi-supervised, and unsupervised learning methods. The method used depends on the characteristics of the available time-series data [5]. In this section, we introduce various methods for detecting anomalies, including the classical machine learning approach and deep learning.
Classical Machine Learning Anomaly Detection
Classical machine learning approaches for anomaly detection depend on the type of dataset and training algorithm; the datasets are either labeled, unlabeled, or partially labeled. Most supervised learning approaches for anomaly detection use label datasets as a binary classification task to distinguish between normal and anomalous data. Salman et al. [15] proposed linear regression and random forest to detect anomalies in a multi-cloud environment. They used a publicly available dataset to train and test the model for detection and categorization, and achieved a high accuracy of 99%. Deahyung et al. [16] proposed a data-driven classifier that identified representative anomalies from multimodal features. The classifier used conditional log-likelihood from sequences of input signals that are below a time-varying limit. However, this method often encountered the problem of class imbalance due to the smaller number of anomalies than the number of normal data in the training dataset [17]. Providing accurate labels, especially for the anomaly class, requires domain experts; as a result this process is time-consuming and expensive. Additionally, it can be challenging to scale high dimensional dataset. For this reason, unsupervised learning approaches are preferable.
In unsupervised learning, the spatial closeness of data points is utilized, which includes density-based and distance techniques to detect anomalies. Emadi et al. [18] proposed density-based spatial clustering applications with noise (DBSCAN) and support vector machine (SVM) to detect anomalies in wireless sensor networks using three features(voltage, humidity, and temperature). In their approach, it is necessary to evaluate the accuracy of the input parameters before using DBSCAN to detect anomalies. The authors assumed that two clusters are required for density-based detection of anomalies, and determined the relationship between input and output with coefficient correlation before labeling high-density clusters as normal. They then performed training with the SVM. Mistra et al. [19] presented a method for detecting outliers with local outlier factor (LOF) in a data stream. They assigned a real number to each data point, known as an outlier factor. The LOF value for data points that are deeply integrated into a cluster is given 1. For other points, the LOF value is larger than 1. Mistra et al. compared different LOF models and concluded that memory efficient incremental LOF algorithm is the most accurate and scalable in terms of detecting outliers in data streams.
Zhaolu et al. [20] proposed an integrated approach combining K-prototype clustering with K-nearest neighbor (KNN) classification algorithms to detect anomalies from massive system logs. This approach utilized a novel framework for feature extraction, clustering, and filtering. The authors examined the system based on session data and performed K-prototype clustering to divide the dataset into several groups depending on the extracted attributes to accurately define user activities. After filtering out normal occurrences, which typically appeared as highly coherent clusters, the remaining events are considered anomalies that warranted further investigation. The authors created two additional distance-based features to assess the local and global anomaly levels. They used KNN classifiers to evaluate the accuracy of their approach. In another study, Gerhard et al. [21] presented a novel traffic anomaly detection technique using K-means clustering. K-means clustering is a grouping approach that divides features into in K disjoint groups depending on their characteristic values [22]. In their study, unlabeled traffic flow records from training data are divided into clusters of regular and abnormal traffic. Then,matching cluster centroids are used as patterns for distance-based anomaly detection in the monitoring data.
Principal component analysis (PCA) is a method for reducing the dimensionality of datasets, improving interpretability, and minimizing information loss [23]. Takanori et al. [24] proposed a robust PCA method for detecting anomalies using daily or weekly periodicity in traffic volume. This method utilized a covariance matrix that is derived from normal traffic of the preceding period. The proposed method solved the problems of subspace contamination and a false negative ratio in anomaly detection. Amor et al. [25] introduced a method for detecting anomalies in medical measurements using PCA to examine physiological data gathered from sensors and identify multivariable abnormalities based on the squared prediction error in real-time. Their experimental results on an actual medical dataset revealed a high recall rate and low false positive rate.
Deep Learning-Based Anomaly Detection
Deep learning has been used to solve anomaly detection problems. Yunli et al. [26] introduced a deep learning approach for health monitoring and cooling equipment monitoring. Their method involved a two-stage approach that consisting of data prediction using an LSTM network and anomaly detection with the exponential weighted moving average using the prediction errors from the LSTM model. Zhao et al. [27] proposed an LSTM network for machine health monitoring. Their method provided promising results on raw sensor data with a shallow and deep LSTM network. Their experimental results indicated that the LSTM network is capable of learning meaningful representations from raw sensor signals in comparison to other deep learning model evaluated by the author.
Kim et al. [28] proposed convolution long short-term memory (C-LSTM) based web traffic anomaly detection to effectively model the spatial and temporal information in traffic data. They used an LSTM network to model temporal information, a convolution neural network to minimize the frequency fluctuations in spatial information, and a deep neural network for data mapping into a more separable space. They identified the best model by comparing their proposed approach with existing machine learning models through parametric trials, model comparison studies, and data analysis. There is a latency in detecting anomalies in real data because their proposed model used a sliding window to prepossess data. Anomaly detection in unsupervised multi-high dimensional datasets is frequently hindered by decoupled model learning with inconsistent optimization targets and the inability to preserve crucial information in low-dimensional space. Zong et al. [29] proposed a method of solving this problem using a deep autoencoding gaussian mixture model for unsupervised anomaly detection. The proposed model employed a deep autoencoder to generalize a low-dimensional representation and reconstruction error for each input data point, which is then fed into the gaussian mixture model.
Malhotra et al. [30] proposed the long short-term memory autoencoder model (LSTM-AE) to detect outliers using the reconstruct output of sensor data in mechanical devices. The training data contained only normal samples without outliers; and a higher reconstructed output in the test set sample data is an anomaly. Son et al. [31] reported the significance of structural health monitoring in ensuring the durability and safety of buildings and bridges. They presented a two-stage approach to anomaly detection of a cable-stayed bridge using an LSTM-autoencoder model. In the first stage, they classified the anomalies into continuous anomalies due to damaged structural damage and temporary anomalies due to inaccurate data. The cables causing temporary anomalies are selected, and the anomalous data are replaced by interpolated values. In the second stage, the authors examined the replaced inaccurate data to evaluate whether it is accurately categorized and substituted with acceptable values.
Workflow of EPS Anomaly Detection
This section introduces the overall architecture of our proposed method. Further details of the LSTM-autoencoder for anomaly detection are described in subsection three. Figure 1 illustrates the framework of our proposed method for detecting anomalies in EPS data. We applied a deep learning model on time series torque data. The input data are preprocessed before being input into the LSTM-autoencoder model, and the model is trained with preprocessed data containing only normal samples from the collected datasets. The final stage of anomaly detection is based on reconstruction errors of the model on test samples that included both normal and anomalous samples.
Data Preprocessing
The general approach to deep learning is to investigate dataset for initial pattern discovery, handle missing or erroneous values, and normalize or standardize the data before feeding them to an anomaly detection algorithm. This approach is essential to prevent a data sample from skewing the objective function, especially in algorithms that compute the distance between features, if the datasets does not follow a normal distribution [32,33]. We scaled our dataset with normalization techniques in our proposed framework to prevent this scenario, using the scikit-learn MinMaxScaler to normalize values ranging from 0 to 1. The training, validation, and testing datasets are all scaled under the same conditions to validate that the actual value has not diverged from the scaling procedure. The equation for normalization is as follows: where Z i represents the normalized value, and x i indicate a data point from the actual dataset.
(LSTM)
The LSTM network is an improved form of RNN. The LSTM network provides a long-term memory cell to regulate the flow of information, capture long-term dependencies in time-series data, and determine the temporal correlations.. LSTM is an extension to RNN that allows for the use of long-term memory compared to ordinary RNN with short-term memory only [34]. There are various types of LSTM networks,with the most common one being the vanilla and peephole. The peephole networks have not been widely adopted in the literature, since recent studies provided contradictory results [35,36]. Working memory connections is introduced to achieve precise control of the gates and to address instability during training that is often encountered with peephole connections [36]. However, the proposed working memory connections model is limited due to the lack of significant performance improvement when training the stacked LSTM model. Our proposed framework adopts the popular vanilla network described in [35]. As illustrated in Figure 2, the vanilla LSTM is composed of a memory cell, input gate, output gate, and forget gate. The memory cell retains values over arbitrary time intervals, and the three gates control information flow in and out of the cell. Each of the gates is composed of a sigmoid layer followed by a pointwise multiplication operation. The sigmoid layer outputs integers ranging from zero to one, depending on which elements from the vector of the previous hidden state and new input data are allowed to enter the network.
where σ is the activation function, w f and b f are the weight and bias of the forget gate. The input gate has two primary objectives. First, it verify whether the new information (current input and previous hidden state) is important in the new cell state. Second, it updates the cell state. The input gate achieves these objectives in two stages. Similar to the forget gate, the first step is to determine which information is relevant in the current cell state. This process is defined as follows: where σ is the activation function, and w i and b i are the weight and bias of the input gate. The next stage is to generates a memory cellC t by combining the previous hidden state with the current input data. In this process, a tanh activation function is used to generate the vector of the memory update, whose element values range from [1,−1]. This memory cell is defined as follows:C where w c and b c are the weight matrices and the bias of the memory cell state. The two processes in Equations (2) and (3) are pointwise multiplied to update the old cell state C t−1 . Then, the combined operation of C t−1 f t and addition by i t C t results in the new cell state C t of the network being updated, as defined in Equation (4): The final gate is the output gate, which is updated with the new cell state. The output gate decides the new hidden state using the new cell state, previous hidden state, and current input data. The new input data and previous hidden state are fed through a sigmoid activated function to produce the output, as defined in Equation (5): where w o and b o are the weight matrices and the bias of the output gate. As illustrated in Equation (6), the resulting output from filter vector o t and the cell state c t , which is passed through the tanh activation function, are pointwise multiplied to obtain the new hidden state: The new hidden state h t and current cell states c t become the previous hidden state h t−1 and previous cell state c t−1 in the next LSTM unit. This process continues until the entire input sequence has been processed.
Autoencoder
An autoencoder is a type of unsupervised neural network that efficiently encodes and decodes unlabeled input. The encoding process produces a latent representation of the inputs through dimensionality reduction, and then the decoder uses this representation to reproduce the original inputs [37]. As illustrated in Figure 3, the components of an autoencoder include an input layer, encoder, latent vector, decoder, and output layer. The objective of the encoder is to convert the input data x of a high dimensional vector [x ∈ R m ] into a low-dimensional latent space representation z after removing insignificant data often known as noise, from the features. The input equation is as presented in Equation (7): where f is the activation function, and W and b are the weight and bias of low dimensional data sequence z, respectively. In the decoder, the latent vector is processed to generate the outputx which is the reconstructed values of the input data.The output equation is as follows: where f is the activation function, W and b are the weight and bias of the reconstructed inputx, respectively. In a typical autoencoder model, the reconstruction loss is minimized to reduce the difference between the input and output data. We used the mean absolute error (MAE) as our loss function to calculate the reconstruction loss. The reconstruction loss function is given is given as follows: where n is the number of samples in the training set, and x andx represent the input and output data, respectively. The autoencoder can be used as a feature extractor and fed into a supervised learning model, in addition to reducing the dimensionality of features [38]. Therefore, in our proposed model, we combined both the attributes of an autoencoder and LSTM. We employed the autoencoder to extract important features from data and inject them into the LSTM network to capture temporal dependencies.
Anomaly Detection
After training the model using normal datasets, the anomaly decision function can be used to evaluate the reconstructed test dataset and determine whether an anomalous event has occurred. As illustrated in Equation (10), we use MAE as the reconstruction loss. Other metrics for calculating the reconstruction loss include the mean square error; however, we use MAE since it has been proven in several studies to be more robust and minimize the false-positive rate in model predictions [39]. Our anomaly detection threshold is set as the maximum value of the MAE. Therefore, when sensor data are normal, reconstruction loss for the testing set is less than the anomaly threshold and more than the anomaly threshold when anomalies are present.
Experiment and Results
This section describes the experimental setup, including the data collection, training environment setup, and performance metrics. The results are also analyzed and discussed.
Data Collection
To experimentally verify the performance of our model, we collected torque sensor data from a test jig for an EPS using a 12-bit analog-to-digital converter. The experimental environment is illustrated in Figure 4. The running speed varied between 0 and 50 km/h, and the data acquisition board used is a field-programmable gate array. Through the board's general purpose input and output pins, we transferred the sample data at 10-ms intervals. In total, we collected 80,000 samples.
Experimental Setup
We trained our model on a dataset of normal data, which has been demonstrated to provide satisfactory results with deep learning models in other studies [30,31,40]. Our test dataset included both anomalous and normal data. Before training the model, we divided the dataset into training, validation, and test sets. Training is performed using the training and validation sets to minimize the reconstruction loss. To evaluate the performance of our model training, we plotted the training and validation error versus the number of epochs, as illustrated in Figure 5. As can be seen, the loss stabilized quickly at approximately 10 epochs, and as expected, the validation loss stabilized thereafter. Table 1, presents the hyperparameters used for training the model. The encoder and decoder had two LSTM layers with 128 units each.
Evaluation Metrics
To evaluate the performance of the proposed methods, we used the classification accuracy (A), precision (P), recall (R), F-score (F), and area under the receiver operating characteristic curve (AUC). These are defined as follows: True positive (TP) refers to the number of correctly detected anomalies, while true negative (TN) refers to the number of correctly detected normal data without anomalies. False positive (FP) refers to the number of normal data that are incorrectly classified as anomalies, while false negative (FN) refers to the number of anomalies that are not classified as anomalies. The accuracy of the anomaly detection algorithm is the proportion of correctly detected anomalies to all data in the dataset, while the precision is the ratio of true anomalies to predicted anomalies detected by the model.Recall is the percentage of anomalies predicted by the model out of the entire set of anomalies. The F-score is the harmonic mean of precision and recall. Therefore, the higher the accuracy is, the more accurate the model is at detecting normal and abnormal data. In addition, the higher the recall and precision are, the large the number of detected anomalies and the smaller the number of false alarms are, respectively. We conducted the experiment with three models on the EPS dataset and the metric scores are reported in Table 2.
Anomaly Detection
The test sets are utilized to identify anomalies through inference from the trained model. The reconstruction error of the LSTM-AE model is used to measure the model's performance. We set the anomaly threshold using Equation (10) and the method of scaling magnitude applied in [41] to simulate anomaly data of 501 samples from the test set. These samples are labeled as ground truth anomalies to evaluate the model's anomaly detection capability. The maximum acceptable range of our EPS sensor data is illustrated in Figure 6, with the red dashed line representing the threshold and the red points representing the anomalies point. Generally, the data score are in the range of 0.00 and 0.01. Anomalies in sensors included drift, outlier, gap, and break anomalies, and the degree to which each anomaly type occurs varied. This is conducted by scaling the magnitude value from 1 to 1.5. A magnitude of 1 represents no anomalies and a magnitude of 1.5 indicates the value increased by 50%. We considered drift anomalies and outliers to be present when the overall distribution or individual points of the sample data changed, as this can be associated with mechanical structural failure or wear [42].
Performance Analysis
To evaluate the performance of our model, we computed the confusion matrix and comparison table of the three models including our proposed model, gated recurrent unit autoencoder, and bi-directional longs short term memory autoencoder (LSTM-AE, GRU-AE and BiLSTM-AE). The bold text represents the model with the highest score in the performance analysis.
Additionally, we plotted AUC to evaluate the classification accuracy of the three models in Figure 8. AUC indicates the degree of separability between the TP and FP rate of our model. The curve demonstrates the ability of our model (LSTM-AE) to detect anomalies by achieving a score of 0.99. The GRU-AE model score was 0.95, which was lower than that of the LSTM-AE, this is due to the absence of control memory cells. The output gate in the LSTM unit controls the amount of memory content that is accessed or used by other units in the network. The gated recurrent unit, on the other hand, discloses its entire content without any restriction. For the BiLSTM-AE, we obtained a score of 0.92, where the TP rate was observed to be low. The main reason for the difference in the performance of BiLSTM-AE and LSTM-AE is the direction of information. The BiLSTM-AE considers both directions when training the network, running the input from past to future and vice versa. It is computationally expensive to train a bidirectional LSTM,in addition, more data are required for the performance to be equivalent to that of a unidirectional LSTM. Table 3 assesses our model's performance compared to similar techniques for anomaly detection that employ unique attributes of LSTM, AE, or a combination of both.
Conclusions
In this paper, we propose an LSTM-autoencoder-based anomaly detection framework for EPS sensor devices. The autoencoder uses an encoding function to capture the input representation and is implemented with an LSTM network to capture the temporal dependencies in the dataset. This method does not require expert knowledge of advanced feature engineering or complex preprocessing to discover meaningful features from the sensor signal. The proposed framework follows a semi-supervised learning approach that only requires normal data to train the model. Therefore, the proposed framework captures the expected distribution of the sensor signal. The maximum MAE of the reconstruction loss of the trained model is used as the anomaly score to detect anomalies during inference on the test data samples. We demonstrate that the proposed model can accurately detect the simulated drift and outlier anomalies. This study only simulated two possible EPS sensor anomaly scenarios. In future work, we plan to investigate anomalous conditions collected from EPS systems, such as gaps anomaly when there is a high difference between primary and secondary signals of a non-contact torque sensor coil and abrupt drops from high to low of the speed sensor. To improve the robustness of the proposed framework for diagnosing EPS system anomalies. | 2022-11-23T16:13:53.020Z | 2022-11-01T00:00:00.000 | {
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218978532 | pes2o/s2orc | v3-fos-license | Neutrophil FcγRIIA availability is associated with disease activity in systemic lupus erythematosus
Background Immune complexes (ICs) are detectable in a variety of inflammatory diseases, including systemic lupus erythematosus (SLE), reflecting autoantibody binding to antigens. Though ICs are the main contributors to disease pathogenesis through FcγR-mediated inflammation and organ damage, IC levels are not part of the clinical assessment of SLE. The aim of this study was to explore the clinical utility of analyzing levels of ICs in SLE patients using a novel technology, IC-FLOW. Methods Paired serum samples, at the time point of high and low disease activity (n = 92), were analyzed using two assays: an IC ELISA from a commercial company and a novel in-house flow cytometry-based method, IC-FLOW. IC-FLOW measures FcγRIIA availability on the neutrophil cell surface by flow cytometry, whereas the commercial ELISA measures IC binding to C1q. Results Using IC-FLOW, 90% of SLE patients with active disease had elevated levels of circulating ICs (p < 0.0001). Using the commercial assay, only 17% of SLE patients had elevated levels of circulating ICs. For both assays, levels of ICs reflected active disease as determined by SLEDAI (r = 0.45, p < 0.0001) and were associated with type I IFN activity (r = 0.37, p = 0.001), and complement consumption (p = 0.0002). Levels of ICs measured with IC-FLOW, but not with the commercial ELISA, were associated with active lupus nephritis (p = 0.004). Conclusions This novel FcγRIIA-IC assay can detect levels of circulating ICs in patients with SLE. Analyzing IC levels may facilitate monitoring of disease activity, as well as identify patients at risk of lupus nephritis, allowing for early preventive interventions.
Introduction
Circulating immune complexes (ICs), formed upon recognition of antigens by antibodies, are detectable in a variety of systemic diseases, including autoimmune and infectious diseases. In systemic lupus erythematosus (SLE), an autoimmune rheumatic disease, ICs deposit in tissue, including skin and kidney, to induce complement activation and organ damage. Further, ICs are engulfed by immune cells causing inflammation including induction of type I interferons (IFNs) [1,2], as well as the formation of neutrophil extracellular traps (NETs) [3,4]. Although central to the disease pathogenesis, levels of circulating ICs are rarely measured in reference laboratories due to the lack of specificity in current assays with a broad array of assay measuring different features of ICs [5]. Early studies detected circulating ICs by their capacity to bind to neutrophils as determined by immunofluorescence techniques [6][7][8]. Current technologies, however, primarily rely on the capacity of circulating ICs to either fix complement C1q or bind complement C3 antibodies and/or precipitate using polyethylene glycol (PEG) [5]. Comparing C1q-based ELISAs, the overall agreement was about 50% between four commercially available assays [9]. The main concerns relate primarily to the ability of anti-C1q antibodies and rheumatoid factor (e.g., anti-IgG antibodies) to interfere with the ELISAs. Finally, though complement opsonization is an important event in the clearance of ICs, complement opsonization, as shown by us and others, neutralizes the ICs, reducing their inflammatory capacity [2,10]. Thus, assessing complement-bearing ICs will primarily target non-inflammatory ICs and not the harmful ICs. The inflammatory trigger relies on ICs engaging FcγRs on immune cells through the Fc portion of the IgG molecule.
We recently found that FcγRIIA is the main FcγR responsible for the uptake of ICs by neutrophils [3]. In the current study, we investigated whether our novel technology, IC-FLOW, assessing the availability of FcγRIIA on a neutrophil cell surface, could provide a novel, clinically meaningful method of assessing levels of circulating ICs in SLE. Several methods have been suggested for the analysis of circulating ICs, including PEG-dependent precipitation, binding of ICs to C1qcoated plates, or binding of ICs to anti-C3 antibodies [5]. We here demonstrate that the analysis of neutrophil FcγRIIA cell surface expression, and not IgG levels, by flow cytometry accurately captures levels of circulating ICs in patient samples. The advantages of our novel technology are the lack of influence from rheumatoid factor and anti-C1q antibodies and the assessment of "pathogenic" ICs, e.g., ICs signaling through inflammatory FcγR-mediated pathways rather than through complement receptors.
Materials and methods
Patients SLE patients (n = 92), recruited at the Skåne University Hospital, Lund, Sweden, were retrospectively selected from our biobank with stored samples to include patients with matched samples at time points of both high and low disease activity. Healthy controls (n = 100) were recruited at the Skåne University Hospital, Lund, Sweden. The study was approved by the regional ethics boards (LU06014520 and LU 378-02). Informed written consent was obtained from all participants according to the Declaration of Helsinki. The patient cohort is presented in Table 1 and has been described in great detail previously [12][13][14][15][16][17][18]. Nephritis was defined as the new onset of urinary casts, hematuria, pyuria, and/or proteinuria according to SLEDAI-2 K [11].
Type I interferon assay
Type I IFN activity was measured as previously described assessing the capacity of circulating type I IFNs to signal through IFNAR [19][20][21]. Briefly, WISH cells were cultured with patient serum (50%) and analyzed for the induction of six IFN-regulated genes (LY6E, MX1, OAS1, ISG15, IFIT1, EIF2AK2) and three housekeeping genes (GAPDH, PPIB, B2M) using the Quantigene Plex 2.0 assay as described by the manufacturer (Panomics, Inc.). Increased type I IFN activity was defined as a 2fold increase in type I IFN-regulated genes as compared to healthy controls.
Autoantibodies and complement components
Autoantibodies, including dsDNA (Crithidia luciliae immunofluorescence test) and complement levels in the serum, were measured according to routine analyses at the Department of Clinical Immunology, Skåne University Hospital, Lund, Sweden, performed at the Department of Laboratory Medicine, Lund, Sweden. Immune complex assays Levels of immune complexes were analyzed using an inhouse method, IC-FLOW. Briefly, neutrophils were isolated through density gradient (Polymorphprep, Axis-Shield) and incubated with sera (10%) for 90 min in RPMI-1640 medium. After serum incubation, and potential IC-binding and internalization of FcγRIIA, neutrophils were analyzed for cell surface expression of FcγRIIA by flow cytometry using FITC-conjugated IV.3 (STEMCELL Technologies) and PE-conjugated FUN-2 (BioLegend) antibody clones. The results are presented as micrograms per milliliter using heat-aggregated IgG of known concentration as a standard curve. For the commercial ELISA, levels of ICs were analyzed as per the company's instructions (Quidel).
Statistics
For non-paired sample sets with non-Gaussian distribution, the Mann-Whitney U test and Spearman's correlation test were used, as applicable. In some analyses, logistic regression analysis was used for dichotomized variables. As a cutoff for positivity, the 95th percentile of the healthy controls was used. GraphPad Prism and IBM SPSS were used for the analyses. All analyses were considered statistically significant at p < 0.05.
FcγRIIA internalization is a marker of IC binding
We recently found that FcγRIIA was the main FcγR involved in the uptake of circulating ICs by neutrophils [3]. Upon binding of ICs, the IgG-binding domain of FcγRIIA is occupied, and the receptor is subsequently internalized. Consistently, upon addition of heataggregated IgG (HAGG) to neutrophils, we found a dose-dependent reduction in the levels of cell surface FcγRIIA as determined by flow cytometry using two distinct FcγRII antibody clones: IV.3 and FUN-2 (Fig. 1a).
Of note, the two FcγRII antibodies strongly correlated (r = 0.94, p < 0.0001, Fig. 1b), suggesting that they detect the same process, i.e., binding of ICs to FcγRIIA.
SLE patients have elevated levels of circulating ICs
After establishing that the assay, IC-FLOW, was able to quantify levels of circulating ICs in an FcγRIIAdependent manner, we next asked whether patients with SLE had elevated levels of ICs. As determined by IV.3 staining, levels of circulating ICs were highly elevated in SLE patients as compared to healthy individuals (p < 0.0001, Fig. 1c). Similar to our in-house assay, elevated levels of ICs were found in SLE patients also with the commercial assay (p < 0.0001, Fig. 1d), consistent with what has been described for C1q-and C3d-based ELI-SAs [22,23]. The two assays only partially correlated (r = 0.42, p < 0.0001, Fig. 1e). Using the 95th percentile of healthy controls as a cutoff, 90% of SLE patients had elevated levels of circulating IC at the time point of active disease as determined by the IC-FLOW technology.
In contrast, only 17% of the SLE patients were positive using the commercial ELISA. Similar results were seen also for patients with low disease activity (78% vs 9%, respectively, for the different assays).
Levels of circulating immune complexes are associated with disease activity
As demonstrated in Fig. 1c, d, levels of circulating ICs were elevated in patients with active disease as compared to patients with low disease activity. Further, levels of circulating ICs correlated with SLEDAI at the time point of active disease as measured by IC-FLOW as well as the commercial assay (r = 0.45, p < 0.0001; r = 0.38, p = 0.0004, respectively, Fig. 2a). ICs are commonly found deposited in a tissue, including joints, skin, and kidney, promoting local inflammation and organ damage in SLE patients. Given the association between levels of circulating ICs and disease activity, we next asked whether levels of circulating ICs would reflect disease activity in specific organ systems, including the skin, joints, and kidneys. In contrast to early studies [24,25], comparing paired patient samples at the time point of high disease activity with corresponding patient samples at the time point of low disease activity, levels of circulating ICs were found to be elevated in patients with the following organ manifestations: nephritis, rash, oral ulcers, and arthritis (p = 0.004, p = 0.0007, p = 0.02, and p = 0.002, respectively, Fig. 2) as compared to patients without those disease manifestations. Patients with vasculitis did not have statistically significant elevated levels of IC (Fig. 2c). In contrast to IC-FLOW, the commercial ELISA could not distinguish between active and inactive nephritis (Fig. 2b). Further, levels of ICs were associated with the presence of anti-C1q antibodies, known to be associated with renal disease (Fig. 3b).
Levels of ICs are associated with immunopathological markers of disease
Asking whether levels of ICs were associated with serological markers of disease activity, we found that IC-FLOW, but not the commercial ELISA, was associated with the presence of anti-dsDNA antibodies (Fig. 3a).
We have shown that nucleic acid-containing ICs are potent inducers of type I IFN production by plasmacytoid dendritic cells in vitro [2]. However, whether levels of ICs in vivo would relate to ongoing type I IFN activity in SLE patients is not known. As shown in Fig. 3c, type I IFN activity correlated with levels of circulating ICs as measured by both assays (Fig. 3c). ICs, both circulating and tissue-deposited, are potent activators of the classical pathway of the complement system. Consistently, levels of ICs were associated with complement consumption for both assays (p = 0.0002 and p = 0.01, respectively, Fig. 4a). Further, levels of ICs were inversely correlated with complement C1q, C3, and C4 (r = − 0.48, p < 0.0001; r = − 0.53, p < 0.0001; and r = − 0.51, p < 0.0001, respectively, Fig. 4b-d).
Discussion
Immune complexes play a central role in many autoimmune diseases, contributing to inflammation and organ damage often through FcγR-mediated mechanisms. Though an abundance of assays has been developed to quantify levels of circulating ICs, they have failed in their specificity as well as due to technical properties, including interactions with autoantibodies such as rheumatoid factor and anti-C1q antibodies. In the current study, we propose that analyzing IC binding to FcγRIIA using flow cytometry may be a novel and superior approach to assess levels of inflammatory and pathogenic IC relevant to disease progression. Several methods have been suggested for the analysis of circulating ICs, including PEG-dependent precipitation, binding of ICs to C1q-coated plates, or binding of ICs to anti-C3 antibodies [5]. Though the precipitation assay has the least specificity, precipitating also other proteins, the ELISA assays, e.g., the complement-dependent assays, are challenged by the presence of anti-C1q antibodies as well as rheumatoid factor, either blocking or amplifying IC levels. Further, we and others have demonstrated that . c Levels of ICs were correlated with type I IFN activity score. Statistical analyses were performed using the Mann-Whitney U test and Spearman's correlation with *p < 0.05,**p < 0.01, and ***p < 0.001 complement-opsonized ICs are not inflammatory, with C1q signaling through complement receptors such as LAIR-1 to induce silent clearance [2,10,26]. Instead, ICs devoid of C1q, that is, signaling through FcγRs, are highly inflammatory, particularly if containing nucleic acid material [2]. Early work in the 1970s investigated IgG binding to neutrophils, capturing both circulating ICs and antineutrophil antibodies [6][7][8]. We here demonstrate that analysis of neutrophil FcγRIIA cell surface expression, and not IgG levels, by flow cytometry accurately captures levels of circulating ICs in patient samples. The advantages with our novel technology are the lack of influence from rheumatoid factor and anti-C1q antibodies and the assessment of "pathogenic" ICs, e.g., ICs signaling through inflammatory FcγR-mediated pathways rather than through complement receptors.
Prior work, pioneered by the group of Lars Ronnblom, clearly demonstrated that nucleic acid-containing ICs, in vitro, promote induction of type I IFNs by plasmacytoid dendritic cells in an FcγRIIA-and TLR-dependent manner [1]. These cytokines are central in SLE pathogenesis, with a majority of the patients having a "type I IFN signature," in particular, in active disease [27,28]. To our knowledge, our investigation is the first to demonstrate a direct association between levels of circulating ICs and type I IFN activity in SLE patients. This is important as other inducers of type I IFNs, including cGAMP, a second messenger acting in the cGAS-STING pathway, have recently been implicated in SLE [29]. Further studies are needed to investigate which interferogenic pathways are activated in SLE patients.
Upon IC formation, complement C1q will bind to the Fc region of the IgG antibody and initiate the activation of the classical pathway of the complement system, leading to the consumption of complement components, C3 and C4, and generation of complement split fragments, C3dg, as also demonstrated in our investigation. Complement activation is commonly observed in SLE, with complement activation fragments, in particular, C3a and C5a, acting to recruit inflammatory cells to the area of IC deposition. Upon recruitment, neutrophils will be activated, through FcγRIIA, and release inflammatory and cytotoxic components, including reactive oxygen species (ROS) and proteases, causing tissue damage. Consistent with these mechanisms, levels of ICs, as determined by IC-FLOW, identified patients with complement consumption and, not least, nephritis. These results clearly demonstrate that our novel assay, IC-FLOW, captures events involved in IC-mediated organ damage and inflammation, key processes in lupus pathogenesis. Finally, levels of ICs were associated with disease activity, including the skin, joints, and kidney. Long term, we anticipate IC-FLOW to stratify SLE patients, identifying patients with an IC-driven disease, likely to benefit from B cell-targeted therapy.
Conclusions
In all, we have developed a novel method, IC-FLOW, to assess levels of circulating ICs in patients with autoimmune diseases, with a focus on SLE. This assay identified SLE patients with an active severe disease, including ongoing nephritis. Of note, the assay performed better than a commercial assay. Future studies aim to determine whether levels of ICs can predict disease progression. | 2020-05-29T14:54:04.471Z | 2020-05-29T00:00:00.000 | {
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252335481 | pes2o/s2orc | v3-fos-license | Opening Pandora’s Box? Joint Sovereignty and the Rise of EU Agencies with Operational Tasks
This article problematises the proliferation of European Union (EU) agencies with operational tasks as a new phenomenon capturing the exercise of joint sovereignty in European integration. While joint decision-making has been a feature of EU politics for decades, joint sovereignty is a broader category that additionally involves the creation of EU bodies able to intervene ‘on the ground’ alongside national public actors. We argue that the choice for joint sovereignty opens a Pandora’s box of implementation deficiencies which undermine the ability of both national and supranational actors to conduct operational activities effectively. We subsequently identify two frequent dysfunctions in policy implementation and connect them to ambiguity and conflict at the decision-making stage. Empirically, we illustrate the systemic link between decision-making and implementation problems in the functioning of two agencies with operational tasks active in the fields of border management (Frontex) and police cooperation (Europol).
Introduction
In modern governance, 'agencification' is a phenomenon describing the transfer of government powers from classic ministerial departments to new bodies delegated with specialised tasks and various degrees of autonomy (Trondal, 2014). In the European Union (EU), agencies proliferated in the 1990s in many policy areas (Levi-Faur, 2011). At the outset, EU agencies fulfilled mainly (semi-)regulatory functions such as providing information and expertise-based rules in the single market (Egeberg & Trondal, 2017;Majone, 1994). However, the last two decades have also witnessed a strengthening of EU agencies with operational tasks in fields like border management (the European Border and Coast Guard Agency, known as Frontex), asylum (the European Asylum Support Office, EASO), police cooperation (Europol), or civil and criminal justice (Eurojust and the European Public Prosecutor's Office). The development is significant for two reasons. First, agencies with operational tasks go beyond the EU's classical 'regulatory state' model because they require supranational capacity-building in terms of money, personnel and coercive ability (Bremer et al., 2020;Genschel & Jachtenfuchs, 2016). Second, operational tasks presuppose the physical presence of EU officials on member states' territories in order to limit, enhance, or even replace the actions of national public authorities. While EU institutions had already been involved in policy enforcement vis-à-vis private actors (Scholten, 2017), operational tasks are substantively different because they target the activities of public actors.
In this article, we conceptualise this new empirical trend as the exercise of 'joint sovereignty' in the EU. We focus, in particular, on the domestic sovereignty of member states (Krasner, 1999;Thomson, 1995) and its two main components: decision-making authority (the sole right to make rules in a given territory) and coercive capacity 1 (the sole capability to physically enforce said rules through a centralised bureaucratic apparatus). Borrowing from the new intergovernmentalism, we first show the proliferation of EU agencies since the Maastricht Treaty as a process of 'integration without supranationalism' which created 'de novo bodies' (Bickerton et al., 2015) under joint national and supranational authority. Then, we examine the case of operational agencies as a specific institutional form through which national and supranational actors share not only decision-making authority but also the capacity to enforce rules 'on the ground' in different policy areas. When operational agencies participate in activities like law enforcement, border management, or inspections of national infrastructure, member states come to exercise sovereignty jointly with them and thus no longer hold the monopoly of coercive capacities on their territories. Given its emphasis on physical policy enforcement, the resulting joint sovereignty model is broader than shared authority in multi-level governance (Hooghe & Marks, 2001) and goes beyond notions of 'pooled sovereignty' in decision-making (Moravcsik, 1998). Beyond the EU, similar arrangements can be found in other 'coming-together' polities (Kelemen, 2014;Stepan, 1999) practicing cooperative or executive federalism, such as the United States, Switzerland or Germany (Bolleyer & Thorlakson, 2012;Börzel, 2005).
Second, we tackle the connection between the conditions that facilitate the emergence of joint sovereignty in the EU and the ability of the resulting system to implement its own decisions effectively. Borrowing a metaphor from Scholten (2017Scholten ( , p. 1360, we argue that joint sovereignty opens a 'Pandora's box' of implementation deficiencies when the same conditions that allow the establishment of joint coercive capacities undermine the ability of the system as a whole to enforce policies effectively. In order to establish new institutions or to adopt policies under the joint sovereignty model, member states typically strike compromises with a high level of ambiguity (Matland, 1995) or based on 'incomplete contracts' (Jones et al., 2016;Pollack, 2003, p. 22). This pushes policy conflict further down the line to the implementation stage. Consequently, the same conditions that pave the way for the emergence of joint sovereignty create vertical conflicts over the ill-defined exercise of joint coercive capacities in policy implementation. Which actor on which level of authority is, for instance, responsible for registering, processing and implementing the decision of an asylum application? The EU's systematic reliance on hybrid governance structures equips both national and supranational actors with the ability to enforce policies on the ground, which generates specific deficiencies. Using an adapted version of Matland's (1995) ambiguity-conflict model in policy implementation, we identify responsibility-shifting and obstruction as the most likely dysfunctions of joint sovereignty in the EU. Empirically, we illustrate the deficiencies with two emblematic examples borrowed from the activities of Frontex (responsible for border management) and Europol (tasked with coordinating police cooperation).
Our contribution to the literature is fourfold. First, we analyse the EU agencification phenomenon in a new light. While acknowledging the role of all agencies in the construction of an EU administrative space, that is the 'institutionalisation of common administrative capacity' (Trondal & Peters, 2013, p. 296), we understand the expansion of operational agencies in particular as a new feature of EU governance, that is, the exercise of joint sovereignty. Second, we investigate tenets of the new intergovernmentalism (Bickerton et al., 2015) and the core state powers literature (Genschel & Jachtenfuchs, 2016) by examining the practical consequences of sharing sovereignty in the EU multi-level system. Third, in exploring such consequences, we open up a new area of investigation in the flourishing literature focusing on the challenges of multi-level implementation (Heidbreder, 2017;Thomann & Sager, 2017). Fourth, we expose pathologies of joint sovereignty that apply not only to the EU but also to other multi-level contexts. When novel pressures for centralisation occur in devolved areas of competence, 'coming-together' federations (Kelemen, 2014;Stepan, 1999) might indeed develop similar implementation dysfunctions as the EU.
The article is organised as follows. We start with a description of a new empirical phenomenon in European integration: the expansion of EU agencies with operational tasks (section 2). Next, we conceptualise the development as the emergence of 'joint sovereignty' in the EU, which displays specific features (section 3). We then explain how the conditions that facilitate decision-making on joint sovereignty are bound to lead to systemic implementation deficiencies (section 4). To illustrate the problems, we describe two cases of ineffective implementation: on the one hand, the mechanics of responsibility-shifting as reflected in Frontex's approach to fundamental rights in joint operations; on the other hand, the practices of obstruction visible in the reluctance of national authorities to share police intelligence with Europol (section 5). We conclude by discussing the applicability of our argument to other EU policy areas and institutional arrangements.
Empirical Observation: The Rise of EU Agencies with Operational Tasks
In the past three decades, agencification has become a pervasive feature of EU governance (Egeberg & Trondal, 2017). Since the early 1990s, agencies have proliferated across policy fields and acquired an increasingly important role in the functioning of the EU machinery (Egeberg & Trondal, 2017;Ripoll Servent, 2018). Among the plethora of existing networks and committees (Levi-Faur, 2011), EU agencies are the institutions which have undergone the most impressive development: from only two agencies created in 1975, the EU now counts over 36 of these bodies, which possess a wide range of different implementation tasks (for an overview, see Vos et al., 2018).
The most common understanding of EU agencies is provided by Kelemen & Tarrant, who define them as EU-level public authorities with a legal personality and a certain degree of organisational and financial autonomy, created by secondary legislation in order to perform clearly specified tasks (Kelemen & Tarrant, 2011). All EU agencies are characterised by a shared governance structure (Vos et al., 2018) as their management boards are composed by member states' representatives and the European Commission, and sometimes even the European Parliament (Busuioc, 2012). Therefore, the autonomy of agencies is dependent on the interplay between national and supranational actors. From a delegation perspective, agencies act under the control exerted by multiple principals (Kelemen & Tarrant, 2011;Thatcher, 2011), that is, according to a double delegation logic by the Commission and the member states (Michaelowa et al., 2018). Due to legal limitations set by the Treaties 2 and the Court of Justice, 3 agencies are usually unable to take fully autonomous decisions and/or to organise operations independently from the member states. Nevertheless, various agencies are progressively moving to the forefront of policy implementation (Chamon, 2016;Scholten, 2017), thus unsettling the traditional paradigm of EU executive federalism (Börzel, 2005) and the assumption that policy implementation is chiefly a national concern (Tsourdi, 2021).
Despite having many similarities, EU agencies differ substantially from each other. Existing classifications are based either on the type of function (e.g. Wonka & Rittberger, 2010) or on the kind of powers and level of discretion conferred on them (for an overview, see Chamon, 2016). For the purpose of our argument, we categorise agencies according to the target of their enforcement action: specifically, we identify a fundamental distinction between agencies tasked with regulation and those tasked with operational activities 4 (Chamon, 2016, p. 24;Wonka & Rittberger, 2010). Accordingly, the pursuit of regulatory tasks implies the production and enforcement of common rules vis-à-vis private actors (Scholten, 2017), where enforcement usually occurs 'from the distance', that is, experts monitor compliance with rules and (in some cases) impose sanctions in case of violations. 5 Since regulatory agencies mainly target private actors, they are common in internal market policies. Their role is to provide rules and scientific expertise, to work as an enhancement of comitology committees and to implement detailed rules stemming from complex legislation (Migliorati, 2020b). Such agencies may enjoy considerable autonomy both in setting regulatory standards and in implementing them. For example, the European Medicines Agency (EMA) plays a crucial role in the approval of pharmaceutical products and sets regulatory standards and best practices for pharmacovigilance (Sabel & Zeitlin, 2008). The European Food Safety Authority (EFSA) identifies and ranks public health hazards in food and feed production (Hartley, 2016). In turn, the European Securities and Market Authority (ESMA) sets regulatory and supervisory standards and practices in the financial sector, and even has the right to directly apply and enforce some aspects of EU law vis-à-vis private parties 6 (Scholten, 2017;Scholten & Van Rijsbergen, 2014).
Conversely, operational tasks entail assisting member states in the physical enforcement of rules on their territories; as such, they are associated with public authorities in 'hands-on' activities like law enforcement, border management and inspections. To this end, operational agencies require fiscal, administrative and coercive resources, typically considered 'core state powers' in European integration (Bremer et al., 2020). Different from regulatory tasks, operational activities imply direct contact with state actors, and in some cases, even the ability to enhance the human and material resources of individual member states by drawing from the EU budgetas is the case for Frontex (Tsourdi, 2021, p. 180). In general, operational tasks vary according to the mandate of the agency. They can include the exchange of information to facilitate or realise operational activities (e.g. Europol); providing training for national officials (e.g. CEPOL and EASO); the coordination of joint member state operations (Eurojust, Europol, EASO, Frontex and the European Fisheries Control Agency) or the organisation and execution of inspections (e.g. the European Maritime Safety Authority) (cf. Chamon, 2016 pp. 40-44).
In the past three decades, EU agencies with operational tasks have witnessed a remarkable development. Figure 1 compares the two groups in absolute numbers over time, while Figure 2 compares their economic empowerment as reflected by their EU-funded budgets. 7 Given the pervasiveness of agencification in the post-Maastricht era, it is unsurprising that the number of both regulatory and operational agencies has increased substantially. Moreover, keeping in mind that the role of the EU as a regulatory polity has persisted since the 1990s (Majone, 1994), agencies with regulatory and information-sharing tasks continue to outnumber operational ones. Yet, the more interesting observation can be found in Figure 2: Compared to regulatory bodies, agencies with operational mandates have seen their budgets increase massively, especially in the past 5 years. These budgetary increases concern agencies involved in border management, asylum and law enforcementnamely, Frontex, EASO, Europol and EU-Lisa. As with any public institution, budgetary increases signal an expansion of activities; in fact, the budgets of EU agencies are strongly correlated with their empowerment over time (Migliorati, 2020a). In other words, higher budgets mean not only more money and staff, but also an extension of their original mandateas was the case for Frontex or EASO. With their mandates expanding and their budgets increasing, EU agencies with operational mandates are likely to become more involved in 'hands-on' policy implementation in the member states.
In the next section, we argue that the expansion of EU agencies with operational tasks signals a broader development in European integration since the Maastricht Treaty.
Towards a Joint Sovereignty Model
Why are EU agencies with operational tasks on the rise? The main explanation for this phenomenon has two parts. The first part refers to the general expansion of EU agencies in European integration after the Maastricht Treaty (1993) and the broader trend towards 'integration without supranationalism' (Bickerton et al., 2015). The new intergovernmentalism describes the phenomenon as a deliberate decision by national governments to further advance European integration without empowering the traditional supranational institutionsthe Commission, the European Parliament (EP), or the Court of Justice (Bickerton et al., 2015, p. 705). Instead, member states created new governance structures where decision-making authority remained in the hands of national governments or became shared in a hybrid intergovernmentalsupranational architecture within de novo bodies (which include both regulatory and operational agencies). Given the increased domestic contestation of EU policy-making since Maastricht, national governments felt constrained not to transfer further powers to supranational institutions (see also Hooghe & Marks, 2009).
The second part of the explanation concerns the rise of EU operational agencies in particular. This relates to another development in European integration in the post-Maastricht era, when the EU expanded from market integration to the integration of core state powers (Genschel & Jachtenfuchs, 2016). Core state powers denote key functions of sovereign government 'derived from the state's twin monopoly of legitimate coercion and taxation', including police forces, border patrols, the military, public administration and fiscal institutions (Bremer et al., 20020, p. 58). Unlike in market integration, the integration of core state powers concerns and directly affects state elites as opposed to private economic actors (Genschel & Jachtenfuchs, 2016, p. 52). In such fields, it is generally difficult to use the EU's classic 'regulatory state' model (Majone, 1994), that is, pooling decision-making at the supranational level whilst leaving implementation dependent on member states' capacities.
To an extent, core state powers integration required the establishment of supranational capacities able to intervene directly vis-à-vis or alongside member states within their territories (Genschel & Jachtenfuchs, 2016). This resulted in the establishment (and empowerment) of operational agencies.
Against this background, we argue that the rise of EU agencies with operational tasks illustrates the emergence of a joint sovereignty model in the EU political system. We are specifically interested in the transformation of member states' domestic sovereignty (Krasner, 1999, p. 4) when EU institutions gain the 'authority to intervene coercively in activities within [their] territory' (Thomson, 1995, p. 219). In international law, the term 'joint sovereignty' refers to disputes over territorial borders within condominiums. Condominiums are political entities where two or more states simultaneously exercise sovereignty within a territory in conformity with pre-established rules, for example, Andorra (Samuels, 2008). Transposed to the EU, 'joint sovereignty' moves away from territorial conflict between horizontal units; instead, the concept denotes the vertical exercise of decision-making authority and coercive capacities by two levels of governance (the EU and national actors) at the same time. In the next pages, we outline the building blocks of the term.
Theorising Joint Sovereignty
Two fundamental issues have long haunted the academic debate on the concept of sovereignty, and on sovereignty in the EU in particular. The first concerns sovereignty's very nature. Is sovereignty real, something found 'out there'? Or is it a discursive construct that merely serves to reproduce existing power structures (Krasner, 1999)? Traditional political thought understood sovereignty as something tangible, the 'supreme authority within a territory' (Bodin, 1992), 'the exercise of the general will' (Rousseau and Cranston, 2003, p. 69), or control over the 'state of exception' (Schmitt and Schwab, 2005, p. 5). More recently, scholars from the 'linguistic turn' emphasised the socially constructed character of sovereignty, pointing to the contingency of its meaning and instrumental usage over time (Adler-Nissen & Gammeltoft-Hansen, 2008;Bartelson, 2014;Werner & de Wilde, 2001). A second cause of fundamental disagreement, especially in multilevel polities, concerns the divisibility and the sharing of sovereignty (Waever, 1995, p. 417). If sovereignty describes the ultimate authority in a given territory, how can it be shared across levels of governance?
In our view, sovereignty has practical implications for the workings of the international system and the self-understanding of embedded actors, irrespective of the constructed nature of the term (Bartelson, 2014;Krasner, 1999). In the EU setting, the divisibility of sovereignty is a de facto reality: 'pooled among governments, negotiated by thousands of officials through hundreds of multilateral committees, compromised through acceptance of regulations and court judgements which operate on the principle of mutual interference in each other's domestic affairs' (Wallace, 1999, p. 506). In adopting such a pragmatic approach, we follow recent contributions that demonstrate the continued relevance of the concept in EU multi-level governance. Scholars have shown the evolution of the EU political system has generated conflicts of sovereignty between the national and the supranational level (Brack et al., 2019) that have reconfigured previously established sovereignty practices (Jabko & Luhman, 2019). We take this observation as our starting point and explore the recent reconfiguration of sovereignty in the EU from both an analytical and an empirical perspective.
Our conceptualisation of 'sovereignty' starts from Krasner's notion of 'domestic' or 'internal' sovereignty, denoting the 'legitimate authority within a polity and the extent to which that authority can be effectively exercised' (Krasner, 1999, p. 4). From this perspective, sovereignty comprises two dimensions. First, authority denotes the exclusive right to take legally binding decisions recognised as legitimate in a given territory (Thomson, 1995, p. 223). Second, the exercise of authority presupposes the capability to enforce rules, which in turn requires 'a central bureaucratic apparatus claiming a monopoly on organised coercive forces' (Thomson, 1995, p. 221). The emphasis on physical enforcement overlaps with Weber's definition of the state as the entity holding 'a monopoly of the legitimate use of physical force within a given territory' (Weber, 1946). A sovereign entity thus fulfils both conditions at the same time: it can take authoritative decisions and physically enforce them in practice.
If we accept the premise above that sovereignty can be divided in multilevel polities, then 'joint sovereignty' denotes the simultaneous exercise of decision-making authority and coercive capacities within a given territory by (at least) two levels of governance. In the EU, the Treaties set the division of competences between national and supranational actors and thus diffuse the exercise of legitimate authority (Hooghe & Marks, 2001, p. xiii). In fact, joint decision-making has been a feature of European integration for decades, as seen in the different procedures of the EU legislative process (Scharpf, 2006). The novelty lies in the ability of operational agencies to engage in activities that erode member states' monopoly of coercive capacities within their territories. In this respect, we argue that operational agencies mark a shift from 'pooled' (Moravcsik, 1998) or 'shared sovereignty' (Wallace, 1999) to 'joint sovereignty' in European integration. The difference between the terms is substantive, not only in terms of who is doing the sharing but also in respect to what is being shared. Both Moravcsik and Wallace emphasised the sharing of decision-making authority on the horizontal, among member states; by contrast, 'joint sovereignty' is about sharing both decision-making authority and coercive capacities on the vertical, between national and EU-level actors. For Moravcsik, national governments started pooling their sovereignty once they agreed to replace unanimity with qualified majority voting in the Council (Moravcsik, 1998, p. 67). For Wallace, member states were no longer fully sovereign because they had to constantly negotiate and make compromises with each other in EU decision-making (Wallace, 1999, p. 506). By contrast, 'joint sovereignty' takes root with the emergence of joint national and supranational capacities for the physical enforcement of EU rules.
Following this line of thought, all EU agenciesincluding regulatory ones -fit the category of joint decision-making authority. Since they are governed by member states' representatives, the European Commission, and sometimes even the EP (Busuioc, 2012;Vos et al., 2018), agencies allow national and supranational actors to take decisions together. Conversely, only operational agencies participate in the physical implementation of EU policies 'on the ground' in areas such as border management or law enforcement (see section 2). In fact, their main goal is to limit, enhance, or even replace the coercive resources of member states in a given situation. Unlike regulatory agencies whose role in enforcement targets private actors, operational agencies affect the activities of public actors themselves. Moreover, unlike the Commission in infringement procedures (Scholten, 2017(Scholten, , p. 1350, operational agencies have a direct role in enforcement that typically involves their physical presence within member states' territories. From this standpoint, joint coercive capacities go beyond the horizontal coordination of national public administrations through networks (Heidbreder, 2017(Heidbreder, , p. 1371 or the convergence of national administrative practices in an emerging 'European administrative space' (Olsen, 2003).
The patterns we describe for the EU are potentially indicative of a broader phenomenon found in 'coming-together federations' (Kelemen, 2014;Stepan, 1999). When such polities face novel challenges that exert pressures for centralisation, they occasionally revert to similarly hybrid institutional structures as the EU. This is especially the case if, like the EU, such systems have developed highly institutionalised intergovernmental relations (Bolleyer, 2009) and already practice cooperative or executive federalism, whereby the federal and sub-federal level divide legislative and enforcement functions (Bolleyer & Thorlakson, 2012;Börzel, 2005). One example in recent history concerns the fight against terrorism. In most federal systems (Canada being an exception), law enforcement is a devolved competence. Since the 1990s and 2000s, some federations have resorted to the establishment of similar jointly owned capacities that bring together federal and sub-federal authorities in the enforcement of anti-terror legislation. Already pre-9/11, the Federal Bureau of Investigation established Joint Terrorism Task Forces uniting federal, state and local law enforcement authorities (Kilroy, 2019). In Germany, federal and Land police authorities and intelligence services established jointly owned enforcement structures such as the Joint Internet Surveillance Centre (GIZ) and the National Cyberdefence Centre (NCAZ) (Stüwe, 2019).
Keeping this in mind, in the next section we argue that the proliferation of joint coercive capacities has consequences for the ability of multi-level polities to implement their own policies effectivelyespecially in the EU.
Joint Sovereignty and the Problems of Multi-Level Implementation
The choice for joint sovereignty, which consists of sharing decision-making authority and coercive capacities, does not automatically lead to dysfunctions in policy implementation. When governance structures entail a clear division of decision-making authority and coercive activities between the two levels, joint sovereignty can lead to effective outcomes. Conversely, joint sovereignty can open a 'Pandora's box' of implementation deficiencies when the problems associated with joint decision-making come to undermine the effective use of shared coercive capacities at the enforcement stage.
We examine cases of multi-level policy implementation in an operational context as the locus where joint sovereignty problems are likely to occur. In order to map potential outcomes of joint sovereignty, we employ Matland's famous ambiguity-conflict model of policy implementation (Matland, 1995). The model was originally designed to explore top-down or bottom-up conflicts in policy implementation more generally (Hill & Hupe, 2002;Sabatier, 1986). While the model has been applied to the EU context before (e.g. Bossong, 2008;Heidbreder, 2017), we modify its parameters to capture the simultaneous implementation of operational tasks by two levels of governance.
In line with Matland's model, policy implementation can be understood by examining two dimensions: (1) the ambiguity of goals or means in a policy to be implemented and (2) the level of conflict between implementing actors (Matland, 1995, pp. 156-157). Transposed to joint sovereignty, ambiguity refers to the clarity of rules specifying the division of responsibilities between multiple levels of governance in the use of coercive capacities. When member states decide to create a new policy or institution with joint coercive capacities, they adopt a legislative act that is supposed to clarify the role of different actors in the implementation process. Such clarity is possible in theory. In practice, however, clarity is difficult to achieve when legislative agreements reflect compromises on the lowest common denominator between numerous parties with diverging preferences. In fact, according to Matland, ambiguity 'is often a prerequisite for getting new policies passed at the legitimation stage', overcoming conflict by allowing 'diverse actors [to] interpret the same act in different ways' (Matland, 1995, p. 158). In the EU context, an ambiguous division of competences between levels of governance is known to facilitate agreement in legislative decision-making (Jones et al., 2016;Pollack, 2003), yet it risks generating confusion or conflict at the implementation stage. Eventually, the choice for joint sovereignty via legislative ambiguity will catch up with national and supranational actors, resulting in vertical conflicts over the illdefined exercise of joint coercive capacities.
The other dimension necessary to understand policy implementation under a joint sovereignty model is the degree of conflict between implementing actors at different levels of governance. According to Matland, conflict describes a situation when 'more than one organisation sees a policy as directly relevant to its interests and when the organisations have incongruous views' (Matland, 1995, p. 156). When it is agreed that a policy requires joint coercive capacities, national and EU actors can discover that they hold incompatible preferences about the objectives or means of implementation; moreover, they can enter into jurisdictional disputes about their respective roles in the process (Heidbreder, 2017(Heidbreder, , p. 1372. Oftentimes, the intensity of such conflicts is linked to the decision-making stage, specifically to the strategies that allowed governments to overcome deadlock and adopt a common policy in the first place (Falkner, 2011, pp. 7-8). Such strategies include, among others, incomplete contracting, the use of qualified majority voting and resorting to different types of 'subterfuge' (Falkner, 2011;Héritier, 1999). What these strategies have in common is that they merely suppress conflict, and that political and administrative actors might later refuse to enforce decisions that run counter to their own preferences. Figure 3 captures the possible outcomes of joint sovereignty in multi-level implementation.
Based on the interaction between the adapted dimensions of ambiguity and conflict, we identify four possible outcomes of joint sovereignty in multi-level implementation. Whereas one of these constellations constitutes a case of effective multi-level implementation (division of responsibilities), there are three dysfunctional outcomes (non-implementation, responsibility-shifting and obstruction). We discuss them in turn below.
Division of Responsibilities
Joint sovereignty can result in effective multi-level implementation under conditions of low ambiguity and low conflict. If the EU creates joint coercive capacities with a clear understanding of the responsibilities assigned to implementing actors at different levels and without persistent conflict at the decision-making stage, a functional division of responsibilities is possible during implementation. Hypothetically, let us assume that member states wanted to create a jointly managed system for processing asylum applications to the EU. 8 In this scenario, all national competent authorities would support the instrument because they would see the benefit of EU help in the performance of operational tasks, such as the registration of asylum-seekers, interviews with applicants, or returns of migrants whose application was rejected. Consequently, the EU would be able to adopt a new policy that would clearly define the attribution of tasks between national and EU actors in the implementation of such a system. Once the system becomes operational, implementing actors would have unambiguous guidelines regarding their competences in the process while enforcement would be unencumbered by conflicts between levels of authority. In other words, implementation would run smoothly. More generally, a functional division of responsibilities is likely to occur in systems of administrative or executive federalism in which the federal level legislates and the sub-federal level enforces federal law (Bolleyer & Thorlakson, 2012). One example is the governance of public security in Switzerland, which continues to possess no federal police force (Kelemen, 2014). Consequently, the anti-terror units established in the wake of 9/11 remain fully canton-owned (Mohler & Schweizer, 2019).
Non-Implementation
The second constellation occurs when the EU establishes joint coercive capacities with little ambiguity over responsibilities between levels of governance but under conditions of high conflict among member states. National governments who oppose the policy are either outvoted through qualified majority voting or agree to the instruments in the context of 'package deals' where they strongly support the adoption of other measures (which are conditional on the contested policy). At the implementation stage, national authorities from the countries who opposed the instrument simply defy their respective obligations and refuse to contribute to multi-level implementation. To continue the example above, let us assume that not all member states are in favour of a jointly managed system for the processing of asylum applications. Despite a clear separation of competences between levels of authority, a few national governments object to the instrument because they want to preserve national sovereignty in asylum decisions. However, the objecting governments are not able to veto the measure in the Council due to qualified majority voting. After the instrument is adopted, national competent authorities refuse to cooperate with the supranational actors meant to support the processing of asylum applications on their territories. Consequently, the system as a whole is undermined, as EU actors are dependent on the cooperation of national authorities for the use of joint capacities in multi-level implementation.
While non-implementation is a theoretical possibility, it remains highly unlikely at the current stage in the evolution of the EU polity. In practice, in cases where member states oppose the creation of joint coercive capacities, there is either deadlock in decision-making or the instrument is adopted via differentiated integration, where each government can opt out (Leuffen et al., 2013). For example, only 22 member states participate in the European Public Prosecutor's Office. In any case, under conditions of high conflict, the possibility for the EU to adopt a policy with clear rules for the responsibilities of implementing actors (i.e. with low ambiguity) is virtually inexistent. In other systems, non-implementation indeed occurs on highly politicised issues. Examples include the refusal of various US states to enforce parts of the post-9/11 Patriot Act (Bulman-Pozen & Gerken, 2009) or the proclamation of sanctuary cities and states that refuse the enforcement of federal immigration law (Amdur, 2016). In the EU, by contrast, most current problems of multilevel implementation stem from ambiguous rules at the decision-making stage, which can breed the two types of dysfunctionalities discussed below.
Responsibility-Shifting
This constellation is bound to occur when the EU adopts a new policy which requires joint coercive capacities under conditions of high ambiguity (the legal framework lacks clarity regarding the division of responsibilities between different levels) and low conflict (with no persistent opposition from individual member states). In such cases, implementation responsibilities remain unclear, opening up the possibility for national actors to shift responsibility for the execution of specific tasks to the EU level, or the other way around, for EU actors to claim that responsibility belongs to national authorities. This constellation is deficient because it results in incomplete implementation or outright failures to act in specific cases. In the hypothetical example above on the joint management of asylum applications, responsibility-shifting would be the result of member states agreeing in principle to the utility of such a system, but without discussing the details of the division of tasks between levels of governance in implementation. Once the system becomes operational, it is unclear who is supposed to do what, allowing each level of governance to shift the blame for poorly implemented measures. Responsibility-shifting occurs frequently in multi-level settings (Heinkelmann-Wild & Zangl, 2020), and agencification has even bred new variants of this pathology (Mortensen, 2016). One example in the US context was Hurricane Katrina where, despite disaster management being a competence shared between the federal and the state level (Birkland & Waterman, 2008), Louisiana and federal officials attributed the responsibility for mishandling the crisis to the respective other (Maestas et al., 2008).
Obstruction
The fourth and final constellation is bound to occur when the EU establishes joint coercive capacities under conditions of high ambiguity (lack of clarity regarding the division of responsibilities between levels of governance) and high conflict (unresolved domestic contestation of decisions adopted despite national opposition). In such cases, both national and supranational administrative actors participate in multi-level policy implementation but have not defined a clear division of responsibilities between themselves; moreover, the two levels hold contrasting views of whether and how policies should be enforced. The constellation is deficient because it leads to uncoordinated and contradictory implementing action that wastes resources (money or time) for both national and EU actors. In the hypothetical example of joint processing of asylum applications, these problems emerge when a few member states oppose such a system yet cannot prevent its adoption at the EU level. Instead, they agree to create a system ambiguous enough to allow national authorities to water down their contribution to policy implementation. For example, national asylum authorities can formally invite EU actors on their territories but only grant them partial access to asylum-seekers, thus obstructing the management of the joint processing system. Lower-level obstruction is among the classic pathologies of the literature on top-down implementation (Derthick, 1972;Pressman & Wildavsky, 1973;Stoker, 1991) and remains a common issue in multi-level systems. Recent examples of administrative obstruction include US states that wilfully seek to sabotage the roll-out of the federal Aid to Families with Dependent Children program (Bulman-Pozen & Gerken, 2009) or of Obamacare (May, 2015).
Keeping in mind that non-implementation is empirically unlikely in the current EU context (for the reasons stated above), we argue that responsibilityshifting and obstruction represent the likely systemic deficiencies of the joint sovereignty model. More generally, joint sovereignty is bound to create problems in policy implementation because the conditions that allow its establishment in the EU multi-level system, that is, high rule ambiguity, come to undermine the ability of that very system to effectively use coercive capacities in practice.
From an empirical perspective, how do we recognise the deficiencies of joint sovereignty when we see them? The observable implications of our argument presuppose an analysis of the deliberate inclusion of ambiguity at the decision-making stage and the occurrence of conflict at both the decisionmaking and the implementation stage. Initially, we have to examine the extent to which a new policy fails to specify the division of implementing tasks between national and supranational actors with joint coercive capacities, and the degree of decision-making conflict between political actors at both levels of governance. Next, we move to investigating the effectiveness of policy implementation, with a focus on dysfunctionalities encountered in the process of enforcing operational tasks. Finally, we assess the causes for implementation failure, testing the connection to high ambiguity of coercive responsibilities and/or unresolved conflict at the decision-making stage that continues to manifest itself during multi-level implementation.
Illustration: Joint Sovereignty in Practice
Given the novelty of the phenomenon under analysis, we test the plausibility of our argument by examining two crucial cases (Gerring, 2007) that correspond to the systemic implementation deficiencies of the joint sovereignty model. Our attempt is exploratory (Stebbins, 2001)aiming to empirically identify the occurrence of responsibility-shifting and obstruction in cases that clearly fulfil the scope conditions of our argument. We specifically focus on two examples of EU agencies with operational tasks, namely, Frontex (dealing with border management) and Europol (covering police cooperation). Both agencies are perfect examples of the choice for joint sovereignty in the EU contextnot least because border control and law enforcement are key core state powers (Genschel & Jachtenfuchs, 2016). At the EU level, the mandate of the two agencies has expanded significantly in recent years, in parallel to their budgets (see the Supplemental appendix). In theory, the functioning of these agencies should offer ample scope for the manifestation of joint sovereignty problems in policy implementation; to paraphrase Levy's ideas about the inverse Sinatra inference: if our expectations about joint sovereignty 'cannot make it here, [they] cannot make it anywhere' (Levy, 2008, p. 12). What follows is an empirical illustration of responsibility-shifting and obstruction in relation to Frontex's mandate on the protection of fundamental rights and Europol's mandate on the gathering of intelligence for counterterrorism purposes.
Shifting Responsibility for Human Rights between Frontex and Member States
As outlined above, we expect responsibility-shifting to occur under conditions of high ambiguity (regarding the division of responsibilities between levels of governance) and low conflict (with no persistent opposition from individual member states). In such instances, administrative actors on both levels exploit ambiguous mandates in order to shirk responsibility for deficient policy implementation. The approach to human rights under the joint operations led by Frontex and the Greek border authorities between 2006 and 2020 provides a case in point.
Frontex was established in 2004 through Regulation 2004/2007. Its formal purpose was to ensure effective management of the EU's external borders by coordinating operational tasks implementing relevant EU measures. This included monitoring migratory flows, carrying out risk analyses and vulnerability assessments, as well as coordinating joint operations and rapid border interventions at the external borders (Ekelund, 2017). Since 2004, the agency was reformed four times in order to respond to increasing migratory pressures at the EU's external borders. This was done by means of a more robust mandate (Ripoll Servent, 2018) and an immensely increased budget. However, since the beginnings of its operations, Frontex has been criticised by several scholars and practitioners for its alleged non-compliance with international human rights law (Ekelund, 2017). Following our main argument, we trace these deficiencies back to the high ambiguity of Frontex's mandate and the absence of (open) conflict over human rights violations between the agency and national authorities.
In the regulation establishing Frontex, the issue of human rights was only mentioned in the Preamble in relation to Article 6(2) TEU and the EU Charter of Fundamental Rights. This reflected the limited scope of operational activities envisaged for the agency: there was no conflict regarding its responsibilities for fundamental rights protection because that was not considered a possibility at the time. However, the lack of clarity in this respect will become problematic at the implementation stage with the increase of Frontex activities over time. Since 2006, several border patrol operations known as the 'Poseidon Joint Operation' have been carried out at the Greek borders under the supervision and assistance of Frontex and with the participation of EU member states that provided personnel and equipment. Until 2010, these missions had a rather limited budget and focused on the monitoring of migrants smuggling and border guards training (Frontex, 2011). In 2010, in response to an unprecedented migratory pressure on the Greek-Turkish border, the Greek government requested the direct intervention of Frontex Rapid Borders Intervention Teams (RABIT), a system to 'provide rapid operational assistance, for a limited period of time, to a requesting member state facing a situation of urgent and exceptional pressures' (Regulation 863/2007, Art 1). Over 4 months, Frontex activity increased massively and coordinated the deployment of a total of 567 officers from 26 member states and Schengen-associated Countries (Frontex, 2011). The deployment of this mission together with the steady increase of Frontex operational activities (Keller et al., 2011) raised the first serious concerns regarding the ambiguity of the Frontex mandate as to human rights protection.
Among the most worrisome aspects was the unclear division of responsibilities between border guards from other EU member states, Frontex and the requesting member state (Carrera & Guild, 2010). According to Amnesty International and the European Council on Refugees and Exiles (ECRE), the blurring of tasks and responsibilities in the Poseidon Joint operation's mandate 'potentially permits member states to engage in border management with impunity' (Amnesty International & ECRE, 2010). In 2011, these concerns were echoed by the European Court of Human rights (ECtHR) in a judgement (M.S.S. v. Belgium and Greece, 30696/09) which found that conditions in Greek migrant detention centres at the time were 'inhuman and degrading' (European Court of Human Rights, 2011). During the 2010-2011 RABIT mission in Greece, Frontex also facilitated the transfer of migrants to detention centres in which Human Rights Watch documented similar conditions to those condemned by the ECtHR. Nevertheless, neither the agency nor national border guards were held responsible (Human Rights Watch, 2011). The agency defended itself by stating that it had 'no direct role in the immigration or asylum systems of member states and especially not in detention' (Arias Fernández, quoted in Mann, 2011). From the EU side, the Commission spokesperson for internal affairs claimed that 'Frontex should not be held responsible for the failings of a member state, in this case Greece' (quoted in Traynor, 2011).
In 2011, the agency's mandate was amended to 'rapidly strengthen the competences of Frontex and put more effective tools at its disposal' (JHA Council, 2011). Under pressures exerted by the EP and the Fundamental Rights Agency (European Union Agency for Fundamental Rights, 2013), EU Regulation 1168/2011 also required Frontex to adopt a 'Fundamental Rights Strategy' (Article 26a Regulation), whose implementation was to be actively monitored (Articles 26a [1]) with the help of a Consultative Forum on fundamental rights (Article 26a [2]) and a Fundamental Rights Officer (Article 26a [3]). However, when the EU Ombudsman recommended Frontex to establish a complaints mechanism, the agency rejected the proposal by stating, once again, that individual incidents are the responsibility of the respective member state (European Ombudsman, 2013). In 2012, Frontex eventually sought to develop internal procedures for staff and guest officers to report possible violations. Still, the ambiguity of the legislation limited its practical applicability. According to Amnesty International, 'the lack of a clear mechanism for investigating reports of human rights abuses from joint operations or operational areas where Frontex is present and the inability to handle individual complaints means that this human rights framework is, in practice, of limited discernible impact' (Amnesty International, 2014).
In the following years, border management activities under the Poseidon Joint operation were the stage of further illegal pushbacks perpetrated by Greek authorities in areas of Frontex jurisdiction, that is, on the land border between Greece and Turkey in Evros and a large section of the Aegean Sea (Amnesty International, 2014). In such circumstances, Frontex would have had the power to activate Article 3(1)a of the Frontex Regulation to suspend the operation, yet it failed to do so.
In response to preoccupations voiced by several institutions including the Ombudsman and the EP (European Parliament, 2015), EU Regulation 2016/ 1624/EU incorporated new safeguards with regard to the human rights obligations of the actors participating in the activities of Frontex. Article 72, in particular, established a mechanism to allow migrants and asylum-seekers the possibility to lodge individual complaints about fundamental rights violations committed by staff involved in Frontex activities (Carrera & Stefan, 2018). At the same time, since 2015, Greece has been accused of illegal pushbacks of thousands of refugees (Kingsley & Shoumali, 2020). There is also recent evidence of multiple instances in which Frontex personnel was either present at pushbacks, or sufficiently close to be aware of them (Waters et al., 2020). While denying any involvement, Frontex pointed out that it can suspend officers on its operations, but has neither 'the authority over national border police forces' nor 'the power to conduct investigations' on the territory of EU member states (Euractiv, 2019).
From the outset, it appears that Frontex reforms over time have primarily focused on member states' desire to strengthen border protection, rather than upholding human rights standards (Lavenex, 2015). The insertion of human rights protection mechanisms has run in parallel to the empowerment of Frontex. At the decision-making stage, this may have been rather uncontroversial as the EU legislator is bound to commit to human rights protection in line with principles enshrined in the European Charter of Fundamental Rights. However, at the implementation stage, the reforms have not reduced the ambiguity over responsibilities or indeed curbed human rights violations under Frontex's watch (Carrera & Den Hertog, 2016). Ultimately, the effectiveness of the multi-level implementation of human rights norms in the EU border regime is systematically undermined by the ambiguity of the mandate and responsibilities of Frontex and national border guards. In the absence of any visible conflict between the EU agency and national authorities, this ambiguity has allowed the creation of broad legal grey areas where both administrative levels can shift the responsibility for human rights violations on the respective other. While Frontex continuously shifts its responsibility for pushbacks onto member states, administrative actors on neither level of authority are effectively held accountable for the deficient implementation of their mandate (Fink, 2020).
National Obstruction of Europol's Counter-Terrorism Efforts
In contrast to responsibility-shifting, we expect to see obstruction under conditions of high ambiguity (regarding the division of responsibilities between levels of governance) and a high level of conflict between implementing actors. In such instances, administrative actors on one level dispute the authority of actors on the other level, undermining the efficiency of multi-level implementation through non-cooperative action. The counterterrorism activities of the European Union Agency for Law Enforcement Cooperation, known as Europol, provide a case in point.
In January 1994, Europol began limited operations outside the EU treaty framework as the Europol Drugs Unit, focusing specifically on enhancing the exchange of information between member states concerning the illicit trafficking of drugs (Niemeier & Wiegand, 2010). It was only in 2010 that the Lisbon Treaty integrated Europol into the EU treaty framework, officially rendering it an EU agency (Article 88 TFEU). Over time, Europol's substantive and operational mandate increased substantially. Areas of operation now extend to virtually all types of organised and transnational crime and, since 9/11, have included counter-terrorism activities. Europol has also built up significant expertise beyond information exchange, in areas such as strategic analysis, intelligence analysis, forensics, operational coordination of member state investigations, and by participating in joint investigation teams with the member states (Aden, 2018). As of 2020, Europol disposes of 1300 staff (Europol, 2020d) and an annual budget of EUR 158 million (Europol, 2020c).
Collecting and connecting (Cruickshank, 2017) information on crime and terrorism in (and also beyond) the EU is part of Europol's core business. As the Treaty stipulates, Europol is to engage in the 'the collection, storage, processing, analysis and exchange of information, in particular that forwarded by the authorities of the member states or third countries or bodies' (Article 88 [2] TFEU). To this end, Europol has created the Europol Information System (EIS). Established in 2005, the EIS is to collect all central criminal information and intelligence of transnational relevance in and for the EU. According to Europol, the database 'contains information on serious international crimes, suspected and convicted persons, criminal structures, and offences and the means used to commit them' (Europol, 2020b). Since 2013, the EIS has also contained DNA samples. The EIS is interlinked with SIENA (Secure Information Exchange Network Application), Europol's encrypted messaging environment. By the end of 2019, the EIS contained close to 750,000 data points, over 50% of which were related to terrorist activities (Europol, 2020a).
Despite this record of gradually increasing capacities, the effectiveness of intelligence-sharing in counter-terrorism via Europol has proved persistently low. Deficient multi-level implementation has led some observers to conclude that EU 'anti-terrorism convergence has failed' (Wieczorek, 2018, p. 53). In line with our paper's conceptual framework, we trace this finding to a high level of both rule ambiguity and implementation conflict concerning the sharing of data between Europol and member states. Reticent to disclose what they regard as highly sensitive information and oftentimes preferring more easily controllable bilateral information channels over the EIS, member states have a longstanding record of non-cooperative behaviour that has consistently obstructed the effective implementation of counter-terrorism policies in the EU.
EU legislation that mandates Europol to engage in intelligence-sharing activities has specified member states' data-sharing obligations in a notoriously ambiguous manner. In 2002, the first Council decision (2003/48/JHA) on police and judicial cooperation to combat terrorism stipulated that 'each member state shall take necessary measures to ensure' (emphases added) that all available information on terrorist activities in the respective member state be transmitted to Europol. Despite specifying the type of information to be transmitted to the EIS (i.e. name, date and place of birth, nationality, sex, place of residence, profession, identification documents, fingerprints and DNA profiles), the subsequent Council decisions of 2005 (2005/671/JHA) and 2009 (2009/371/JHA) kept this original wording intact. Consequently, EU member states are legally obliged but cannot be forced to disclose their information with Europol. The same applies to the latest Europol Regulation (2016/794), which depicts Europol as 'a hub for information exchange in the Union' and states that 'clear obligations should be laid down requiring member states to provide Europol with the data necessary for it to fulfil its objectives', but otherwise maintains the original wording of 2002. Despite a sophisticated EUlevel legal and technical infrastructure in place via various decisions, regulations and the EIS, member states retain ultimate ownership of their intelligence and control de facto the extent to which they share sensitive intelligence with others.
The high level of ambiguity in EU rules on counter-terrorism intelligencesharing stems from conflicts among and between member states and EU institutions over the sensitivity of the data to be shared. Since 2002, member states have frequently ignored the various Council decisions that had encouraged them to share particularly the most sensitive categories of intelligence with Europol. As Bureš observes, 'national security and law enforcement agencies are still too often reluctant to share "high-grade", real-time intelligence on terrorism that can be acted on immediately.
[…] Although numerous Council decisions and Commission proposals include an obligation for EU member states to share information, in practical terms, this duty has had little impact because it cannot force member states' authorities to share more information, that is, intelligence that has not previously been disseminated' (Bureš, 2016, p. 61).
Ultimately, member states' deficient sharing of intelligence with Europol has variously undermined the efficiency of counter-terrorism efforts in the EU. Already in the run-up to the 2004 Madrid train bombings, security agencies in France, Germany, Italy, Norway and Spain had kept information about terror suspects to themselves, refraining from sharing it with their EU partners (Gebauer, 2004). Before the 2015 Paris attacks, again, Belgian authorities withheld information they had gathered on the Abdeslam brothers from Europol, depriving French services of important knowledge on the latter's prior criminal activities (Bureš, 2016). Ahead of the 2017 Barcelona attacks, the Catalan police (Mossos d'Esquadra) had repeatedly demanded EIS access, but was hindered by national police forces (Carrera et al., 2017). Ultimately, in an institutional environment characterised by a high level of role ambiguity, persistent conflict about the extent of member states' obligation to share sensitive data with Europol has bred a longstanding pattern of obstructive behaviour. This obstruction has seriously hampered the effectiveness of counter-terrorism efforts in the EU multi-level system.
Conclusion
To sum up, the rise of EU agencies with operational tasks is a new and expanding phenomenon that captures the exercise of joint sovereignty in the EU. Under joint sovereignty, national and supranational actors take decisions together and share the coercive capacities necessary to implement policies 'on the ground' on member states' territories. EU agencies with operational tasks illustrate both features and additionally allow member states to avoid full transfers of competence to supranational institutions while benefiting from resources created at the EU level. At the implementation stage, however, the exercise of joint sovereignty poses specific challenges. Whether intentionally or not, the institutional choice for joint sovereignty opens the way for several implementation deficiencies that undermine the ability of both national and supranational actors to enforce policies effectively. We subsequently identified two likely dysfunctions in policy implementation and connected them to ambiguity and conflict at the decision-making stage. As shown by the case of human rights violations in border management activities, responsibilityshifting occurs under conditions of high ambiguity and low conflict. By contrast, the case of Europol's intelligence-gathering efforts in counterterrorism illustrates obstruction. In this case, high ambiguity and high conflict between implementing actors allow national authorities not to share data with each other and thus undermine the whole system. Overall, joint sovereignty marks a new stage in the evolution of the EU polity but breeds new systemic pathologies that hamper the effectiveness of policy implementation.
In the future, joint sovereignty warrants additional investigation in different directions. First, future research should move beyond the cases currently covered. Both Frontex and Europol provide instructive illustrations of the mechanics of joint sovereignty dysfunctions. The pathologies they describe, however, may apply to other newly established (or reformed) agencies with operational tasks. For instance, the European Labour Authority, established in 2019, has the power to coordinate and support the execution of concerted and joint inspections of companies suspected of labour mobility abuses, together with national authorities (EU Regulation 2019/1149, Articles 8-9). The European Public Prosecutor's Office began its operations at the end of 2020, combining under one roof supranational and national efforts to investigate fraud against the EU budget. The COVID-19 crisis has led to a proposal to turn the European Centre for Disease Prevention and Control into a European Health Agency able to coordinate the management of health threats (Deruelle & Engeli, 2021). In June 2021, the EU legislators have agreed to turn EASO into the European Union Agency for Asylum (EUAA), able to provide stronger operational and technical assistance. Second, scholars should look beyond the EU case in exploring the dynamics of joint sovereignty in multilevel implementation. In this article, we provided various examples of similar institutions and pathologies in cooperative coming-together federations. Systematic comparison could aid in exposing the specificities of joint sovereignty in the EU and in predicting its durability and future development.
Finally, future analyses should move beyond the confines of the here and now. Joint sovereignty is a recent development of the EU's multi-level system, and its mechanics could evolve. For instance, the European Commission's current criticism of human rights violations at sea might encourage Frontex (European Commission, 2020) to take a more conflictual stance vis-à-vis national authorities in the future, which could eventually lead to a replacement of the current pattern of responsibility-shifting with obstructive action. Were this the case, the implementation deficiencies described here would amount not merely to a highly dysfunctional but also a lasting dynamic of European integration. 7. Income deriving from industry fees is excluded as this would distort the picture.
Disaggregated budget figures are included in the supplemental appendix and in the CPS dataverse (Freudlsperger et al., 2021). 8. In fact, the EU attempted to create such an instrument with the establishment of hotspots at the external borders during the refugee crisiswith mixed results (Neville et al., 2016;Tsourdi, 2016). | 2022-01-19T16:28:49.765Z | 2022-01-17T00:00:00.000 | {
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212696721 | pes2o/s2orc | v3-fos-license | Analytical View on the Impact of Foreign Aid on the Palestinian Local Development within the Framework of the Local Government Sector
Citation: Bawatneh S.J. (2020) Analytical View on the Impact of Foreign Aid on the Palestinian Local Development within the Framework of the Local Government Sector. Open Science Journal 5(1) Received: 25 June 2019 Accepted: 26 December 2019 Published: 10 February 2020 Copyright: © 2020 This is an open access article under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The author(s) received no specific funding for this work Competing Interests: The author have declared that no competing interests exists. This paper trying to assess the impact of foreign aid on the Palestinian local development by focusing on the role of PLGS and monitoring the readiness of the Palestinian local development plans to face and manage the future in case foreign aid is cut off permanently from the State of Palestine. The paper poses main question: Is foreign aid “within the framework of the PLGS” being channeled within the proper course of local development? Taking into account the exceptional circumstances of building a State under colonialism and under a centralized system of government. Main results were presented via SWOT analysis which based on deep literature review, interviewing local officials, and identification of performance indicators which used in the assessment. Findings of this study pointed out that despite the fragility of the Palestinian local governance sector which has many internal problems and external challenges; there are many opportunities that must be invested within the available potentials in order to achieve sustainable local development. Besides, reducing the value of foreign aid until do without it is the proper course toward sustainable local development through changing the mentality of consuming into investing. The study presented many valuable recommendations to correct the path of local development in the state of Palestine and how to activate the positive aspects that related to obtaining foreign aid. Developing countries can rely on the results of this study as they are similar in the fragility of their administrative systems.
This paper trying to assess the impact of foreign aid on the Palestinian local development by focusing on the role of PLGS and monitoring the readiness of the Palestinian local development plans to face and manage the future in case foreign aid is cut off permanently from the State of Palestine. The paper poses main question: Is foreign aid "within the framework of the PLGS" being channeled within the proper course of local development? Taking into account the exceptional circumstances of building a State under colonialism and under a centralized system of government. Main results were presented via SWOT analysis which based on deep literature review, interviewing local officials, and identification of performance indicators which used in the assessment. Findings of this study pointed out that despite the fragility of the Palestinian local governance sector which has many internal problems and external challenges; there are many opportunities that must be invested within the available potentials in order to achieve sustainable local development. Besides, reducing the value of foreign aid until do without it is the proper course toward sustainable local development through changing the mentality of consuming into investing. The study presented many valuable recommendations to correct the path of local development in the state of Palestine and how to activate the positive aspects that related to obtaining foreign aid. Developing countries can rely on the results of this study as they are similar in the fragility of their administrative systems.
Introduction
The controversy about the association between foreign aid and local development dates back to the post-World War II period (In'airat, 2007). "The Paris Declaration (PD), 2005" may be seen as the most important attempt between others for many decades, which regarding as an international obligation to guide foreign aid more effectively (Dabelstein, 2013). To reach the poor, improve the quality of life, improve the performance of development, strengthen governance, and achieving sustainability. Five basic principles were developed by Paris Declaration to ensure the effectiveness of the donations; ownership, alignment, harmonization, managing for results, and mutual accountability. Further, twelve indicators were set up based on the mentioned five principles for monitoring the progress of its implementation which are valid up to 2010 (OECD, 2008).
Numerous critics have criticized the implementation of the Paris Declaration on the ground, whence there is a great gap between the high-level items contained in the convention and the reality of chaotic development after implementation (Mirapeix, 2011). Furthermore, the application of Paris Declaration reveals that donors are framing the policies and strategies of recipient countries as a prerequisite for receiving grants, thereby undermining and limiting the development options of their peoples within a specific list that is far from the development goals of the society itself. That is contrary to the Paris Declaration regarding to the preservation of ownership, the democracy of peoples, and building local capacity (Mahmud, 2008).
In addition, there are undeclared targets for the foreign aid, which hide behind its stated goal of influencing the policy making process of recipient countries. (Ex.: "Privatization of Utility"; projects are motivated by poverty reduction but contain the obligation of governments to contract with companies especially for sale the basic services to citizens). Instead of being a support for people's democracy and a source of security, they are a clear violating the democracy and sovereignty of nations, through the conditions accompanying aid (Mahmud, 2008). On the other hand, donors often force inappropriate policies; create transaction costs, control financing without referring to recipient countries, etc. Indirectly they mislead local democratic processes. As a result, donors are outside the local political process of developing country, so they are not accountable according to the people or elected parliaments (Mahmud, 2008).
Some experts explain that donations often enabling bureaucracies in governments; bad governments continue, the aristocratic and elite in poor countries continue enlarging their wealth, or these aids just been lost. Whereas the reality witnesses widespread of poverty especially in Africa and south Asia (Jayathilaka, 2009). Radelet clarified that donations are not necessarily be conducive to development. Aid might be wasted and stimulate corruption or enabling bad governments in power and postpone reform. Another claim that, aid support countries within the war, to fund and preserve the conflict (Radelet, 2006).
Open Science Journal -February 2020 3 Aid fails in its goals because it`s incentive structure is similar to the central planning. Foreign aid sustains poverty, supports and empowers the responsible for this situation and stimulates corruption. Reform foreign aid will not change structural systems; aid conditionality, or aid privatization, foreign aid will remain a waste of resources and counterproductive (Prokopijevic, 2006). The problem of foreign aid is often "corruption". Corrupt regimes impede the normal development of aid, that is, it hinders the positive outcome that aid can produce (Ahmed, 2014). Foreign aid system is broken today; developing countries should try to explore new strategies for poverty alleviation (Kelter, 2018).
Research Problem
The phase of state building automatically means the situation of political and economic instability, therefore; the prospects for establishment an Independent Palestinian State are very limited, especially in light of existence an ambitious Israeli colonization in the region. The problem of this study is concentrated in that, under the mentioned critical phase, The State of Palestine receives a large amount of foreign aid. After twenty-six years of establishing the State of Palestine, and under a centralized system of government, local government bodies are still fully dependent on foreign aid for the implementation of its own infrastructure projects, which increases the concern about the future of the Palestinian state entity.
On the other hand, the priorities of most donors agree to reduce poverty rates; subsequently there is a trend towards supporting projects for the lowincome people within Fragile States 1 . The dilemma, after the implementation of such projects on the ground, the standards fluctuate, whereas these projects become preserved for the highest strata of society and forbidden for the poor, which promote the social segregation 2 within the same society and the same race (Terenzi, 2014). Furthermore, poverty rates as one of the most crucial indicators for assessing the performance of local development, pointed out according to Palestinian Central Bureau of Statistics (PCBS, 2018); the rates of extreme poverty are increasing continuously in the Palestinian context.
Research Questions
The main question: Is foreign aid "within the framework of the PLGS" being channeled within the proper course of local development? Taking into account the exceptional circumstances of building a State under colonialism.
Population is growing in worrying 3 rate, how willing is the PLGS to manage the future of local development if foreign aid is cut off permanently? Why has the Palestinian local government bodies not yet been able to implement infrastructure projects by its own without the donations of other countries? On the other hand, if the poverty rates are increasing continuously in the Palestinian context, then who actually benefits from the foreign aid?
1 Fragile State: The term first appeared at the end of the 20th century. The features of the fragile state are characterized by the weakness of its legitimacy within its borders, its inability to maintain internal security, the inefficiency of its services to the citizens, the weakness of its institutional structure, poor allocation of resources, and existence of a continuous dynamic situation and instability (Brinkerhoff, 2011) 2 Rawabi city as Palestinian example has started as affordable housing project, which funded by different Arabic and foreign donors, but now Rawabi city is customized for the velvet layers and fully empty from the low-income people, which consolidate the meaning of social segregation (Terenzi, 2014
Goals and Objectives
This study aims mainly to: ▪ Trying to assess the impact of foreign aid on the Palestinian local development within the framework of the local government sector. ▪ Monitor the readiness of the local development plans (within the PLGS) to face the future in case the foreign aid is cut off from the State of Palestine.
The limits of the study The nature of a State under construction phase imposes receiving foreign aid. Unfortunately, foreign aid supports all aspects of life in the Palestinian model, including the payment of salaries to public officials. This study focuses on the role of foreign aid that directed to the PLGS, and how they affect local development, especially since the Paris Declaration 4 so far. Why the study focused on the local government sector? Because the local government sector includes local government bodies which are considered as nuclei 5 of local development within the Palestinian model.
Methodology
In order to achieve the objectives of this study, three main stages were adopted serially. The first stage: The priorities of local development for both donors and recipient "PLGS" were reviewed and identified. Then these priorities had compared in order to determine the extent of compatibility and difference of priorities between both parties. That required reviewing the "Strategic Development Plans" of the PLGS, in addition to interviewing officials within this sector, specifically from the Ministry of Local Government, Municipal Development and Lending Fund, and some local municipalities from the other side, interviewing officials from donors.
The second stage: For assessing local development performance by the foreign aid, performance indicators must be identified. Identification the performance indicators were based mainly on a global and local review of the literature with a holistic and analytical approach, guided by the main principles of the Paris Declaration and the Millennium Development Goals, in addition to consulting some local experts and officials within the PLGS, such as; Municipal Development and Lending Fund, etc.
The third stage: An attempt to diagnose the impact of foreign aid on the Palestinian local development within the framework of the PLGS. In order to present a detailed and clear picture about the Palestinian model, SWOT analysis had been done from a Strategic 6 planning perspective, based on all the data of this study whether literature review, or interviews. Besides, reading the Palestinian model through the predefined performance indicators, every tiny idea Open Science Journal -February 2020 5 has employed within the SWOT analysis. This is to enable readers and researchers from different backgrounds and purposes to form their impressions or decisions about the Palestinian experience objectively and not on aspirations and hopes.
Results and discussion
Literature review Foreign Aid Effectiveness -Universal Scale Aid effectiveness is related to its compatibility and reasonability with the critical value which usually determined by the recipient country's absorptive capacity (Asra, 2005). If there are fragile infrastructure, insufficient skilled workers, and controlled transport systems, for sure the effectiveness of aid will reduce for the recipient country (Radelet, 2006). (Elayah, 2016) view developing countries as a closed and empty circle, therefore the ineffectiveness of foreign aid comes from being filled with fragile institutional environment, weak policies and corruption. Numerous attempts were done but the truth that reforming these regimes is a difficult task. Another opinion illustrate that the legal framework must be fortified for the developing countries to ensure effective aid, rather than over dependency on the flow of ODA, which absolutely will lead to negative impacts on the macro level of the development (Yiew, and Lau, 2018).
Furthermore, foreign aid have an adverse result on the economic growth of developing countries that proved by testing 85 sample (Ekanayake, 2010). Both the bilateral aid and multilateral aid are ineffective by its own for enhancing economic development, regardless whether one measures them for GDP of recipient or per capita (Wako, 2011). Several studies explored that there is a Ushape relationship among foreign aid and economic development (Ravallion, 2014). Whereas the impact on development is negative at first, but over the time becomes a positive character (Yiew, and Lau, 2018).
Aid could be effective if given for nonstrategic purposes, good policies, and democratic organizations. While fragmented aid or aid quantity, which exceed the absorptive capacity of the recipient country, for sure will reduce aid effectiveness (Dreher, 2017). However, there has been no evidence so far that the effectiveness of aid is increasing if it is given to countries with good policies, but evidence shows that the effectiveness of aid decreases as the flow of aid increases (LENSINK, and WHITE, 1999).
Universal Cases Study
By highlighting some experiences of developing countries through studying the impact of foreign aid on their local development, literatures review pointed out that Tanzania receives a large amount of foreign aid but still live in high poverty levels with very low economic growth. By studying the impact of foreign aid on GDP results shows that total debt service and foreign assistance have a negative impact on GDP growth while export growth and the net national savings have an optimistic impact on GDP growth (Kabete, 2008).
Kenya has unstable macroeconomic policy environment. Therefore negatively affected public investment and economic development, despite the reformations of Open Science Journal -February 2020 6 macroeconomic policy that had been done. Furthermore, foreign aid has unpredictable features in Kenya so local development also has unpredictable features (Ojiambo, 2009). According to the African continent generally, foreign assistance programs have to complement the national budget of developing countries, reduced the debt burden and achieved local development, but soon proved inefficient. Moreover, the burden of developing countries on conditions those are difficult to achieve in return for such assistance, and therefore not achieve the results of development and increase citizens' damage and exhaustion. The results of local studies have shown the failure of these programs and all initiatives on the African continent to create sustainable development strategies (Niyonkuru, 2016). Despite Africa receiving more than ($600 billion) of foreign aid, large part of Africa remained undeveloped with an extreme poverty (FARAH, 2018).
Foreign aid in the region Sub-Saharan Africa also doesn't have any important effect on economic growth. Despite that, education has a significant result on growth (Ahmed, 2014). In addition, foreign aids do not have any impact on GDP growth in the experience of Ethiopia, while it has a considerable impact on FDI. Furthermore, foreign aid has negatively associated with corruption levels in the country (FARAH, 2018). The negative side of foreign aid in Pakistan that Pakistan's debt load increased over time (Mohey, 2005). Regarding to the Zimbabwe, although aid to Zimbabwe has been conditional by the donors, it has tried to contribute to economic growth. However, it made the government get used to it (Moyo, and Mafuso, 2017).
Foreign Aid Effectiveness -Local Scale (WB)
Regarding to the Foreign aid effectiveness at the local level, in WB aid effectiveness depending on the quality of recipient governance, strategic objectives of donors, and the occupation. Moreover, the culture of corruption, which poses a considerable and harmful factor to the effectiveness of aid (In'airat, 2007). The existing situation should be transformed into Decentralization to empower local governing, but conserving the national unity to counteract the Israeli occupation dynamics (GIZ, 2017).
Palestinian economy is dependent on Israel economy through; the labour market, Israel regarded as the most significant trading partner for the WB and GS. In addition, Israel collects the taxes on behalf of the Palestinian Authority, which usually accounts for about two-thirds of the PA's total revenue (Bennett, 2003). Palestinian economy is below siege (Dajani, 1998). There are three levels of obstacles to the Palestinian economy within explicit policies. At the international level, there are donors and their funding policies. At the regional level, there are Israelis and their closure policy. At the local level, there are Palestinians and their investment policies (In'airat, 2007). Furthermore, in 2017, Trump sought to cut aid to the Palestinians as a tool to pressure them to achieve peace and accept Jerusalem as the capital of Israel (Congressional Research Service, 2018). (Shoukair, 2013) view that Palestinian economy suffering from "Dutch disease" which has been used extensively to explain why external development aid can be ineffective in creating economic growth. Terrorism and economic policy and the restrictions imposed by Israel are the main factors of the disease in this period. Under the current economic and political constraints, foreign aid had little chance of significantly strengthening trade sectors.
Donations are given to the Palestinian either through direct payments or through the Palestinian Welfare Association, which initially had a development aspect. However, recently commitment to a combination of both sides: relief work with a gradual move to the development work. The main problem of Arab funding is the absence of policies to invest in the Palestinian context. However, the Welfare Association experience is regarded as pioneering and outside the Western funding context (Hamdan, 2011).
Local Development Priorities within PLGS Local Development Priorities of the PLGS
The PLGS consists of the Ministry of Local Government, local government bodies, the Municipal Development and Lending Fund and the Federation of Municipalities. Following the review of the sector's "Strategic Development Plans", it is clear that the policies and objectives of the PLGS have been built within the considerations of the Palestinian national policy agenda. Top priorities of the sector are; to move towards a decentralized local governance sector, rehabilitation of local government institutions and development of their human and institutional capacities, improving the quality of provided services to the citizens, achieving democracy, transparency and community participation, and strengthening partnership with the private sector. In addition to activating local economic development, achieve financial sustainability of the local bodies, enhance their creditworthiness, enable them to borrow, activate the lending of municipalities from the Municipal Development and Lending Fund, and exert more efforts to attract funding from international development partners and encourage and sustain foreign aid to the sector (Ministry of Local Government, 2016).
The latest Palestinian strategic plan of the local government sector explained the reason for the decline in the Palestinian investment rate after 2013, due to the restrictions and siege of Israeli on the movement of goods and personnel through control of crossings (Ministry of Local Government, 2016). Nevertheless, Firas Al-Zaghal, as local financial analyst and expert, believes that the recent drop in investment is due to the lack of innovative investment ideas for individuals that are in line with the requirements of the times, despite the huge saving of individuals in banks as stagnant funds (Al-Zaghal, 2018 (Lawson, 2018). Regarding to the USAID priorities in general, usually distributing within five classifications and goals; bilateral development aid, humanitarian aid, military aid, economic aid supporting U.S. political and security objectives, multilateral economic contributions, while the bilateral Open Science Journal -February 2020 8 development assistance has the largest share (Nowels, 2005). The USAID priorities at local level within the Palestinian model are channeled via "Communities Thrive Project" which focuses on the Local Government Bodies, specifically supporting 55 municipalities. The Communities Thrive Project aims primarily to achieving financial sustainability of local councils, by increasing their revenues and reducing unnecessary expenses. In addition to raising the level of service offered to the citizen and increasing citizen satisfaction. Furthermore, increasing the participation of citizens of all segments of the quality, age and professional, the issue of community participation is a prerequisite for the donor and not an optional subject. Also, develop community accountability, and finally, capacity building and rehabilitation of individuals (Al Mbayyed, 2019). The MDLF is a semi-governmental institution which has been in place since 2005, but was legalized in 2016 after it was formally ratified by a presidential decree. The formation of this fund aims at empowering local Palestinian bodies to achieve self-sufficiency, achieving financial sustainability and raising their creditworthiness (Al Budeiri, 2019 Achieving financial sustainability of local councils (increasing their revenues and reducing unnecessary expenses).
2
Rehabilitation of local government institutions and development of their human and institutional capacities.
Raising the level of service offered to the citizen and increasing citizen satisfaction. 3 Improving the quality of provided services to the citizens.
Increasing the public participation, this is a prerequisite for the donor and not an optional.
4
Achieving democracy, transparency and community participation. Develop community accountability.
5
Strengthening partnership with the private sector.
Capacity building and rehabilitation of individuals.
6
Activating local economic development.
7
Achieve financial sustainability of the local bodies and enhance their creditworthiness and enable them to borrow.
8
Activate the lending of municipalities from the Municipal Development and Lending Fund.
9
Exert more efforts to attract funding from international development partners & encourage and sustain foreign aid to the sector.
Compare Priorities
The Paris Declaration recognizes the importance of the ownership 7 and the democracy of recipient countries, besides the alignment of priorities within policies and strategies of developing countries (Mahmud, 2008).
Before comparing the priorities of PLGS and donors, it is necessary to take a reflective and analytical view on the priorities and objectives of the PLGS that shown briefly in the table (1). There are some fundamental observations have to be clarified: Open Science Journal -February 2020 10 ▪ The achievement in the priorities of the Palestinian strategic plans within the PLGS focus on building and empowering the institution and staff rather than focusing on the citizen himself and his needs. Most of the problems of the plans revolve around the problems of coordination between the staff, the problem of distribution of powers, etc. many problems are nascent and new, and there is no any relation for citizens. But in fact, they are an additional pressure on the citizen and burdened with greater burdens than his original problems. ▪ There is a contradiction inside the priorities of the PLGS itself in regards the concept of "sustainability". From one side, the priority of "achieve financial sustainability of the local bodies, enhance their creditworthiness, enable them to borrow". From the other side, the priority of "exert more efforts to attract funding from international development partners and encourage and sustain foreign aid to the sector". While the strategic plan lacks of any policies that support sustainable development by empowering the local economy and infrastructure projects to become an independent sector, independent of external support and able to develop independently. Ambitious roof of the Palestinian strategic plans is the continuity of foreign aid to the sector. ▪ There is a legacy gap from one strategic plan to another within the PLGS.
Represented in preparing the priorities of the plans within the sector and not to implement them at all, then transfer of priorities from one plan to another. The result is lack of achievement. Perhaps because of the lack of a vision and mechanism to implement these priorities within the same plans. ▪ There is a clear absence of leadership role in the PLGS in numerous crucial issues, such as; -Their justification of the lack of progress in the priority "moving towards a decentralized local governance sector" (since 2003 so far) because there is no clear vision about the decentralized local government. -Regarding to the priority of evolving the local development of local bodies, the strategic plan emphasized that the local development will be effective only if Palestinians response to the conditions of donors. -The major challenge that local government sector faces concentrated in "the weak of catalyst environment to the local and sustainable economic development". But, is not it the leader of the local government sector that should create the enabling environment for investment whether by individuals or any other parties? -There are many financial gaps within the local government sectoraccording to the strategic plan-for instance: at the level of capacity building, there is need for $ 12 million that estimated in the first three years of the current plan (Ministry of Local Government, 2016). But how long will it take to spend heavily for capacity-building? Why there are no indicators to measure expenditure performance in this regard? Especially, that donor indicators, point to the lack of preparedness and inefficiency of local bodies to obtain grants. Consequently, many grants are wasted for internal reasons in the sector (Al Mbayyed, 2019). Thus, many problems in the sector are exacerbated, such as; marginalization of citizens' interests and rights, the marginalization of community participation, the stagnation of local development, and surrender to the conditions of the donors.
Regarding the compatibility of donor priorities with the priorities of the local Palestinian bodies, according to (Al Mbayyed, 2019) he stated that the USAID priorities are determined by holding several meetings with random sample selected from the 55 municipalities to monitor their needs and determine their priorities. Thence, the principle of competition prevails according to the efficiency of the municipality and its readiness to win the support of donors. While the view of Ramallah municipal planners regarding external support that allocated to the municipality; support is often directed to specific projects according to the directions of the donor, without reference to the municipality and matching their priorities and their strategic plans (Al-Sayegh, 2019). Al-Sayegh added that the MDLF is currently supervising and monitoring these projects.
Practically, the coordination process between donors and projects implemented within the PLGS is carried out at multiple levels: coordination is carried out through the Local Government Sector Working Group, which in addition to donors includes Palestinian institutions such as the Ministry and the Fund. This type of coordination is nothing more than a protocol; information and presentation of ideas and projects only. The other and most effective type is what happens among the donors themselves, through regular monthly meetings sponsored by Denmark as the lead donor in this sector. There is also bilateral coordination among donors as needed, and the subject (Rajab, 2019).
As regards the extent to which donors' priorities are aligned with MDLF's own priorities to support local development within Palestinian municipalities, Mr. Al-Qawasmi said that the Fund originally derives its priorities from the Palestinian national policy agenda, and there is no conflict between the implementation of MDLF's priorities and donors. On the other hand, an important role of the Fund is the coordination of projects among municipalities and donors to avoid any conflict (Al Qawasmi, 2019). From an academic point of view, it is easy to find a correlation between donor priorities and Palestinian priorities, especially as donors are usually keen to align their priorities with national strategies, as Palestinian national strategies are comprehensive and general (Rajab, 2019).
However, not all donors offer their support to the Palestinian municipalities through the MDLF. Some countries offer their support directly, such as China, Japan, Italy and America. According to the director of operations of the MDLF, there are supportive countries that impose conditions on the Fund that deviate from the Palestinian priorities, so they undertake directing their support away from the Fund. For instance the USAID is demanding that Gaza Strip not to be supported. Anyhow, USAID now has effectively stopped supporting all Palestinians, in both GS and the WB, since the end of last January. Germany also requires from the MDLF to take all official approvals by the Israeli occupation before funding any Palestinian development activity. But the current Palestinian situation cannot obtain Israeli approvals for the implementation of Palestinian projects, especially in Areas C, where the bulk of the MDLF's projects are implementing within a risk and a kind of the Palestinian challenge under the siege of Israeli colonialism and without obtaining the required implementation permits, that's in order to meet the urgent Palestinian needs in the place and to be survived (Al Qawasmi, 2019).
From the point of view Palestinian expert in infrastructure projects (Abu Madi, 2003); the majority of foreign aid is conditional, donors control disbursement within their considerations and priorities rather than the Palestinian's priorities. Whereas Palestinians must appoint foreign experts with salaries that are depleting the value of the assistance itself, and the remaining ones are used to purchase equipment from the donor country. Subsequently, infrastructure projects do not reduce the cost to the citizens and reduce poverty, but rather promote the concept of privatization.
Performance Indicators of the Local Development
Most researches on the impact of foreign aid and its effectiveness on development often have been measured within a very short period of about 4 years. This is illogical to observe the expected impact in such a short time (Clemens, 2004). On the other hand, assessment of aid effectiveness at micro level usually offers positive results while the results at the macro level are ambiguous. The impact of foreign aid depending on the stability of macroeconomic policy environment, allocation of aid, income level, and geographical location (Durbarry, 1998).
Aid could have qualitative or quantitative effects (Wamboye, 2012). Generally, development objectives by donations as per Radelet, are channeled through certain fields; support in providing food or other commodities during wars crises or relief operations, support economy post-crises for stability, or support health, education, environmental issues, or political structures. Besides, support in building infrastructure, or productive sectors like agriculture, or inserting new technologies to stimulate economic (Radelet, 2006). Some empirical studies proved that the concept of lending is more effective than just providing donations, because the amount of funding should be returned (Jayathilaka, 2009).
In the aftermath of the Arab Spring revolutions, voices within that region began to query foreign aid. Foreign aid can be effective on condition of the absence of corruption, with sound economic policies and institutions, in addition, more coordination among donors themselves. Sometimes aid in the technical form can have greater impact (Rady, 2012). However, there is no indication that government corruption limits and reduces its chances of receiving external aid (Alesina, and Weder, 1999).
Palestinians receive a relatively high proportion of aid on the basis of political considerations rather than solidarity and development issues (PMA, 2011). Main indicators of high foreign aid in WB were reflected in; increasing unemployment, the decline in GDP growth, the increase in the non-tradable sector share, the declining share of the tradable sector and the declining share of exports of GDP (Shoukair, 2013). If we check the effect on micro level of development, for instance, water and sanitation sector is receiving a large amount of donations, but there is still a significant shortfall in meeting the basic needs of the population in the Palestinian territories (Abu Madi, 2003).
PLGS has developed many indicators in order to measure the extent in which their policies and priorities have been implemented. For instance; measuring the gradualization forward decentralization, and development of the local government bodies law to support that, additionally the increase in the revenues of local bodies. However, success has not been achieved in this regard based on the latest strategic plan analysis. Furthermore, lending aspect of the MDLF has not been activated because there are no clear policies on lending to the local bodies. The MDLF has become a funding channel instead of a tool to strengthen the decentralization of local bodies (Ministry of Local Government, 2016).
According to the general director of the MDLF and the current strategic plan of the MDLF, both emphasize that; it is not possible to activate the lending aspect of the Fund, because there is no current need for this. The lending system Open Science Journal -February 2020 13 cannot be implemented in light of the inability of the Palestinian municipalities in the current circumstances to apply and adhere to the loan procedures. MDLF`s efforts are being directed towards enabling institutionalizing municipalities to reach financial sustainability as a first step (Al Budeiri, 2019). However, in the long run, there are intentions to apply some 10 pilots among the highest municipal performance ratings for studying the credit worthiness of the Palestinian municipalities to borrow from the Fund, but it is not clear yet.
Certainly, if the external support stops today, the MDLF will be closed on the same day (Al Qawasmi, 2019). However, all Palestinian municipalities in the WB and GS receive support from the MDLF within two grants of the Fund: a basic grant based on population and needs within the municipality that representing 50%, and the other grant is the performance grant. This grant is followed by number of performance indicators at the Fund. The evaluation mechanism follows the municipality's classification under (A, B, C, D). Performance indicators are based on the municipality's financial performance, institutional performance, and the transparency and community accountability. Therefore, the minimum level for any Palestinian municipality to obtain the MDLF's support, however low its performance indicators is at least 50%, which is the basic grant. On the other hand; the MDLF is the only institution that supports local development in Gaza Strip within the local government sector (Taha, 2019).
According to the USAID indicators within Palestinian experience, results points out to the lack of readiness and efficiency of the majority of Palestinian local bodies to obtain grants. Consequently, many grants are wasted and lost for internal reasons in the sector (Al Mbayyed, 2019). As a result, the local government sector focuses on self-rehabilitation and capacity building, and allocating annual budgets for these purposes, but without internal indicators to measure the effectiveness of this trend, thus dispersing Palestinian efforts and budgets ineffectively.
Open Science Journal -February 2020 14 Table (2), including crucial indicators to be considered during assessment of local development performance by foreign aid. Identification the performance indicators were based mainly on the literature review with a holistic and analytical approach, guided by the five main principles of the Paris Declaration, besides the Millennium Development Goals, in addition to consultation of some local experts and officials within the PLGS. These indicators according to the researcher's vision could be applicable for all recipient countries and not exclusively for the Palestinian model.
SWOT
(Strengths, Weaknesses, Threats and Opportunities) analysis of the foreign aid impact on the Palestinian local development In order to diagnose the impact of foreign aid on the Palestinian local development within the framework of the PLGS, it is necessary to display a detailed and clear picture about the Palestinian model. Broad ideas are shown here via SWOT analysis context from a Strategic planning perspective.
Strengths
Numerous international initiatives and efforts that have been exerted since World War II, and still being made to rectify the direction of foreign aid to become more efficient and effective within local development concepts. The most important of them is the Paris Declaration 2005. Furthermore, Palestinians are entitled to development assistance under the international law (Hamdan, 2011) despite that most of received aids on the basis of political considerations rather than development issues (PMA, 2011). It should be noted that there are many international institutions and donors located in the Palestinian arena that seek to support local development and empowerment of local government bodies, such as; European Union, The World Bank, UNDP, UN HAPITAT, OCHA, USAID, JICA, MDLF, etc.
Weaknesses
In spite of the MDLF is one of the most important official channels of external financing that directed to support local government bodies within the PLGS, but lending side of the MDLF has not yet activated. At the same time, most Palestinians officials recognize the importance of this aspect in creating a real challenge of the Palestinian's municipalities, whereas pushing them towards sustainable local economic development and reducing its dependence on external support. Unfortunately, until now, no serious steps have been taken to prepare deliberate plans to face the future if the foreign aid is cut off permanently, although there is a full conviction, understanding and attempts by the officials in this sector to prepare for this scenario by encouraging local economic development, increase revenue, and capacity building of local bodies to be more effective and sustainable (Rajab, 2019).
Foreign aid constitutes the largest proportion of the development budgets (projects) of the local bodies, which may reach in some bodies to more than 80% (Rajab, 2019), nonetheless most foreign aid is consumed rather than invested, which means that so far, the PNA cannot rely on such assistance to achieve sustained economic growth (PMA, 2011). Moreover, there is a contradiction between the priorities of the Palestinian local governance sector itself in regards the concept of "sustainability". From one side, the priority of "achieve financial sustainability of the local bodies, enhance their creditworthiness, enable them to borrow", and from the other side, the priority of "exert more efforts to attract funding from international development partners and encourage and sustain foreign aid to the sector". Thus, ambitious roof of Palestinian strategic plans is the continuity of foreign aid to the sector.
In fact, there is a clear absence of leadership role in the PLGS in most crucial issues, (Decentralization, response to the donor's conditions, weak of catalyst environment, and financial gaps). Thus, problems are exacerbated in this sector, such as; marginalization of citizens' interests and rights, the marginalization of community participation, the stagnation of local development, and surrender to the conditions of the donors. Then transfer of priorities from one plan to another without any achievement.
Donors often force inappropriate policies; create transaction costs, control financing without referring to recipient countries, etc. Indirectly they mislead local democratic processes. As a result, donors are outside the local political process of developing country so they are irresponsible to the people. On the other hand, foreign aid creates a dependency, enabling bad governments in power and postpones reform thus increase citizens' damage and exhaustion, continuity of conflict and corruption. Besides, it has no clear relationship with investment and development, plus it can increase the costs of basic commodities, and promoting the principle of privatization. Additionally, in the Palestinian context foreign aid promote the concept of job dissatisfaction in certain functional segments as a result of creating a situation of functional differentiation due to the different advantages of working within foreign institutions (Abu Madi, 2003).
Threats
Without a doubt, the formation of a state under the umbrella of external colonization is not a normal mode; this is a major challenge on all levels of Palestinian development, especially that Palestinian economy is below siege. On the other hand, possibly poverty rates would be higher in the absence of aid, but studies have shown that conditional aids are framing the policies and strategies of recipient countries, limiting their development options. Besides, increases the burdens of developing countries more than their capacities. And, the most importantly, that is contrary to the Paris Declaration regarding to the preservation of ownership, the democracy of peoples, and building local capacity.
Taking into considerations there are many factors that prevent the achievement of the least benefit of foreign aid generally, the most important of which is the weak policies (fragile infrastructure) and the corruption of the ruling regimes of developing countries and the existence of political and economic instability caused by wars and so on. Further several studies explored that there is a U-shape relationship among foreign aid and economic development, whereas the impact on development is negative at first, but over the time becomes a positive character. Therefore, sufficient time must be given to monitor development results.
Open Science Journal -February 2020 17
Opportunities
We have to remember that Local Economic Development (LED) is a partnership between all segments of the society, including the public and private sector, civil society organizations, local investors, donors, research and educational institutions led by the local government sector through the local government bodies. The theme is not limited to the role of external funding to create successful local development of the receiving country. Besides, there is a key role of the local government bodies in local development, through creating conducive environment for other parties in various fields, rather than for instance focusing on the housing sector randomly, which is outside the capacity of municipalities to provide the infrastructure services associated with investment in the thoughtless housing and exceeds the current Palestinian need.
Positive aspects of foreign aid include the introduction of basic concepts and tools in planning for the Palestinian community, such as; obligating municipalities to prepare annual budgets and external audits. They also oblige them to prepare annual strategic plans to monitor local development goals and measure their achievement. In addition to activating community participation and accountability concept. Sustainable local development couldn't be achieved without public participation, adopting "Agenda 21" 9 as one of planning tools enabling local municipalities to obtain more donations from UNDP and other donors. As well as implementing a large number of infrastructure projects that primarily serve the Palestinian citizen. Furthermore, aid is not necessarily to be financial only; financial aid is easily manipulated and routed away from local development, while technical assistance, training programs and others qualitative aid have more assured results practically.
The State of Palestine still receiving foreign aid through several official channels. This assistance must be utilized to support priorities more strategic and comprehensive dimension to the concepts of local development. On the other hand, the percentage of Palestinian learners, experts and analysts is very high compared to other developing countries. Therefore, efforts should be made to use these capacities to guide foreign aid more efficiently and towards more sustainable local development.
Conclusion
Developing countries, including Palestine are suffering from weak policies and fragile institutional structure, thus they have to be well aware that foreign aid is not a magic wand that can transform the poverty into wealth and well-being. Besides, perhaps the traditional system in developing countries which representing in central government systems (top-down approach) is not the ideal system to manage local development within local government bodies. Maybe it's time to try another administrative alternative especially that poverty rates are increasing continuously in the Palestinian context despite the continuity of getting foreign aids. Moreover that infrastructure projects are fully funded by foreign aids so far.
On the other hand, Foreign aid system is a fake and misleading system of real local development in developing countries. In fact, foreign aid is a drug that generates a state of addiction to the loss of mind and will of the recipient populations in order to meet their basic needs in life. Although initially looks good, it is an anesthesia process for local development. This form of colonization programs keep the recipient communities within a time-bound schedule that directs the individual's energies towards meeting the financier's requirements and finally; neglecting real local development concepts. Therefore, treatment should be initiated by gradually reducing the value of the aid until it is eliminated. Or diverting the grants into loans within possible terms to recipient countries, that carries a glimmer of hope toward sustainable local development, via changing the mentality of consuming into investing.
Recommendations
Perhaps Palestinian local development should take an example and this is not something wrong. The Palestinian experience lacks guide and a good example, there are many countries around the world that were living worse than the Palestinian situation and are now referred to as leading countries in most areas of life. Moreover, a most important issues on the Palestinian level that, to activate the role of the leader of PLGS, so that control and guide the local development priorities, monitor the results continuously through performance indicators. As well as, activating lending side of the MDLF in order to increase the responsibility of Palestinian`s local government bodies and stimulate them to achieving financial sustainability.
Finally, most studies and literature from various developing countries agree to explain why foreign aid is inefficient by the corruption of the ruling regimes of their countries. All researchers are seeking to the idea of "Self-flagellation". But, is not the time for developing countries to ask themselves why donors support corrupt policies despite their knowledge of corruption? Why have not donor countries yet launched programs to educate people about the corruption of their powers and how to reduce of them? Why not fingers pointing and accountability of donors as a key player influencing in making of developing countries' policies through their conditional donation whether from local population or from elected parliaments? Many questions should be raised for future researches within this context, whether at the level of the Palestinian model or any other developing country. | 2020-02-13T09:22:18.940Z | 2020-02-10T00:00:00.000 | {
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229162875 | pes2o/s2orc | v3-fos-license | Modulation of Muscle Pain Is Not Somatotopically Restricted: An Experimental Model Using Concurrent Hypertonic-Normal Saline Infusions in Humans
We have previously shown that during muscle pain induced by infusion of hypertonic saline (HS), concurrent application of vibration and gentle brushing to overlying and adjacent skin regions increases the overall pain. In the current study, we focused on muscle-muscle interactions and tested whether HS-induced muscle pain can be modulated by innocuous/sub-perceptual stimulation of adjacent, contralateral, and remote muscles. Psychophysical observations were made in 23 healthy participants. HS (5%) was infused into a forearm muscle (flexor carpi ulnaris) to produce a stable baseline pain. In separate experiments, in each of the three test locations (n = 10 per site)—ipsilateral hand (abductor digiti minimi), contralateral forearm (flexor carpi ulnaris), and contralateral leg (tibialis anterior)—50 μl of 0.9% normal saline (NS) was infused (in triplicate) before, during, and upon cessation of HS-induced muscle pain in the forearm. In the absence of background pain, the infusion of NS was imperceptible to all participants. In the presence of HS-induced pain in the forearm, the concurrent infusion of NS into the ipsilateral hand, contralateral forearm, and contralateral leg increased the overall pain by 16, 12, and 15%, respectively. These effects were significant, reproducible, and time-locked to NS infusions. Further, the NS-evoked increase in pain was almost always ascribed to the forearm where HS was infused with no discernible percept attributed to the sites of NS infusion. Based on these observations, we conclude that intramuscular infusion of HS results in muscle hyperalgesia to sub-perceptual stimulation of muscle afferents in a somatotopically unrestricted manner, indicating the involvement of a central (likely supra-spinal) mechanism.
INTRODUCTION
For most individuals, it is relatively easy to distinguish between innocuous and noxious stimuli. However, in a subset of individuals afflicted with chronic pain, there is a disturbance of normal somatosensory function, such that a normally innocuous stimulus can evoke pain, for example, the emergence of tactile allodynia in patients with sciatica (1). This can have a debilitating impact on both the individual and society (2,3). Studies using hypertonic saline (HS) infusions have shown a touch-evoked pain (allodynia) that extends to overlying (4) and adjacent (5,6) skin regions. Intramuscular HS administration produces a deep musculoskeletal pain that often extends or refers to distal regions (7)(8)(9). Repeated intramuscular injections of HS reveal plastic processes with a decrease in the area and intensity of local pain and an increase in the expression of referred pain (10) in addition to the emergence of pain hypersensitivity that extends bilaterally (11). These complex interactions cannot readily be explained by changes in peripheral circuitry and appear to mimic characteristics of chronic pain conditions such as fibromyalgia. Within such chronic pain conditions, current arguments favor an explanation based on a central change in, or sensitization of, the neural function that results in the observed widespread and diffuse musculoskeletal pain, pressure-pain hypersensitivity, cutaneous allodynia, and tactile dysesthesia (12)(13)(14).
In the current study, a HS infusion model was used to examine whether the interaction previously observed between muscle and skin (4,6,11) can be replicated between adjacent and remote muscles. We hypothesized that the presence of background nociceptive activity using HS infusion would produce a state of central sensitization resulting in an exacerbation of the overall pain (hyperalgesia) to the application of a normally innocuous stimulus (normal saline, NS). We also hypothesized that this effect would occur regardless of whether the NS was infused into an adjacent or a remote muscle.
METHODS
Twenty-three healthy naïve participants aged 18-28 years (six females), with no reported history of musculoskeletal or neurological disorders, were recruited for this study. Participants were asked to abstain from intensive bouts of exercise for 48 h preceding the experiment so as not to sensitize the target muscles (15). Six participants took part in multiple arms of the study across different experimental sittings (30 experiments total), the inclusion of these participants in multiple study arms was random. One participant took part in all arms of the study, whilst a further five participated in both the contralateral and remote testing procedures. To minimize the risk of a placebo effect or familiarization with the protocol, repeat participants did not undertake experiments in any prescribed order with control recordings in the absence of HS-infusion (i.e., no-pain) obtained at the commencement of each separate experiment session across each of the test locations.
Informed written consent was obtained from each participant prior to the experiment. This study was approved by the Human Research Ethics Committee (approval numbers: H9190 and H13204) of Western Sydney University in accordance with the revised Declaration of Helsinki.
Participants were comfortably seated in a chair throughout the experiment. HS and NS were infused using a Syringe Infusion Pump (Harvard Apparatus, South Natick, Massachusetts, USA) and a 25G winged infusion set. Importantly, the Syringe Infusion Pump used for NS-infusion was obscured from sight and did not include any audible cues. Pain ratings were continuously recorded using the ADInstruments Response Meter connected to the ADInstruments PowerLab (ADInstruments, Dunedin, New Zealand). The Response Meter had a slide control, and the pain scale was divided into ten equal segments within a range of 0 (no pain) to 10 (worst pain). In addition, participants were asked to verbally report the location of pain during the course of the experiment.
Infusion of Hypertonic Saline
Across all parts of the study, 5% HS was infused into the belly of the flexor carpi ulnaris (FCU) muscle of the forearm for ∼10 min to establish a stable baseline pain. The muscle belly was palpated whilst the participant performed light flexion and adduction of the wrist to identify the boundaries of the FCU muscle. The needle was inserted ∼0.8-1 cm into the center of the muscle belly at an angle perpendicular to the skin at the site of insertion. The infusion rate of HS in the FCU varied between subjects (30-175 µl/min) to establish a moderate pain intensity preferably between 4 and 6 (out of 10) on the pain scale. Once a stable baseline pain was achieved, no further changes were made to the infusion rate.
Infusion of Normal Saline
After a stable baseline pain was maintained for at least a minute, NS (0.9%) at room temperature was concurrently infused at the rate of 50 µl/min for 1 min per trial (tested in triplicate). This duration was chosen based on the data collected in a pilot study which indicated a delay of several seconds before the onset of an increase in pain levels. The delayed response has been reported in previous studies (6,16). The triplicate NS trials were performed at 1-min intervals.
Participants were asked to continuously rate the overall pain intensity, and any changes thereof, on the pain scale. Care was taken to avoid the use of suggestive language with participants informed that the HS-induced pain could remain the same, increase or decrease during the co-infusion with NS.
In addition to concurrent HS-NS infusions, NS alone was infused in triplicate trials prior to the commencement and upon cessation of HS-evoked pain in all experiments. Typically, the HS-evoked pain disappeared over a time course of under 10 min. After a 3-to 5-min wait following cessation of pain, NS infusion was repeated at each site. Collectively, ∼450 µl of NS was infused per muscle.
Part 1: Interactions With Adjacent Muscles
NS was infused into the ipsilateral abductor digiti minimi (ADM) muscle of the hand to examine potential interactions between adjacent muscles in response to HS-induced acute muscular pain. The muscle belly of the ADM was identified by palpation whilst the participant abducted the fifth digit of their hand. The infusion needle was inserted to a depth of ∼0.5 cm into the center of the ADM muscle belly. The ADM muscle was chosen as it shares the same peripheral innervation (ulnar nerve) as the HS-infused FCU.
Part 2: Contralateral Interactions
NS was infused into the belly of the contralateral FCU muscle of the forearm to test whether the HS-NS interactions were limited to muscles within the same nerve territory or spread to contralateral muscles as well. The needle location and insertion for the contralateral FCU were identical to the HS-infusion site described prior.
Part 3: Remote Interactions
NS was delivered to the belly of the tibialis anterior (TA) muscle of the contralateral leg to determine the spatial extent of inter-muscle interactions in an acute pain state. The muscle belly of the TA was identified by palpation during dorsiflexion of the ankle. The needle was inserted into the middle of the belly of the TA muscle perpendicular to the skin to a depth of ∼1 cm.
Statistical Analysis
Repeated measures two-way analysis of variance (RM 2-way ANOVA) was used to compare pain ratings at baseline (HS infusion alone) with evoked responses (co-infusion of NS and HS) at each location (adjacent, contralateral, and remote). Where a significant change (P < 0.05) was found, individual comparisons were made using Tukey's multiple comparison test. The normal distribution of data was confirmed in all groups using D'Agostino and Pearson omnibus normality test. Pain scores for the baseline (HS) and co-infusion (HS and NS) conditions are presented as mean ± standard error of the mean (SEM) for all parts of the study. Statistical analysis was performed using GraphPad Prism (version 7.04, La Jolla, California, USA).
RESULTS
Prior to the induction and following the cessation of HS-evoked muscle pain, all participants reported NS infusion (50 µl/min) to be innocuous (i.e., rated as 0 out of 10 on the pain scale) and imperceptible regardless of the NS infusion site ( Figure 1A). The infusion of 5% HS into the FCU always resulted in a diffuse, deep pain in the muscle that extended down the medial aspect of the forearm. This baseline pain remained stable in the absence of NS co-infusions ( Figure 1A) and did not significantly differ between the different parts of the study (P = 0.66).
At all three test locations (adjacent, contralateral, and remote), the co-infusion of NS significantly increased the overall pain in all trials (T1-3, P < 0.0001, Figures 1B-D left-hand panel). All observed increases in pain scores during co-infusion were transient and time-locked to the NS-infusion, with the pain returning to baseline (HS) within 1 min of the cessation of NS co-infusion (example shown in Figure 1A). Further, the increases in pain scores did not vary in amplitude based on the location (adjacent, contralateral, and remote) of the NS co-infusion (P = 0.30).
The pooled mean response of all participants in each part of the study, with respective HS and HS + NS data points linked, are shown in the right-hand panel of Figures 1B-D and described further in the following sections.
Part 1: Interactions With Adjacent Muscles
The infusion of HS into the FCU resulted in a pooled mean score of 4.3 ± 0.5 (n = 10). When NS was co-infused into the adjacent ADM in the presence of this background pain, the pooled mean score increased to 5.0 ± 0.4 ( Figure 1B). This constitutes a pain increase of ∼16% and when comparing baseline and co-infusion pain scores the increase in pain ratings was significant [P < 0.0001, F (1,27) = 318.5]. This indicates that muscle pain can be modulated by low-threshold/sub-perceptual stimulation of an adjacent muscle.
Part 2: Contralateral Interactions
The infusion of HS into the FCU resulted in a pooled mean score of 4.3 ± 0.1 (n = 10). The co-infusion of NS into the contralateral FCU increased this pooled mean score to 4.8 ± 0.2 ( Figure 1C). This represents a ∼12% increase in the pain scores during co-infusion, an effect found to be significant [P < 0.0001, F (1,27) = 156.7]. This demonstrates that muscle pain can be modulated by normally sub-perceptual stimulation across contralateral muscles, thereby suggesting a central (spinal/supra-spinal) phenomenon.
Part 3: Remote Interactions
Within this aspect of the study, participants reported a pooled mean score of 4.0 ± 0.1 in response to infusions of HS into the FCU (n = 10). During concomitant infusion of NS into the contralateral TA, participants reported a pain increase of 15% with the pooled mean score increasing to 4.6 ± 0.1 (Figure 1D). A comparison of the baseline and co-infusion pain scores revealed a significant difference [P < 0.0001, F (1,27) = 97.84]. The observed interaction between the site of noxious muscle stimulation and remote innocuous muscle stimulation alludes to the involvement of a supraspinal mechanism.
In Figure 2, triplicate responses for each individual (n = 10 per test location) at all three test sites (n = 90) to transient NS infusion during HS infusion (i.e., HS + NS) have been plotted as a function of the baseline pain evoked by HS alone. When plotted in this manner, all data points fell to the left of the line of equivalence (x = y or HS = HS + NS) indicating that the NS infusion evoked a reproducible pain increase across a broad range (pain scale 1.4-6.7) of baseline pain levels.
When participants were asked about the location of pain, all participants-except 2 in part 1 and one each in parts 2-3-ascribed it to the forearm where hypertonic saline was infused with no discernible percept attributed to the sites of NS infusion. This was true not only for HS-evoked pain but also for pain increases during HS-NS co-infusions. The four subjects who did not ascribe the pain increase to the HS-infusion site instead ascribed it to the NS-infusion site. Importantly, these subjects always reported NS-infusion as imperceptible at the local site under control and recovery conditions (no HS-pain). This suggests that NS infusion was almost always nonpainful regardless of whether there was HS pain or not, but in the presence of HS pain, the NS co-infusion resulted in hyperalgesia at the HS site, and this modulation of HS pain was not somatotopically restricted.
DISCUSSION
The current study has provided evidence that muscle pain can be modulated (hyperalgesia) by sub-perceptual stimulation of muscle afferents in a somatotopically unrestricted manner. This finding not only builds upon the previous observation that an intramuscular HS infusion can result in allodynia in the overlying and adjacent skin regions (4, 6, 17) but the spatial extent of this modulation, spanning several spinal segments, suggests the involvement of a central, likely supra-spinal, mechanism.
The sub-perceptual nature of repeated intermittent NS infusions (50 µl over 1 min) under control (no HS-pain) condition suggests that localized muscle distension does not activate the nociceptors (18) but may activate low-threshold stretch-sensitive receptors within the muscle. In this respect, these weak mechanical stimuli resemble the inability of weak (micro) intraneural electrical stimulation to produce a discernible pain sensation at recording sites dominated by muscle spindles (19,20). We have also previously shown that intradermal infusions of NS (50 µl/min for 2 min) do not produce a percept (5).
The conversion of the sub-perceptual NS stimulus to one that enhances pain, during HS infusion in the FCU muscle, is unlikely to be due to peripheral sensitization given the anatomical separation (forearm vs. hand, >15 cm) and the small volume of intermittently infused NS. Likewise, the increase in pain evoked by NS-infusion into the contralateral forearm is more consistent with a central involvement. Furthermore, the interaction between the FCU and the contralateral TA suggests that the central involvement likely extends to supraspinal structures. Assertions as to the exact location of this central involvement cannot be resolved by this study, but the acute/short-lasting and reversible nature of these interactions do suggest that the requisite circuitry may already be present, and thus an elaborate anatomical reorganization need not be necessary for these to occur.
The broad-ranging muscle-muscle interactions observed here appear to be in marked contrast to the somatotopically constrained interactions observed in the skin; for example, the confinement of secondary hyperalgesia to the region immediately surrounding intradermal capsaicin injection (21)(22)(23) or the inability of microstimulation of large-diameter mechanoreceptors innervating a skin region beyond the site of secondary hyperalgesia to produce a painful percept (16).
The effects observed in the current study are most likely driven by a transient and reversible episode of central sensitization (increased excitability and synaptic efficacy of central nociceptive pathways) (24) in response to the HS-induced muscle pain. The HS infusion alone was run for ∼10 min prior to the commencement of NS co-infusion, and this may have resulted in a state of central sensitization. Indeed, the clinical correlates of central sensitization (25,26) are apparent in a HS-infusion model with hyperalgesia and allodynia reported in this and previous work (4,6,11,17).
The generalized modulation of the exacerbated pain response at the HS-induced muscle site during NS-infusion in the adjacent, contralateral, and remote muscles is noteworthy and warrants further study using more quantitative measures of pain localization than verbal reporting. Further, the quality and temporal characteristics of this hyperalgesia need further investigation. In addition to the prerequisite of ongoing nociceptive input (HS infusion), we observed that the onset of NS-evoked increase in pain tended to be delayed by several seconds, which suggests a possible need for temporal summation.
Previous findings in humans have shown that repeated intramuscular HS injections in the TA result in a pressurepain hypersensitivity developing across both the ipsilateral and contralateral TA muscles (11). Further, it has been shown in animals that a unilateral forelimb injury can produce sensory perturbations in the contralateral limb (27). In the case of intramuscular HS, the evidence for centralized effects necessitates the need for control data collection prior to any HS administration and warrants an investigation into other commonly used pain models.
In the current study, the co-infusion of sub-perceptual NS resulted in increased HS-pain (i.e., hyperalgesia). In HS and other experimental models as well as chronic pain conditions, tactile and thermal stimuli can produce allodynia (pain to a normally nonpainful stimulus) and hyperalgesia (increased pain from a painful stimulus) (1,4,6), but paradoxically, these modulatory stimuli-both painful and nonpainful-can also reduce pain with slow gentle brushing of the skin shown to reduce cutaneous heat pain (28). Conditioned pain modulation is a well-recognized phenomenon in which a painful stimulus can be inhibited by a second painful stimulus applied to a different body site (i.e., pain inhibits pain) (29)(30)(31). The underlying mechanisms are not fully understood but likely involve a complex interplay between excitatory and inhibitory circuits in the central nervous system.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Human Research Ethics Committee of Western Sydney University. The patients/participants provided their written informed consent to participate in this study.
AUTHOR CONTRIBUTIONS
SN and DM contributed to the conception and design of the study. JD performed the experiments and organized the database and wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version. | 2020-12-15T14:14:29.783Z | 2020-12-15T00:00:00.000 | {
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247314318 | pes2o/s2orc | v3-fos-license | The change in the researcher's position in the study of shamanism
Since the 1960s, shamanism has become one of the landmarks for a new way of life and a more respectful relationship of humans with nature in the Western world. Both researchers and practitioners regard the foundation of shamanism as being animism – an understanding of the connection of all living beings. The role of the community is highlighted in shaman ism as the shaman is expected to work for his community. Shamanistic techniques vary according to the society or place where it is practised. In the study of neo-shamanism in the post-modern world, autoethnographic research has been seen as necessary, especially in order to explore the experience of the shaman. In this article we ask what has changed in the perceptions of the community in the study of shamanism and how this change has influenced the position of the researcher. Our article reflects on the resolution of the diversity and sameness through autoethnography, where the researcher is located not only in the experience, but also in its interpretation – which highlights great challenges in contextualizing the study, in writing on the concepts concerned, and indeed in the entire research process.
Introduction
Shamanism as a phenomenon has drawn researchers' attention since the nineteenth century. Anna-Leena Siikala (2002: 43), a scholar of traditional Finnish shamanism, writes that 'shamanism is not a religion, but rather a complex of rites and beliefs existing within different religions' . There is no single inherited shamanism, nor one current shamanism, but rather shamans operating in different cultures, communities, and time. Therefore, it is preferable to talk about different forms of shamanism. The basis of the shamanic customs and beliefs is in the culture and community where the shaman works. As Graham Harvey (2005: 139) writes, 'shamans are performers of particular roles, skills and arts that require the participation of others' . We concur with him that even though 'shamans shamanise, that does not make them, or the groups for whom they shamanise, members of a religion called Shamanism ' (p. 139).
Over the last century, the societies and the contexts in which we can find shamanism have changed drastically. Through this lens we examine shamanism as a historic al phenomenon with great local varieties while our focus is on the Finnish context. As there are many shamanisms, they have their own histories. However, science has sought to retrieve common traits and denominations, and thereby a common history. In fact, when we write the history of shamanism, we write the history of the study of shamanism. The two are difficult, even impossible, to keep apart, especi ally when we are interested in how the position and methods of the researcher of shamanism has changed in the study of shamanism.
Research methods have also evolved and developed. To understand the change in the position of the researcher, we have to understand the change both in the context of the research topic, shamanism, and in the scientific methods and concepts linked thereto. The variety of cultures and societies where shamanism is practised, as well as the diversity of shamanisms and the research into them, are extensive. However, in one article it is impossible to conduct an extensive review or comparative research into all the communities or cultures in which shamanism has been practised or researched. We therefore hope that the reader will note that our view of the changes in the communities is intended to serve mainly as an introduction for our more important research issue in this article; that is, how changes in the field should be reflected within the researcher's role.
As the perspective in this article isto some extent -historical, we start with defin itions, moving on to the historic al perspectives of the study of shamanism. We first open up discussions about the concepts shaman, shamanism and animism, and the historical context of where these come from. We then briefly examine the research of so-called traditional shaman ism by taking our examples from the Finnish history of researching shamanism. Only after this do we move on to look at the context of contemporary shamanism in more detail.
There is still a paucity of research on contemporary Western shamanisms; especi ally the examination of the experience of practitioners is quite limited (see e.g. Wallis 2003: 1, 30;Høst 2001). In reflection of the researcher's role, we will discuss the method, autoethnography, used in our study in the later stages of this article. We will reflect on our experience-based knowledge in practising shamanism. In gender studies, positioning is proportionate to the concept of experience. Experience is defined as inclusion in rele vant practices, discourses and institutions (Koivunen and Liljeström 2004: 271, 278). In this article, we locate ourselves in this way in experience. In addition to the interpretation of experience we open up these concepts. By doing this we emphasize the role of the researcher as an interpreter between two fields -the field of researching shamanism and the field of the research topic, namely contemporary shamanism. As the shamanic practitioner and teacher Jonathan Horwitz from the Scandinavian Center for Shamanic Studies (SCSS) says in an interview (Kelly 2017: 98), shaman ic knowledge can only be acquired through individual experience, which is 'different from a dogma or a belief system' . He explains that 'this is because shaman ism is not a matter of belief, it is a matter of experi ence' and 'a spiritual path' . It is a 'healing spiritual practice' .
The research material used in this study is composed of studies of shamanism, anthropological and religious studies on shamanism, shamanistic literature and years of fieldwork experience amongst shamanic practitioners in Europe. The choice of writings for quotation is based on our own shamanistic education. For ex ample, Jaana Kouri, a researcher of the study of religion, has participated in the workshops of the SCSS, whose methods and teachings are based on core shamanism, even though they differ from the teachings of Michael Harner, the anthropologist and founder of core shamanism, in some perspectives. 1 Elina Hytönen-Ng, an
Animism and shamanism
The word shamanism holds within it the history of the research. When shamanic practices in Siberia were presented to Western academic audiences by researchers of Siberian shamanism, the Tungus word shaman became attached to similar practices from other cultures (Eliade 1972: 3;Harvey 2005: 139). Different cultures did not necessarily have a single or similar word describing the person performing shamanic rituals. Yet as Harvey (2003: 3) has noted, the word 'shaman' has now become a 'part of various other languages' . It is therefore important to realize that shamans appear in different cultural contexts, and they are not alike. In Finland the words used for a shaman, or someone who works like a shaman, could be tietäjä, näkijä and noita. In English the first word would be translated as 'a sage' , the second word as 'a seer' and the latter one as 'a witch' . The word 'shaman' was taken into the academic discussions to describe the rituals in different cultural contexts and the word then became popularized. During the eighteenth century, research had already developed a more-or-less fixed image of 'shamanism' as a specific type of religion (Stuckrad 2014: 159-60).
Zara Waldebäck joined the team of the centre . They are all teachers of shamanism.
As Susannah Crockford has pointed out, while the word 'shaman' came from Tungus, it has metamorphosed from its original meaning. Therefore, she argues, we need 'to be aware of the etic and emic distinctions, sensitive to cultural differences and biases of interpretation, and understand the positive aspects of Western shamanism for those who participate in it' (Crockford 2010:142). The dichotomy between different kinds of knowledge is commonly expressed by means of the contrast between the 'etic' level of objective description and the 'emic' level, in which cultural subjects make the environment meaningful. Tim Ingold writes that 'if the emic level is understood as reality constituted in relation to the beings whose environment it is, it is apparent that the world becomes a meaningful place for people through being lived in, rather than by having been constructed along the lines of some formal design' (Ingold 2000: 168, ital-ics in the original).
It is also worth remembering that the community of neither traditional nor modern shamanism is a pre-existing entity that expresses itself via a fixed set of symbols. Community is a formation that comes into being through the circulation and use of shared cultural forms, and that is never complete (see Meyer 2009: 4). Moreover, as Robert J. Wallis (2003: 31) has noted, 'critics of contemporary shamanisms tend to fall into a methodological trap of comparing them with indigenous shamanisms, when the plurality of both, and their engagements, indicate they may or may not be commensurable' . Also, the 'hom ogenized' core or neo-shamanism, which we will return later, becomes diverse through the activities and reciprocity of users and the spirit world, as an outcome of each person's own shamanic path. This path varies according to the cultural context of the shamans and their spiritual experiences. It also changes the discourses of shamanism among practitioners and researchers.
Researchers of shamanism have viewed it as essential to define the concepts used: not only shamanism but also animism. Most teachers, practitioners and researchers of shamanism see animism as the basis of shaman ism (see Harvey 2005: 139). Animism as such is, as a scientific concept, one of the oldest terms in the study of religions. Harvey (2005) has explored development in the concept of animism and notes that its definition has changed over time. The older scientific ideas of animism, especi ally Edward Tylor's, were bedded in the idea of the evolutionary stages of religion, where animism represented the primi tive origins of religiosity. According to Harvey the older use of the concept of animism referred to 'a putative concern with knowing what is alive and what makes a being alive' (p. xi).
Harvey suggests a new definition for animism based on his studies in contemporary paganism and indigenous studies. According to his definition of new animism, animists are 'people who recognize that the world is full of persons, only some of whom are human, and life is always living in relationship to others' (Harvey 2005: xi). He specifies that 'persons are those with whom other persons interact with varying degrees of reciprocity, and may be spoken with in contrast regarding objects, which are usually spoken about' (p. xvii). According to him, this is the newer usage, referring to widespread indigen ous and increasing ly popular 'alternative' understanding that humans share this world with a range of persons, only some of whom are human (p. xi). Similarly, Horwitz (2017: 14) says that 'a lot of the time when people say "shamanic", they actually mean animistic -apperception of the world as it truly is with all things alive and in connection. "Animism" is the awareness of our connection to the world. These things are insepar able' .
Practitioners have defined a shaman by what he does (Horwitz 2017). According to contemporary shamanism as taught by Michael Harner, a shaman is the one who goes into the spirit world and brings power, healing and teachings to whoever needs it. When entering the spirit world, a shaman has a task for which he seeks a solution. Likewise, he also has a task to do when he comes back to his human community. A shaman does not exist without the community or the spirits, as the shaman is the servant and mediator of the human and spirit communities alike. Horwitz specifies that 'just because one is aware of the presence of spirits doesn't mean that they are practicing shamanism; one is practicing shamanism when one comes in contact with them -literally [there is] some kind of intimate relationship going on where humans and spirits are consciously helping each other out' (p. 15).
The work of a shaman is based on the connection of all living things and the possibility of communicating with other beings, who are understood as spirits or persons. Perceptions of the essence of beings vary, depending on the scholar, as well as the shaman, and his culture. The definition of a shaman also has its historic al context, depending on the time of the research history, and cannot be fully judged by the today's yardstick. The definitions are reflections of their time and objects. Mircea Eliade's (1964) idea of shamanism as an archaic trance technique does not accommodate an understanding of contemporary or even all traditional shamanism, but it may still be a useful definition in some particular kinds of shamanism.
An example of the complexity of Finnish words referring to shamanic prac tices is well revealed in the discussions by the shamanic community. While Kouri was on the board of Shamaaniseura, the Shamanic Centre of Finland, the network of shamanic practitioners in Finland in the 1990s, the board considered changing the name of the society to an equivalent word in Finnish, such as tietäjä, for ex ample. However, those Finnish words seemed to refer more specifically to only one task of the shamans (Kouri 2020: 235). The use of the 'shaman' or 'shaman ism' was also well established among practitioners at the time, both in Finland and elsewhere, and the centre wanted to be part of the inter national networks of shamans . Some contemporary practitioners also oppose the use of the word 'shaman' and prefer to use culture-specific emic names, such as tietäjä in Finland, or use terms alongside each other (see p. 234). For example, in its homepage a few years ago Pielisen Tietäjäkeskus, a centre for the 'wisdom cultures around Lake Pielinen of North Karelia, Finland' , announces its purpose to be 'to promote the continuum of wisdom and shamanic trad ition of Finland' (Pielisen Tietäjäkeskus).
Facing especially the diffusion and diversity of modern shamanism, one might ask whether it makes sense to assume a common ground or shared worldview with such varied references to shamanism. In Europe, for example, contemporary shaman ism, such as shamanism practised in pagan or neo-shamanism circles, usually does not relate to a homoge neous tradition or culture. As Wallis writes, 'it is impos sible to examine contemporary or "neo-shaman ism" as if it were a single entity, since the diversity of practices and practitioners resists such a metanarrative, and there are no fixed boundaries; as well as convergences between indigenous and neo-practices, there are no clear similarities between aspects of neo-shamanisms and a number of traditions, for example in contemporary Paganisms' (Wallis 2003: 32-3). Practitioners prefer to talk not about neoshamanism but for example about modern Western or modern European shamanism (Høst 2001;Stuckrad 2014: 159-60). In contemporary Finland we find a variety of people shamanizing. Some have participated in the internationally run courses of core shamanism following Harner's teachings and others in the more local tradition of Finno-Ugric people or wicca, to give a few examples. In this variety of contemporary shamanism it is as well to pay attention to what is relevant to a particular group and its historical and geographical context where a shaman works.
From studying traditional shamanism to researching contemporary shamanism
Traditional research on the native cultures has existed since early on, already being in evidence during the nineteenth century. The researchers usually emphasized the rural or uncivilized ways of the natives, colonialism setting the background for the interest. Ethnographic observations were usually made with the pretense of civilizing and educating the natives. These views were very prominent in early anthropology (see e.g. Frazer 1998/1890), while religion was also another matter that influenced the way that the natives were approached.
In the Nordic countries Lars Levi Laestadius was a fine example of this trend. Laestadius, a Lutheran priest living at the beginning nineteenth century, was a Swedish Sámi, also known for establishing the Christian revivalist movement Laestadian ism. During his field excursions he also made notes about the mythic al worldviews of the Sámi people and their religious beliefs, but also about the shaman ic practices of the Sámi (Laestadius 2002). During the nineteenth century the Sámi drums were also largely destroyed or collected into European museums (see Joy 2018b). Research on the drums was still being made during the 1990s (Hultkranz 1991).
The tradition created by colonialism continued into the early twentieth century. While the European ethnologists were focusing on the colonies in Africa, Asia and the Americas, the Finnish researchers were directing their attention to ethnic groups closer to home, the Finno-Ugric and other groups, including the Sámi. The research on groups that are ethnically close to Finns continued up until the 1970s and 1980s; fine examples of this are Anna-Leena Siikala's (1978) and Juha Pentikäinen's work. Pentikäinen has continued to work on shamanism as well as publish on the topic into the twenty-first century (see e.g. Pentikäinen 1998). One of the newest studies of shamanism is the thesis of Francis Joy (2018a) on Sámi shamanism.
In the 1960s, people associated with the 'New Age movement' discovered shamanism. As Kocku von Stuckrad (2014: 16) has summarized, 'the shaman became an indication of a new understanding of humanity's relation to nature, the human's ability to access spiritual levels of reality, and leading a respectful life in the "sacred web of creation" ' . The phenomenon was soon to become known in academic parlance as 'neo' or 'modern Western shamanism' . The so-called neo-shamans that emerged in the 1960s and 1970s enthusiastically consumed popular anthropology books on shamanisms, such as Mircea Eliade's and Carlos Castaneda's books. In emphasizing the symbolic and cosmological aspects of 'shamanism' and downplaying socio-political diversity, Eliade paved the way for academic interest in shamanism and universalism. Castaneda, on the other hand, was the first person to make the shamanic perspective accessible to Westerners. Castaneda's work encouraged Westerners to become shamans themselves, making shamanism more personal and approachable, even though there is little to warrant belief in Castaneda's work as anthropologic al 'fact' . Michael Harner (1929-2018, a professor of anthropology, provided a safe and simple technique for making neo-shamanisms a pragmatic possibility, via an easily access ible set of procedures which did not involve the danger of entheogens: 'core shamanism' (Wallis 2003: 38-9, 41, 43, 45).
Jan Svanberg (2003) argues that in understanding the origin of neo-shamanism it is essential to consider the intradiscip linary changes of the time, the American counterculture and the ambitions of certain authors to bring about interaction between science and its applications. A particular feature of modern shamanism is that many of the teachers of shamanism had an academic background (p. 27). Therefore, in research on shamanism, there is a mingling of practical terms and concepts from the field and academic textualization. It is good to remember that the role of the authoras a shaman or researcher -influences the choice of certain central approaches and concepts in describing the experimental contents of shamanism. The question is how experience-based knowledge is mediated verbally to one's audience. Researchers participated in the shamanistic practices of their objects of study, and conveyed their experiences to scientific and other audiences. The idea that the researcher is able to participate in the lifestyle of the subject holistically, that is the so-called 'going native' idea, reflects the colonial mindsets and attitudes (Wallis 2003: 4-9). Researchers became spiritual experts and combined both cultures of knowledge in their work. They attri buted meanings to shamanism that were legitim ized and turned them into religious practice (Stuckrad 2014: 161, 171-2).
After extensive research both in the field and through anthropological literature, Harner elaborated what he felt to be the common cross-cultural denominators of shamanism. In 1980, he published The Way of the Shaman, which soon became a handbook used around the world for those interested in making a shamanic journey. He also began to give courses on shamanism. From the very beginning, the technique of core shamanism spread throughout the Western world (Harner 1980: xv;Stuckrad 2014: 171-2). As von Stuckrad reminds us, we have to remember that even though Harner's work has been influential, not all contemporary centres and teachers of modern shamanism see themselves as followers of Harner. Their ideas vary, for example according to how their shamanic practice is related to local traditions. Horwitz can be seen as an example of this diversity, as he was first a colleague of Harner, but in 1986 established his own school with the teacher of shamanism, Annette Høst (SCSS). Their teaching differs in some ways from Harner's, especi ally in the way they describe working with spirits (Stuckrad 2014: 174-6). At the end of the twentieth century, Horwitz also held his first basic courses in shamanism in Finland with a Finnish anthropologist and teacher of shamanism, Heimo Lappalainen, on whose initiative the Shamanic Centre of Finland was also established.
The transportation of shamanisms from indigenous to Western contexts since the 1960s is part of a larger process of globalization. Wherever shamanism originated, it is now readily available by electronic means. In the spiritual marketplace thus created it is no surprise that an inevitable individualization is inherent in contemporary shamanisms (Wallis 2003: 58).
Nevertheless, when anthropologists de scribed shamanism, some people recognized similarities and resonances with familiar experience and ideas. Harvey, who examines the period from the viewpoint of paganism, reminds us that 'anthropology gave people a name that could be attached to things they were already doing, and then they added different techniques' (Harvey 2007: 106). Also, the spread of shamanic religious practices as well as ideas to other countries and habitats outside the United States is not a uniform process but involves adaptations to local cultural and historical climates. Neither the studies of the dynamics nor special characteristics of contemporary shamanism in one country, for instance, are necessarily directly transferable to other local contexts. Modern shamans nonetheless study and attend various shamanistic circles as well as cere monies other than in their homelands. The goals of some shamanistic networks or communities are local or work to strengthen the local culture, while those of other shamanistic networks do not recognize national borders.
As noted already, for example in the case of Harner and others, some researchers of shamanism turned into practitioners and left the academic world. It should also be remembered that rather than simply reading books as 'armchair shamans' , many practitioners were -and are -active seekers of shamanic knowledge and the anthropological literature on it (Wallis 2003: 34). Yet as Elina Hytönen-Ng (2016) points out, anthropological literature has usually only been influential at the beginning of the shaman ic path, losing its significance as practice strengthens. Dialogue between scholars and shamanic practitioners nonethe less has been advantageous on both sides. In contemporary shamanism the personal experience of a religion often outweighs the historical or cultural accuracy for practitioners (Crockford 2010: 154). This has even been viewed as the main doctrinal feature of North American and European shamanism (Stuckrad 2014: 176). Crockford (2010: 156) argues that it is the religious experience which for the participants of a ritual constitutes authenticity, 'because only experience is truly our own and provides our own way of flourishing or being fulfilled' .
Changes in the shaman's role
Individuals are practising shamanism when they come into contact with the spirits, as already noted. This is seen in the intim ate relationship where humans and spirits are consciously helping each other out. When shamans shamanize, they mediate spiritual knowledge to their community. In shamanic ontology the community includes human and non-human persons, including animals but also other spirits of nature and spiritual beings.
Somewhat stereotypically, a shaman in an indigenous culture has been understood as a game-resource manager, who understood -as Harvey (2005: 146) notes -that the animals used for food also 'have souls' , or perhaps are souls. Consequently, culturally appropriate forms of respect are offered, and further respectful acts promised at and after death. According to Harvey, 'all this is predicated on the ability of humans and animals to communicate, on the reciprocity of their relationships, and mutual intelligibility of their notions and performance of respect' (p. 147). Harvey writes: Shamans and shamanising provide particularly powerful tests of the bound aries of human attempts to find appropriate ways to live alongside other persons, i.e. of animism. Two major problems recognising one's ontological similarity with others, knowing the necessity of naming them persons, and of attempting to relate respectfully to all who live, are (a) for one some such persons (human or otherwise) are aggressive and even predatory, and (b) must eat at least some of them in order to live. (Harvey 2005: 139-40) There are not only animist cultures but also cultures within which it is possible to act occasionally as an animist (Harvey 2005: xv). One such is the modern Western culture. Although almost all members of the community in modern society do not deal with the killing of animals or the ethical problems associated with it, an unbalanced relationship of man with his environment is still a basic reason for the shaman's role in their society. Common to all shamanisms is that ill-health is often understood as a state of imbalance of power, and a result of inadequate interaction with other persons, both human and other-than-human (see e.g. Harvey 2005: 149).
Horwitz defines shamanism 'as a spiritual discipline which enables one to directly contact, use, and willingly be used by the spirit power of the Universe, generally for the purpose of healing or restoring balance in some way' (Horwitz 2000). Besides the concepts of balance and power, to understand the idea of spirits and spirit ual knowledge the key is the role of a shaman in his or her community, be it a traditional or modern, animist or non-animist, local or global one. It is a fundamental fact to understand that the role of shamans is usuallyeven in modern Western society -a servant of their community, and even shamans nowadays deal with the same 'daily facts of violence and intimacy' (Harvey 2005: 140), which may be different from those of traditional societies. The term 'servant' is to be understood as indicating that shamans often act for the benefit of their community. In modern shamanism, the fact that they possibly manage their own well-being through shamanism is also considered to result in common, even universal, good.
Modern society, within which contemporary shamans live, still has the boundaries of life and death, and the questions of health as well as well-being to face. Some of those may be caused by indifference or direct violence to other persons (human or non-human). Shamans are seen as mediators in this human-non-human communication. Their roles and performances, and sometimes their everyday lifestyle, gender, habits and even their very ontologies, mediate between the diverse oppositions and possibilities of their culture (Harvey 2005: 149). Wallis (2003: 70) points out that where neo-shamans critique society and its conven tional religious and social abuse, for in stance, they are different from indigenous shamans because their stance is not integrated into a wider community. Yet they may work also for improvement in the conditions of the wider society (p. 70). Persons practising shamanism often have another job to support themselves. The animistic worldview and the advice of spirit guides is seen to transform shamans' worldview and behaviour into a more ethic al and spiritual one. The shamanic journey is seen to affect their everyday life and duties in a personal way.
The journey of the shaman is at the centre of shamanizing. In our experience, the holding of various communal ceremonies such as rites of passage or environmental ceremonies is also part of contemporary shamanism. These also connect with the spirit world through various shaman istic techniques. However, their principle is the same. In the rituals or other practices, one or more of the partici pants are in contact with the spirit world in order to balance some situation or bring advice or healing to someone who needs it, which may also be a non-human target, such as a nature area.
Scholars have debated the authenticity of modern shamanism compared to the so-called traditional forms of shamanism. It is essential to conceive shamanism as a historical phenomenon that has changed and changes as a result of interaction between both humans and material as well as spiritual environments. When we look at shamanism , we must also realize that the community around the shaman has changed. The community in traditional societies was created by the society or village around the shaman, as well as the patients that the shaman worked with. In the contemporary setting the community may now even be a virtual community that never meets in person, and patients may be met only online. The Covid-19 pandemic has emphasized and accelerated this movement into the online environment. As we have noticed during our fieldwork, some shamans or shamanic practitioners work with the spirits for the community and the environment. Some do not work at all with humans but only with the environment. The community has also become a global community as practitioners are trained outside their home country. 2 While research on neo-shamanism has emphasized the role of the literature in the learning process (Svanberg 2003), the passing on of silent knowledge comes to be questioned. Hytönen-Ng (2016) has pointed out that anthropological literature does not have a very prominent role in the longer learning process within contemporary shamanism. When the role model of a practising shaman is missing in real life, the literature gives the practitioners a first impulse to discover the practices. Later on, the literature's role diminishes as the spirits that the person is working with take over. The non-human community's role is therefore emphasized.
If the context of traditional shamanism, a particular and relatively cohesive culture, is compared to contemporary global culture, one can also think that control or aspir ation for security is necessary, when we note that a basic course can only give the student basic tools for making a shamanistic journey. There is seldom an intensive teacher-apprentice system in place and the learning depends on the students' own willingness to take further courses. Their primary teachers are teachers of the spirit world. The fellow students can also act as teachers, reflecting the taught course material and giving a different perspective.
Contemporary shamans in the Western world often conduct their practices alone and in private (Wallis 2003: 60). We stress that contemporary shamans often work separated from like-minded animists, and shamans find their way of life, patients and shamanic vocation in many varied societies. Their often lonely practice is made possible by their connection with the spirit world, which represents their non-human community, and which is actualized in the liminal space of ritual. Whether the shaman works in a traditional or modern society, at the centre of shamanic ritual is the shaman 'journey' . The shaman brings help, advice or healing from the spirit world into this world. 3 The mediating of this spiritual knowledge takes place through ritual.
The researcher's role in contemporary shamanism and the challenges of carrying out autoethnography in shamanisms
The study of contemporary shamanism is still rare in Finland, which follows the general pattern of modern shamanism research. 4 Some research does exist, including the doctoral dissertations of Jan Svanberg (2003) and Marjo Remes (2005) on contemporary shamanism, as well as research articles by Kouri (2020) and Hytönen-Ng (2016), and one that we have written together (Hytönen-Ng and Kouri 2020). Some master's theses on the topic have also been written (e.g. Aarnio 2018; Metsähinen 2020).
As Kouri (2017: 34-5) has summarised, writing as such, interpreting experience in the field and understanding it as a fundamental medium of knowledge has been at the heart of anthropology and ethnographic approaches for decades. After the 1970s and 1980s and the crisis of representation, reflexivity and making the researcher's position evident in the research text have been an accepted part of ethnography. Hitherto, this was not expected; the researcher observed things only from the outside, objectively. The background of this change was researchers' growing awareness, especially of the relationship between method, language and social reality.
3 During the shamanic journey a shaman travels into the spirit world or the other world. This journey is usually done with the help of a drum (for a closer look at the use and importance of the drum in making a shamanistic journey, see Hytönen-Ng and Kouri 2020). 4 For more detailed information on the Western world, see Wallis 2003. Research was now seen as reflexive: while investigating, the researcher was also creat ing reality as much as producing a description of it. Ethnography was not just a method; ethnographers were now authors, and ethnographies were their literary product. Auto-ethnography seems like the ideal development in this context. As Kouri (2017: 37-8) notes, autoethnog raphy originally referred to the way ethnog raphers wrote down autobiographic al material or personal fieldwork experiences by talking about 'others' . Thereafter, the autoethnographic research method has evolved in diverse directions. Auto-ethnography stems from a wide and far-reaching 'post-modern' shift in academic thought, which has prompted researchers to be politically self-conscious and self-reflexive (Wallis 2003: 2-4, 9). Overall, autoethnography opposes the dualism of the insiderout sider paradigm. As researchers, we are part of our modern culture, a standpoint that should be regarded as a resource. When studying new religious components in our culture we are already, in a sense, native, and hence insiders within contempor ary shamanism.
Usually, it is thought that the researcher as an autoethnographer makes observations from outside the material world, and as a practitioner takes notes from the internal experiences of the spirit world. While carrying out autoethnography the researcher studies his or her own experi ences (Tienari and Kiriakos 2020). In reality, internal and external observations and experiences happen at the same time, and this demands self-reflection. Karen McCarthy Brown (2001: 14) describes the work of a researcher, especially in writing, as having an interpretation of different meanings and the networks of meanings that arise from them. The researcher weaves one (researcher's) network of meaning together with another, aesthetically different (research object's) network of meaning (p. 14).
It has been seen that producing ethnographic insights into modern shaman ism necessitates a certain degree of qualitative autoethnography and autoanthropology, or 'anthropology at home' (Wallis 2003: 3-6). However, in autoethnog raphy, the researcher's experiences become part of the research material. Firstly, at shamanic rituals, involvement is all or nothing . Shamanic journeys cannot easily be 'observed' or 'revealed' . Whether, for ex ample, the shaman is merged with the spirit helpers is not necessarily visible to the outside observer. Thus, in 1994 Kouri participated in a basic shamanic course in Sweden organized by the SCSS and taught by Horwitz. Over an extended four-day course it took her several days to accomplish the shamanic journey. Over the last days she followed the teachers' advice to change her position so that her feet, not head, were towards the centre of the circle, where the drummer was. It made a difference. When the drumming began, Kouri felt that the sound of the drum went through her soles into her whole body, and at the same time she heard birches singing and her journey started. She 'put her head on the self ' for a while, as Horwitz advised, and stopped the logical dialogue inside her head. For Hytönen-Ng the merging appeared only in a slight shift of balance. Studying one's own bodily experiences in her example highlights the small internal changes within the ritual instead of major externally visible characteristics.
Auto-ethnography also makes use of tacit knowledge that is usually hard to pinpoint with words. Tacit, or silent, knowledge is part of experience-based knowledge. Tacit or silent knowledge refers to the nat ural knowledge, experience and competence that people in their practices and activities depend on, but which is difficult to enunciate. According to Hannele Koivunen (1997: 78) silent knowledge includes all genetic, archetypal and experiential knowledge that humans possess. It is present holistically: it is hand skill, skin knowledge and knowledge of the deep layers of the brain (p. 9). Michael Polanyi distinguishes between two dimensions of knowledge. One is personal, tacit knowledge, and the other is explicit, focusing on 'something' knowledge. Awareness of tacit knowledge he calls an auxiliary or awareness 'of something' . Focused knowledge always needs tacit knowledge as its constituent. Tacit knowledge is not static, but intentional imagining. These auxiliary elem ents jointly carry the meaning of a thing (Polanyi and Prosch 1975: 33-5, 57). In spiritual practices, such as shamanism, part of the tacit knowledge is or has been born as a result of co-working in and with the spirit world and its non-human actors, spirits.
During the shamanic ritual the autoethnographer of shamanic experiences is in a liminal space (see van Gennep 1960). In order to understand the experience of liminal space, one should dismantle the idea of the inner and outer world. Firstly, during the shamanistic ritual the shaman is both on the journey in the spirit world and also in the material place where the ritual is held. Secondly, the direction of each step in the shamanic path is determined by what happens to the shaman in his or her everyday, often animistic, way of life, as well as by the individual experiences in the spirit world, which reciprocally affect the shaman's perceptions and way of life.
Here autoethnographic biography and the study of the oral history of shaman circles is useful for research.
Auto-ethnography allows us to identify meanings shared among subjects and within the research community, and also, in the case of the making of spiritual meaning, as in shamanism, to verbalize interactions, for example during the ritual and especially during the shaman journey between the human and the non-human (Kouri 2020: 230-1;Wallis 2003: 6). The shaman works in a community with both human and nonhuman actors. Spiritual knowledge is conveyed in the ritual of the shaman's journey between the two. Also, autoethnographers who study shamanism move between several different communities; thus their role as interpreters of their spiritual experiences are emphasized.
In autoethnography, the target is to understand the general by examining the individual and specific. Therefore, autoethnography questions the individual self and requires rewriting of the self and community, as Deborah E. Reed-Danahay says (1997: 4). At the same time, for example in this article, as researchers we move from generalizations of shamanism towards the context, experience and interpretation of the contemporary practitioner of shamanism.
Discussion
In this article we have examined how changes in the field should be reflected within the researcher's role. To get a grip on this, we have reviewed how the position and methods of the researcher have changed in the study of shamanism over the years. When looking into the historic al developments within the study of shamanism, it is possible to see a shift in the perspectives. While a century ago the focus was on observing the indigenous cultures and the habits as well as beliefs that the natives had, the current trends in research emphasize a more self-reflexive take on study. One approach is to trace this back to the major shift that Michael Harner with his contemporaries forged on the study and practice of shamanism in the Western world. As contemporary shamanic practices became well known and accepted within Western societies, the society that had traditionally surrounded the shaman also changed. Neo-shamanism found its ways and meanings within contemporary societies that were radically different from the societies that were encountered in traditional shamanism.
At the same time the research methods were developing as the study of the 'other' became questionable while the criticism of colonialism shifted the focus closer to home. More recently the focus in the study of one's own culture has turned towards the researchers themselves. They could now look at their own society and study it, but also -in the later stages of this development -start to study their own experiences as part of a particular society. This leads to the use of autoethnography. With the help of this inside knowledge, which includes corporeal knowledge, a deeper understanding of certain cultural forms could be reached.
In addition to the fact that in reflexive ethnography researchers are seen to be located within their experience, in autoethnography, researchers are located in their interpretation of the experience, concepts and other material of their research, both their own and of their field more widely. It could be said that in auto-ethnography, researchers are in two fields at the same time: the field of their subject and the field of research. They are insiders in both. Therefore, the meaning of interpretation is emphasized: auto-ethnographers interpret the understanding of the same concepts used by scholars and practitioners for themselves and other scholars alike.
Here is the signficance of auto-ethnography in modern shamanism and other spiritual movements to researchers and other readers. Auto-ethnographers act as interpreters of the experience-based knowledge of the field and the spiritual world they convey. The description of experiences of auto-ethno graphers can highlight the contribution of different human and non-human actors in the meaning-making process in which they are involved.
The challenge of modern shamanism is in its high diversity, and the personality as well as variety of the experiences of practitioners. In a situation like this, the import ance of contextualization is emphasized. It is realized in autoethnography in locating the researcher within his or her own body and shaman istic life path. The more that contextualized knowledge is obtained through studies and also through nonscientific biographies and other materials, the more accurately we can understand the specificities of modern shaman ism. Jaana Kouri, Ph.D. and M.Ed., is a researcher in the study of religions at Åbo Akademi University. She is interested in the relationships between humans and their environment. Her research areas include on the one hand the Baltic Sea region's environmental heritage, and on the other hand shamanism. She is also a word-art instructor and explores the possibilities of creative writing as part of research. Her latest publication on shamanism is the article 'Matka henkimaailmaan ja takaisin. Nykyshamanismi kuvittelun tekniikkana' (2020). She is the co-editor (with Aila Viholainen and Tiina Mahlamäki) of Uskonto ja kuvittelu. Taustoja, tulkintaa ja sovelluksia (SKS 2020) and (with Tuomas Räsänen and Nina Tynkkynen) Muutoksen tyrskyt ja kotirannan mainingit.
Kulttuurisia näkökulmia merentutkimukseen (SKS 2020). Her latest article is written with Savitri Yetoo, 'Voicing and visualizing change: perceptions of environmental heritage in the Baltic Sea region' in Heritage (2021).
Elina Hytönen-Ng is an ethnomusicologist and cultural researcher, who holds a doctorate in culture research, specializing in musicology, at the University of Eastern Finland and is a docent in ethnomusicology at the University of Turku. She has specialized in research on musical experiences and performance venues in the contemporary British jazz scene, but has also studied musical experiences related to contemporary shamanism. She has been an academic visitor at the Faculty of Music, University of Oxford, and a visiting research fellow at King's College London. She is currently the primary investigator on a three-year project (2021-4) focusing on lament rituals in contemporary Finnish society. | 2022-03-08T14:50:16.447Z | 2022-03-01T00:00:00.000 | {
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1404447 | pes2o/s2orc | v3-fos-license | Purification and characterization of an alpha1,2,-L-fucosyltransferase, which modifies the cytosolic protein FP21,from the cytosol of Dictyostelium.
A novel fucosyltransferase (cFTase) activity has been enriched over 106-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galβ1,3GlcNAcβ-paranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity behaved as a single component during purification over DEAE-, phenyl-, Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel filtration resins. The purified activity possessed an apparent M of 95 × 103, was Mg-dependent with a neutral pH optimum, and exhibited a K for GDP-fucose of 0.34 μM, a K for pNP-LNB of 0.6 mM, and a V for pNP-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identified a polypeptide with an apparent M of 85 × 103, which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-I-salicylate. Based on substate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fucα1, 2Galβ1,3GlcNAcβ-pNP. The cFTase preferred substrates with a Galβ1,3 linkage, and thus its acceptor substrate specificity resembles the human Secretor-type α1,2-FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent K of 0.21 μM and an apparent V of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fucα1,2Galβ1,3GlcNAcβ-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations.
The glycosylation of proteins traversing the secretory pathway of eukaryotic cells has been well studied (1)(2)(3). Evidence has been steadily accumulating that some amino acid side chains of proteins of the cytosol and nucleoplasm are also modified, with either a single sugar, O-GlcNAc (3,4), or oligosaccharides (5)(6)(7)(8)(9). The frequency of O-GlcNAc modifications of cytosolic and nucleoplasmic proteins may approach that of phosphorylation (10), and there is evidence that cytosolic glycosylation may be regulatory (5, 8, 10 -12), based on the existence of glycoforms which vary with different physiological states and protein localizations, the presence of specific cytosolic glycosidases, half-lives of sugar modifications which vary relative to the host protein, and potential competition for attachment sites between O-GlcNAc and phosphate groups.
FP21 is a protein found both in the cytosol and the nucleus, and is modified by an oligosaccharide in the cellular slime mold Dictyostelium discoideum (7,8). FP21 is a highly conserved protein whose amino acid sequence shows 68% identity, 78% similarity, and one amino acid residue difference in length between Dictyostelium and humans. FP21 has been detected in the cyclin A/cdcK2 complex associated with the G1 checkpoint of the human HeLa cell cycle (13), the kinetechore complex (14) of budding yeasts, 1 and in association with certain membranes of Dictyostelium. 2 FP21 has a particularly high abundance in the inner ear organ of Corti (15)(16)(17). An FP21 gene is present in a green alga virus genome (18), and two copies are frequently found in eukaryotic genomes, including Dictyostelium. 3 Dictyostelium FP21 possesses a single tetra-or pentasaccharide, which appears to contains Fuc, 4 Xyl, and Gal (8) and is probably O-linked based on susceptibility to release by mild base (7) or hydrazinolysis. 2 Two FP21 isoforms have been purified which vary in their proportions of Xyl and Gal (8).
Since a GDP-Fuc synthesis mutant in Dictyostelium exhibits slow growth which can be partially rescued by exogenous Fuc, 2 we have investigated the fucosylation of FP21 for its possible involvement in this phenotype. Using afucosyl-FP21 from the fucosylation mutant as an acceptor substrate, an ␣-cFTase (cytosolic fucosyltransferase) activity dependent upon GDP--Fuc was detected in the cytosolic S100 fraction but not in the particulate or membrane fraction (7). FP21 accounted for Ͼ80% of the acceptor activity in crude S100 extracts of the fucosylation mutant, as if it were the primary acceptor substrate for this enzyme. This activity was also able to attach Fuc to lacto-N-biose (Gal1,3GlcNAc-) attached to a hydrophobic aglycone moiety, as determined by cross-competition studies. This enzyme activity appeared to mediate attachment of a peripheral Fuc, and was novel in terms of its apparent cytosolic, rather than Golgi, compartmentalization, and its submicromolar K m for GDP-Fuc. As shown here, a 1.2 million-fold purified preparation of the enzyme activity retains its high affinity for GDP-Fuc, displays a high affinity for afucosyl FP21, links Fuc in a ␣1,2 linkage to a 1,3-linked Gal on type 1 and 3 acceptors, and copurifies with a polypeptide which can be photoaffinity-labeled with a donor substrate analog.
Cells
Strains Ax3 and HL250 were grown at 22°C in HL-5 medium on a gyratory shaker. In 6-liter flasks, cells grew to a density of 6 -10 ϫ 10 7 per ml, which is referred to as stationary phase. Ax3 is a normal strain and HL250, derived from Ax3 by chemical mutagenesis, is unable to synthesize GDP-Fuc from GDP-Man and thus incorporates negligible Fuc into protein (7,8). Cells were developed by washing logarithmically growing cells in 17 mM KH 2 PO 4 neutralized to pH 6.0 with NaOH (KP) and shaking in 6-liter flasks at 2 ϫ 10 7 cells/ml in KP for 12 h. Cells were synchronized with respect to the cell cycle by diluting stationary phase cells in a 500-ml flask to 10 6 cells/ml of fresh HL-5 in a 6-liter flask, as described previously (19).
Fucosyltransferase Assay-cFTase activity was usually assayed as the transfer of [ 3 H]Fuc from GDP-[ 3 H]Fuc to pNP-LNB, which was determined by adsorption onto a C 18 Sep-Pak cartridge followed by elution with methanol (20). GDP-Fuc was at a concentration 3-fold above its K m value, and pNP-LNB was at slightly below its K m value. Each assay tube routinely contained 9 g of pNP-LNB (equivalent ot a final concentration of 0.36 mM) dried from a 1 mg/ml aqueous solution in a 1.5-ml polypropylene microcentrifuge tube by vacuum centrifugation, 5 l of 20 mg/ml BSA in buffer D, and 40 l of enzyme sample diluted, if appropriate, in buffer D. During pilot studies, column fractions were assayed after desalting on PD-10 columns (Pharmacia) equilibrated in buffer D. These columns contain 9.1 ml of Sephadex G-25 medium. The reaction was initiated by the addition of 5 l of a mixture prepared in buffer H and containing, after dilution to 50 l, 1.0 M GDP-Fuc (1-8 ϫ 10 5 dpm), prepared from a mixture of GDP-[ 3 H]Fuc and unlabeled GDP-Fuc, 15 mM MgCl 2 , 2.0 mM MnCl 2 , 0.10 mM EDTA, 1.0 mM DTT, and 15% glycerol. The mixture is stable for Ն3 days at 0°C, and thus divalent cation-promoted degradation of GDP-Fuc (21) did not occur. When assaying crude cell extracts and DEAE column fractions, the mixture was supplemented with sodium fluoride and ATP, at final concentrations in the assay mixture of 2.0 mM and 1.0 mM, respectively. Assays of crude extracts containing sucrose lacked glycerol. Reactions containing enzyme preparation A were supplemented with 0.05% Tween 20. The reaction mixture was incubated for 10 -120 min at 30°C, and stopped by the addition of 1 ml of cold water. The reaction mixture was either immediately filtered over the C 18 Sep-Pak cartridges, or frozen at Ϫ80°C until filtration. Cartridges were reused until flow rates became unacceptable. Samples were filtered in groups of 10 on a Visiprep manifold (Supelco), which was alternated between a waste receptacle and a second receptacle containing 20-ml scintillation vials. Incorporation was determined as described previously (7).
Reactions involving protein acceptor substrates were conducted similarly, except that protein acceptors were introduced from concentrated stock solutions, Tween 20 was present at 0.05% (v/v), BSA was reduced to 0.5 mg/ml, and the reaction was initiated by the addition of the cFTase. Incorporation into protein substrates was determined after SDS-PAGE of the samples, within 1 day of fixation with Coomassie Blue and destaining, by excising the appropriate region of the gel and scintillation counting, as described (7).
Protein Determination-Protein concentration was determined by a commercial modification of a Coomassie Blue dye binding method (22), or from A 280 values, assuming an extinction coefficient of 1 ml/mg protein for a 1-cm path length.
Cell Lysis-1-2 ϫ 10 11 cells were grown to near maximum cell density. After washing once in ice-cold water, cells were resuspended in buffer A, suctioned through a bed of glass wool, and lysed by forced passage through a 47-mm diameter Nuclepore filter with 5-m diameter pores mounted on the end of a 60-ml syringe. The lysate was centrifuged at 4,000 ϫ g for 2 min, and the supernatant was centrifuged at 100,000 ϫ g for 60 min. All procedures were performed at 0 -4°C.
DEAE-Sepharose Fast Flow Chromatography-The final S100 supernatant was pumped onto a 450-ml DEAE-Sepahrose Fast Flow column (4.8 ϫ 28 cm) pre-equilibrated in buffer B at 250 ml/h, and the column was washed with buffer B until the A 280 dropped below 1% of its maximum. cFTase was eluted in a linear gradient of 0 -0.25 M NaCl, consisting of 1.25 liter of starting buffer B and 1.25 liter of limit buffer C. Protein was determined based on A 280 or the Coomassie Blue dye binding assay. Fractions from the main activity peak were pooled and frozen at Ϫ80°C.
Phenyl-Sepharose 6 Fast Flow Chromatography-DEAE pools from 1.5 ϫ 10 12 cells were thawed and pumped onto a 175-ml phenyl-Sepharose 6 Fast Flow (high-sub) column (2.6 ϫ 31 cm) at a flow rate of 275 ml/h. The column was washed with buffer D until the A 280 dropped below 1% of its maximum. cFTase was eluted by application of a linear 0 -60% gradient of ethylene glycol, consisting of 400 ml of starting buffer D and 400 ml of limit buffer E. Protein was determined based on A 280 and the Coomassie Blue dye binding assay. cFTase activity was determined after 5-fold dilution of fractions with buffer D. Fractions from the activity peak were pooled and concentrated 20-fold by ultrafiltration using a PM-30 membrane, and then diluted 5-fold in buffer D to reduce ethylene glycol to Ͻ10% (v/v). The DEAE and phenyl columns were reused after in situ washing with 8 M urea and 0.1 N NaOH.
Dye Column Affinity Chromatography-The phenyl-Sepharose pool was diluted to 10% (v/v) ethylene glycol with buffer D and pumped onto a 25-ml column (1.6 ϫ 25 cm) containing Reactive Blue-4 coupled to cross-linked 4% agarose beads (5.2 mg dye/ml), which had been sequentially precycled with 0.5 mg/ml bovine serum albumin in buffer D, buffer F, buffer D, and 10% (v/v) ethylene glycol in buffer D. After sample loading, the column was washed in buffer D until the A 280 dropped below 1% of its maximum, and eluted with a linear gradient of 0.1-1.5 M NaCl, consisting of 60 ml of starting buffer D and 60 ml of limit buffer F. Protein was determined based on A 280 and the Coomassie Blue dye binding assay. cFTase activity was determined after 5-fold dilution with buffer D. Fractions from the activity peak were pooled, concentrated 2-3-fold on a Centriplus-30 centrifugal concentrator, and diluted with buffer B to a final calculated concentration of 0.1 M NaCl.
GDP Affinity Chromatography-The Reactive Blue-4 pool was passed over a 1.2-ml GDP-adipate-agarose (1.6 mol GDP/ml packed resin) column connected in series with a 4.0-ml GDP-hexanolamineagarose (2.8 mol of GDP/ml of packed resin) column. The GDP moiety of GDP-adipate is linked via the 2Ј-or 3Ј-hydroxyl of the ribose, whereas the GDP moiety of GDP-hexanolamine is attached via its -PO 4 , which corresponds to the linkage with Fuc in the native donor substrate GDP-Fuc. After washing in buffer D, the GDP-hexanolamine column was disconnected and eluted with a 0 -2 mM gradient of GDP, formed from buffers D and G. 97% of the activity was eluted before 1 mM GDP as determined by HPLC gel filtration, and was pooled and concentrated to Ͻ1 ml in a Centriplus-30 concentrator.
The final Superdex 200 activity pool, purified 1.2 ϫ 10 6 -fold, is referred to as preparation A. Activity purified 317-2200-fold through DEAE, phenyl, and Superdex resins is referred to as preparation B, activity purified 106-fold through DEAE, phenyl, and Reactive Blue-4 resins is referred to as preparation C, and activity purified 27-fold through the DEAE-resin is referred to as preparation D.
Fucosyltransferase Characterization
Photoaffinity Labeling-GDP-hex-ASA, generously provided by Eric Holmes, was iodinated in its salicylate moiety as described (23). Superdex 200 column fractions, 30 -300 l, were preincubated in a quartz 1-ml cuvette for 15 min in dim red fluorescent illumination in the presence of 30 M GDP-hex-125-I-ASA, Ϯ 700 M GDP-Fuc, in buffer D. Photolysis was effected by illumination at 254 nm with a 30-watt Mineralight for 30 s on opposite sides, as described (23). Each sample was centrifuged through two concentration/dilution cycles in a Microcon-10 centrifugal ultrafiltration concentrator. Laemmli sample electrophoresis buffer at 70°C. was added directly to the concentrated sample in the Microcon-10 device, and transferred together with a rinse aliquot to the SDS gel sample well.
SDS-PAGE-SDS-PAGE was performed on 7-20% linear acrylamide gradient gels using the Laemmli discontinuous buffer system, as described previously (8). 80-l aliquots from the Superdex 200 fractions were reduced in 5 mM DTT, treated with with 40 mM iodoacetamide to reduce artifactual bands (24), and then diluted with 4 ϫ concentrated Laemmli sample buffer. Gels were silver-stained according to Ref. 25.
pH Variation, Divalent Cations, and Inhibitors-Effects of pH variation, divalent cations, and potential inhibitors were determined on preparation B by diluting the enzyme 10-fold from buffer D into appropriate solutions. MES was used to buffer the pH range from 5.5 through 6.5; Bis-Tris propane was used from pH 6.5 to 8.5; and CAPSO was used from pH 8.5 to 9.5. Activities did not vary more than 10% between buffers at the crossover pH values 6.5 and 8.5. Inhibitors were preincubated with the enzyme for 15 min prior to the start of the assay.
Kinetic Studies-Reactions were conducted as described for FTase assays. Unless otherwise indicated, reaction mixtures contained 125 pg of preparation A protein as enzyme, 1 M GDP-Fuc, and the indicated amount of acceptor substrate, and were incubated for 1 h at 30°C.
cFTase Reaction Product Characterization
Synthesis of Fucosyl-pNP-LNB-The cFTase assay reaction was scaled up to synthesize suitable quantities of the reaction product for fucosidase digestion. To remove salts and proteins, the reaction product was adsorbed to a C 18 Sep-Pak, eluted with MeOH, dried, redissolved in water, and passed over a PD10 gel filtration column equilibrated in water. The radioactive peak was collected and the concentration of reaction product was calculated from the specific activity of GDP- Exoglycosidase Digestions-Reaction mixtures contained 120 pmol of [ 3 H]fucosyl-pNP-LNB (140,000 dpm), and the indicated amount of enzyme, in a final volume of 50 l of buffer composed according to the supplier's recommendation. Reactions were incubated at 37°C, and aliquots were assayed by determining the percent of total disintegrations/min which did not adsorb to a C 18 Sep-Pak cartridge.
FP21 Isolation
Purification of Dictyostelium FP21-FP21 isoforms-I and -II were purified over DEAE, phenyl, and monoclonal antibody 3F9 affinity columns from strain Ax3 S100 extracts, as described (8). Purification of FP21 from strain HL250 also yielded two isoforms similar in chromatographic behavior to the Ax3 isoforms, and are referred to as GP21-I and GP21-II. Protein was dialyzed against 50 mM HEPES-NaOH (pH 7.4), and concentrated in Centriprep centrifugal ultrafiltration concentrators (10-kDa molecular mass cut-off). Protein concentration was determined by amino acid composition analysis of phenylisothiocyanatederivatives after acid hydrolysis, using norleucine as an internal standard (26).
Preparation of recombinant Dictyostelium FP21-The open reading frame of FP21 was amplified using Dictyostelium DNA as the template and fp15 and fp14 as primers (see Table IV in Ref. 8) in a polymerase chain reaction. These primers contained BamHI restriction sites which were used to clone the amplified DNA into the BamHI restriction site of the inducible (27, 28) expression vector pET19b (Novagen, Madison, WI), downstream of the T7 RNA polymerase transcription element, such that an oligo-His tag was introduced at the NH 2 terminus. The deduced NH 2 -terminal sequence of rP21 is MGHHHHHHHHHHSS-GHIDDDDKHMLEDP followed by the natural FP21 sequence, which was verified by sequencing of the plasmid DNA (8). Expression host Escherichia coli BL21 (DE3) cells carrying a lysogen with a copy of the T7 RNA polymerase gene under lacUV5 control were transformed under carbenicillin selection. After induction with isopropyl-1-thio--Dgalactopyranoside, expressing colonies were examined. Inclusion bodies were not observed. rP21 was isolated from clone rP21A under nondenaturing conditions using an affinity column consisting of nickel cations immobilized on Sepharose 6B, essentially as described (29). Purified protein was exhaustively dialyzed against 50 mM HEPES-NaOH (pH 7.4), and concentrated in a centrifugal ultrafiltration concentrator (10-kDa molecular mass cut-off). Purified protein (Fig. 7) was shown to be recognized in a Western blot by monoclonal antibodies 3F9 and 4E1, and anti-FP21(68 -82)/L97 (8), confirming its identity as rP21. Protein concentration was determined by amino acid composition analysis (see above).
Pilot Studies on Partially Purified
Enzyme-Pilot studies on partially purified enzyme established the conditions of the standard assay. After DEAE-ion exchange chromatography (27-fold purified; preparation D) or a combination of ion ex-change chromatography, phenyl-Sepharose hydrophobic interaction chromatrography, and gel filtration (2200-fold purified; preparation B) enzyme activities using pNP-LNB as substrate were linear with respect to time (0 -48 h) and protein concentration (data not shown). 70% of the activity maximum at pH 7.2 was retained over a broad pH range of 5.5 to 9.0, beyond which activity dropped rapidly. The activity was similar in Tris, Bis-Tris propane, MES, HEPES, or CAPSO buffers. Activity was optimal at concentrations of NaCl from 100 to 300 mM, but was stable for short times at concentrations up to at least 1.5 M NaCl, or (NH 4 ) 2 SO 4 at 20%. The enzyme was significantly less active or less stable in KCl. Of the divalent cations tested, as chlorides of Mg 2ϩ , Mn 2ϩ , Ca 2ϩ , Zn 2ϩ , Fe 2ϩ , and Co 2ϩ , only MgCl 2 and MnCl 2 supported activity. 0.5-2.0 mM Zn 2ϩ , Fe 2ϩ , and Co 2ϩ inhibited activity from 50 to 95% in the presence of 0.5 mM MgCl 2 . Optimal activity occurred in 15 mM MgCl 2 and 2.0 mM MnCl 2 . Sodium fluoride and ATP stimulated activity 20 -40% in crude extracts but inhibited activity 10 -30% in purified preparations, and thus were included only in the former preparations. Activity was inhibited 47% by 1.0 mM N-ethylmaleimide, and only 18% by 1.0 mM pyridoxal-5phosphate, using 0.27 M GDP-Fuc and 0.36 mM pNP-LNB as substrates, suggesting that the active site may contain an essential cysteine (36). Glycerol (up to 30% (v/v)), 1 mM DTT, and 0.1 mM EDTA (in the presence of a concentration excess of Mg 2ϩ ) did not inhibit activity, and thus were included as potential stabilizing agents. NaN 3 at 0.02% (w/v) led to a 25% reduction in activity and so was not used. Optimal temperature was 30°C, with lower activities detected at 37 and 24°C. These properties of the partially purified enzyme activity were similar to those previously determined for the unfractionated activity (7).
To test the effect of detergents, enzyme purified through the Reactive Blue-4 step (preparation C) was diluted 100-fold in buffer D and incubated in varying concentrations of 16 detergents as described under "Experimental Procedures." Only Tween 20 and Tween 80 sustained activity at all concentrations tested. Activity losses ranged from 30 to 99% for the other detergents; in general, greater losses were observed as detergent concentrations diminished below the critical micelle concentration. At later stages of purification, 0.05% Tween 20 was superior to BSA in preserving enzyme activity at 4°C.
Enzyme Purification-cFTase present in the S100 fraction was remarkably stable, with 90% activity remaining after 10 days at 4°C, or after 10 freeze (Ϫ80°C)/thaw cycles. Although activity could be recovered from desalted (NH 4 ) 2 SO 4 precipitates, the broad range (30 -70%) over which activity precipitated was not useful (data not shown). Activity quantitatively adsorbed to DEAE resins from the crude extract, and Ͼ98% of the eluted activity emerged at about 30 mM NaCl as a monodisperse peak ahead of the major protein peaks (Fig. 1A), thus resulting in a 25-30-fold purification ( Table I). The remaining activity emerged later as a small peak. The Fast Flow resin was superior to DEAE-Sephadex and DEAE-cellulose (fibrous or granular) because of rapid flow, resistance to clogging, and resistance to shrinkage at higher ionic strengths. The column was stable to multiple cycles of use and washing in 0.1 N NaOH. Enzyme also adsorbed at pH 6.8 but enrichment was reduced 2-fold. Enrichment was also diminished by Ͼ2-fold if the cell lysate was brought to 5 mM MgCl 2 and centrifuged for 1 h at one-half the g-force, compared to the standard method. As determined by HPLC gel filtration (see below), activity exhibited a single apparent M r of 95 ϫ 10 3 . 5 The pool of the DEAEpurified enzyme was stable to storage at 4°C and tolerated repeated freeze/thawing.
The DEAE pool adsorbed quantitatively to phenyl-Sepharose 6 Fast Flow (high sub) and Ͼ99% of the eluted activity emerged as a single peak with slight trailing close to the end of a 0 -60% gradient of ethylene glycol (Fig. 1B). cFTase activity was assayed in eluted fractions after dilution in buffer D, and recovery ranged from 50 to 90%, or 53% for the trial reported (Table I). Quantitative adsoprtion to phenyl-Sepharose 6 Fast Flow (low sub), or to phenyl Sepharose CL-4B, required addition of 10 -20% (NH 4 ) 2 SO 4 to the DEAE pool, and thus these resins were not used.
The pool of phenyl-purified enzyme was concentrated by ultrafiltration. Recovery of activity after concentration varied from 10 to 90%, or 27% in the reported trial (Table I), and activity was not detected in the ultrafiltrate. At this stage of purification, the activity typically displayed an apparent M r value of 95 ϫ 10 3 after HPLC gel filtration (see below), and thus poor recovery appeared to be associated with insolubility or denaturation. In early trials with the other phenyl resins involving long column residence times, substantial activity with an M r value of 40 ϫ 10 3 was correlated with poor recovery of activity, 5 as discussed further below.
The phenyl-pool activity adsorbed efficiently to the the Reactive Blue-4 dye resin, and 97% of the eluted activity emerged as a monodisperse peak early in a gradient of 0.1-1.5 M NaCl (Fig. 1C). The remaining activity emerged later as a small peak, and overall activity recovery was 92%. cFTase activity could also be adsorbed to RB-1, RB-72, RG-5, RR-120, RG-19, and CB dye resins, but the Reactive Blue-4 resin was selected on the basis of more selective binding relative to BSA. RY-3 and RY-86 dye resins did not adsorb activity. Concentration of activity by ultrafiltration resulted in a 33% loss of activity.
The Reactive Blue-4-pool activity was unretarded by GDPadipate Sepharose CL-4B. 290 g of protein was recovered from the column after elution with 2 mM GDP, but was not examined further. Activity in the flow-through fraction was, however, quantitatively adsorbed to GDP-hexanolamine Sepharose. The selective binding to GDP-hexanolamine was predicted based on the inhibitor characteristics of a series of GDP-Fuc analogues (see below), and is a behavior typical of mammalian ␣1,2-FTases (30 -33). The GDP-hexanolamine column was eluted with a 0 -2 mM GDP gradient, and the activity eluted in the 0 -1 mM range of the gradient. Ͻ5% additional activity was eluted by various combinations of high GDP and NaCl concentrations, Tween 20 and ethylene glycol. After ultrafiltration, the enzyme activity was stable at 4°C for at least 1 days.
The GDP affinity pool was applied to a Superdex 200 gel filtration column by HPLC. 95% of the recovered activity eluted as a monodisperse peak (peak I) centered at a M r position of 95 ϫ 10 3 (Fig. 1D). An additional 3.7% eluted at M r position 40 ϫ 10 3 (peak II), and the remaining activity eluted near the void volume. In pilot studies, Ͼ90% of activity applied to the GDPhexanolamine column was recovered after gel filtration. The 35% recovery in the reported trial (Table I) did not seem to be due to retention on the column (see above), and may have been due to 3 days storage of the GDP-hexanolamine fractions at 4°C prior to further processing. After gel filtration, activity decayed with a 12-h half-life at 4°C, but was stable for 3 days in 0.05% (v/v) Tween 20 at 4°C. Activity was only partially stabilized by 2.0 mg/ml BSA, and strongly destabilized by purified cytochrome c (data not shown). Finally, activity was stabilized by freezing at Ϫ80°C.
HPLC gel filtration fractions were analyzed by SDS-PAGE and silver staining (Fig. 2). Three major silver-stained bands were visible in the fractions of highest activity (peak I), centered at 30.4-ml elution volume. Only the amount of the most 5 T. Scott-Ward and C. M. West, unpublished data. heavily stained band, at M r position of 85 ϫ 10 3 , correlated precisely with the activity of the fractions. To obtain further evidence that this polypeptide, FT85, was equivalent to the enzyme, selected HPLC gel filtration fractions were UV-irradiated in the presence of the photoactive donor substrate analogue GDP-hex-125 I-ASA, at 30 M, in the presence or absence of 690 M GDP-Fuc. Photolabeling under similar conditions has previously been useful in identifying fucosyl-and mannosyltransferases (23,35). SDS-PAGE followed by autoradiographic analysis of a fraction from peak I (30.4-ml elution volume) revealed that the most intensely photolabeled polypeptide, PL85, migrated at the position of FT85 (Fig. 3, lane e), and labeling of this band was inhibited by GDP-Fuc (lane f). PL85 was not detected in the fraction eluting at 28.6 ml (lane c), which possessed Ͻ0.2% of the peak activity, but was faintly detectable in the fraction eluting at 33.1 ml, which possessed 3.1% of the peak activity (not shown). Two additional, much less intensely photolabeled bands were also detected in the major activity peak at M r positions 40 and 29 ϫ 10 3 , and are discussed below. The relative intensities of these bands is clearer in Fig. 3, lane i, which is a shorter autoradiographic exposure. Multiple minor bands were also photoreactive in lane e, but in each case labeling was only slightly inhibited by GDP-Fuc (lane f), showing that their reactivity was nonspecific and that photoprotection by GDP-Fuc was not due to general UV absorbance by the nucleotide moiety.
The minor cFTase activity peak eluting at 37.7 ml (Fig. 1D, peak II; M r position of 40 ϫ 10 3 ) contained a single, specifically- Table I. Protein, which was monitored by absorbance at 280 nm using a 0.5-cm path length flow cell, and cFTase activity, which was assayed as dpm in the presence of 1 M GDP-Fuc and 0.36 mM pNP-LNB, are plotted as a function of elution volume. Panel A, typical example of an S100 fraction which was pumped onto the DEAE-Sepharose Fast Flow resin. After washing, the column was eluted with a 0 -0.25 M gradient of NaCl, whose concentration at the entry point of the 450-ml column is plotted. Fractions from the main DEAE column activity peak which contained Ͼ500 dpm activity/aliquot were pooled and frozen. Panel B, activity pools from 10 DEAE columns were pooled and pumped onto the phenyl-Sepharose 6 Fast Flow (high sub) resin. After washing, the column was eluted with a 0 -60% (v/v) gradient of ethylene glycol, whose concentration at the entry point of the 175-ml column is plotted. Panel C, fractions from the main phenyl column activity peak which contained Ͼ4000 dpm/aliquot were pooled, concentrated, diluted to reduce the ethylene glycol concentration, and pumped onto the Reactive Blue-4 agarose dye resin. After washing, the column was eluted with a 0.1-1.5 M NaCl gradient, whose concentration at the entry point of the 25-ml column is plotted. Fractions from the main peak which contained Ͼ2000 dpm/aliquot were pooled, concentrated, and diluted to 0.1 M NaCl prior to application to the GDP-adipate and GDP-hexanolamine columns (not shown). Panel D, the GDP-eluate from the GDP-hexanolamine column was concentrated and applied to the Superdex 200 HPLC gel filtration column and eluted isocratically. 95.0% of the activity emerged as peak I centered at 30.4 ml, and 3.7% emerged as a minor peak centered at 37.7 ml, as peak II. Calibration of the Superdex column with M r standards (see "Experimental Procedures") indicated that peaks I and II had apparent M r values of 95 ϫ 10 3 and 40 ϫ 10 3 , respectively. labeled photoreactive species, referred to as PL40, at an M r position of 40 ϫ 10 3 (Fig. 3, lane g). Negligible PL85 was detectable in this fraction. The amount of PL40 varied with the level of cFTase activity in neighboring fractions (data not shown). PL40 was not detectable by silver staining (Fig. 2, 37.7 ml), as expected based on its low apparent abundance relative to PL85 (Fig. 3, compare lanes e and g). PL40, together with PL29, were also observed in peak I (Fig. 3, lane e). PL40 appears to be a proteolytic fragment of PL85 based on the following observations. 1) The ratio of PL40 to PL85 in the major peak I was time dependent. After 7 days of storage at 4°C, during which activity diminished by Ͼ90%, only PL40 could be detected (Fig. 3, lanes k-o).
2) The original photolabeling of peak I described above in Fig. 3 had been performed after 4 days of storage at 4°C, during which activity had decreased by 50%. SDS-PAGE analysis of the pooled peak I fractions at this time revealed that the level of FT85 had decreased concomitant with the appearance of the new bands labeled at FT40 and FT29 (compare Fig. 3, lane a, with Fig. 2, 30.4 ml). Thus the appearance of PL40 in the main activity peak is correlated with the appearance of FT40. 3) Pilot studies on phenyl-purified enzyme showed that cFTase activity became smaller as determined by HPLC gel filtration as activity decreased, 5 and that the M r 95 ϫ 10 3 activity peak I was less stable than the M r 40 ϫ 10 3 activity peak II. 6 We conclude that PL85 is equivalent to FT85, and that FT85 is susceptible to proteolytic degradation to FT40, which is equivalent to PL40. FT40 possesses similar K m values for its substrates and similar substrate specificity, but has a reduced specific activity compared to FT85 (see below). This model explains both the time-dependent ap-6 P. Teng-umnuay and C. M. West, unpublished data. The samples in lanes a-j had lost about 50% of their cFTase activity prior to photolabeling and SDS-PAGE, which explains the reduced abundance of FT85 and increased abundance of FT40 and FT29 in lane a relative to Fig. 2. Lanes k-o, peak I fractions from a separate gel filtration of the same cFTase preparation A were photolabeled after Ͼ90% loss of cFTase activity. The amount of original activity which was photolabeled corresponded to 2% of the original activity which was photolabeled in lanes c-j. The relative intensity of labeling of PL40 in the sequential fractions is approximately proportionate to the original activity in the fractions. 2 Together with the results shown in Fig. 2 and other data, this figure shows that FT40, and possibly FT29, are breakdown products of the intact cFTase polypeptide, FT85. These and other results (see text) suggest that reduction of cFTase activity occurs as FT85 is proteolytically degraded to FT40 and possibly FT29.
TABLE I Purification of cFTase activity
Purification of cytosolic GDP-Fuc:lacto-N-bioside fucosyltransferase activity from Dictyostelium cells. S100 extracts were prepared and chromatographed on DEAE-Sepharose Fast Flow. Active DEAE fractions were pooled and frozen at Ϫ80°C. Ten DEAE preparations were thawed and pooled for the subsequent purification steps. The final Superdex 200 activity pool is referred to as preparation A. Activity purified 317-2200-fold through DEAE, phenyl, and Superdex resins is referred to as preparation B, activity purified 106-fold through DEAE, phenyl, and Reactive Blue-4 resins is referred to as preparation C, and activity purified 27-fold through the DEAE-resin is referred to as preparation D.
c Minimum estimate derived from the next stage of purification.
pearance of FT40 in the M r 95 ϫ 10 3 activity peak after gel filtration, as well as the time-dependent appearance of the M r 40 ϫ 10 3 activity peak II before gel filtration. In summary, the cFTase activity was purified 1.2 million-fold at 4.3% yield. Some of the losses could be attributed to specific proteolysis, which may explain the small proportion of activity (Ͻ5%) which could be resolved from the main peak during DEAE, phenyl, Reactive Blue-4, and gel filtration chromatographies. The FT85 polypeptide represents about 30% of the total protein. The cFTase consisted of a single 95 ϫ 10 3 M r activity after the first chromatography step and, aside from inconsistent generation of the apparently proteolytically-derived 40 ϫ 10 3 M r activity, no other molecular species expressing this activity were detected during the purification.
Properties of the Enzyme-Fucosylation of pNP-LNB by the highly purified preparation A was linearly dependent on time and pNP-LNB concentration (data not shown). This preparation exhibited a K m for pNP-LNB of 0.58 mM at 1 M GDP-Fuc, compared to 0.73 mM for the unfractionated activity (7), and a V max of 620 nmol/min/mg protein (Fig. 4). Preparation A exhibited a K m for GDP-Fuc of 0.34 M (Fig. 5) at 2.0 mM pNP-LNB, about 4-fold lower than the value obtained for the unfractionated activity (7). The M r 40 ϫ 10 3 form which was generated in some trials exhibited similar kinetic properties, with K m values for GDP-Fuc of 0.23 M and for pNP-LNB of 0.77 mM, and was probably a degradation product of the higher M r form (see above).
Donor substrate Analogues-Nucleotides are often potent glycosyltransferase inhibitors and, similarly, GDP was a good inhibitor of the cFTase (Table II). Guanosine, GMP, and GTP were also good, but slightly weaker inhibitors, indicating that the 5Ј-phosphates are not important. Substituents on the 5Јphosphates are also not important, because GDP-Man and GDP-hex-ASA were good inhibitors. The ribose moiety is significant, because guanine was not a good inhibitor. The 2Јdeoxy group of ribose is not required for recognition, but bulky substituents are not tolerated, as 2Ј,3Ј-isopropylidene guanosine and 3Ј,5Ј-cGMP were poor inhibitors. Integrity of the ribose ring is essential as the periodate-oxidized 2Ј,3Ј-dialcohol was ineffective. The identity of the guanine base is important because 5Ј-phosphates of adenosine were not significantly inhibitory. Furthermore, the guanine moiety does not tolerate substituents at positions 6 -8. Similar results were observed for the M r 40 ϫ 10 3 form detected in some trials of preparation B. 5 These observations suggested that guanine ribonucleosides modified at the 5Ј-position of ribose may be useful for purifying and identifying the enzyme. These predictions were validated by the affinity chromatography and photoaffinity results described above.
Acceptor Substrate Analogues-The pNP-LNB acceptor substrate consists of Gal1,3GlcNAc attached to a paranitrophenyl aglycone moiety. pNP was an important recognition determinant as Gal1,3GlcNAc at a 12-fold concentration excess inhibited the enzyme by only 50% (Table III). Benzyl-LNB and 8-methoxycarbonyloctyl-LNB exhibited only 15 and 9% of the activity of pNP-LNB at 1 mM (Table IV); these differences were attributable to changes in K m or V max depending on the aglycone (data not shown).
The cFTase preferred a disaccharide acceptor as negligible activity was observed with monosaccharide-pNP substrates. 2-O-methylation or ␣1,2-fucosylation of the nonreducing terminal Gal of pNP-LNB abolished activity (Table IV). The 1,3linkage of the Gal was important for enzyme recognition, as Gal1,4GlcNAc and Gal1,6GlcNAc were at best very weak inhibitors (Table III), and Gal1,4GlcNAc-pNP exhibited only several percent of the activity of pNP-LNB, even above the K m of pNP-LNB (Table IV). Finally, the enzyme was not specific for the identity or linkage of the penultimate sugar of the disaccharide, as Gal1,3GalNAc␣-benzyl was a good substrate (Table IV).
Characterization of the cFTase Reaction Product-The inability of the cFTase to fucosylate the 2-O-methylated derivative of lacto-N-biose suggested that the enzyme catalyzes the formation of a Fuc1,2Gal linkage (37). To test this possibility, the reaction product of pNP-LNB with preparation B of the enzyme, containing 3 H in its fucosyl moiety, was subjected to digestion with fucosidases. As previously observed (7), Fuc was readily released with nonspecific mammalian ␣-fucosidases capable of cleaving Fuc␣1,2Gal and Fuc␣1,3/4/6GlcNAc linkages (Table V). ␣1,2-Fucosidase from A. oxidans released Fuc with similar kinetics, whereas ␣1,3/4-fucosidase from almond emulsin did not show any activity. Although less almond emulsin fucosidase was present in these reactions, the calculated level of activity was still in vast excess of what would be required to hydrolyze an ␣1,4-linkage.
Protein Substrates-To determine whether the cytosolic protein FP21, isolated from mutant strain HL250 in its afucosyl form (GP21), is a substrate for the purified (preparation A) cFTase, GP21 isoforms-I and -II were purified to near homogeneity under nondenaturing conditions (Fig. 7). Since FP21 is not efficiently precipitated by trichloroacetic acid, acetone or ethanol, the reaction mixture was resolved by SDS-PAGE, and the GP21 band was stained and counted. GP21-I and -II were each fucosylated in a time (not shown)-and concentration-dependent fashion (Fig. 8). A Lineweaver-Burk analysis of GP21-II fucosylation (Fig. 8) estimated an apparent K m for GP21-II of 0.21 M, and an apparent V max of 460 nmol/min/mg protein. The apparent V max of the cFTase for GP21-II is about 75% of the apparent V max for pNP-LNB, whereas the apparent K m is over 3 orders of magnitude lower than the corresponding value for pNP-LNB. GP21-I fucosylation appeared to exhibit cooperativity with the Lineweaver-Burk plot tapering to the same y intercept value and slope as GP21-II at higher substrate concentrations.
Fucosylation of the FP21 polypeptide depended on its glycosylation status. Recombinant Dictyostelium FP21 (rP21), purified under nondenaturing conditions from E. coli with a short oligo-His tag at its N terminus (Fig. 7), exhibited no acceptor activity with preparation A (data not shown), consistent with previous evidence that the single Fuc on FP21 is located at the nonreducing terminus of an oligosaccharide (7,8), which is expected to be absent from rP21. FP21-I and FP21-II, isolated from normal Dictyostelium cells, were also not substrates, consistent with previous studies on crude extracts that soluble FP21 is fully fucosylated (7).
pNP-LNB and GP21 would reciprocally inhibit each other's fucosylation if they were substrates of the same enzyme. Both GP21-I and -II inhibited pNP-LNB fucosylation in a concentra- Table I Table I tion-dependent manner (Table VI), as expected based on analysis of crude S100 extracts (7). At a pNP-LNB concentration slightly below its K m , a concentration of GP21-I slightly above GP21-II's K m value inhibited pNP-LNB fucosylation approximately 40%, whereas a higher concentration of GP21-II was required for 40% inhibition. Conversely, much higher concentrations of pNP-LNB were required to inhibit GP21-I than GP21-II fucosylation. Thus both GP21 isoforms and pNP-LNB appeared to be fucosylated by the same enzyme, but GP21-I appeared to have a higher affinity than GP21-II for the cFTase. Recognition of GP21 by the cFTase appeared to depend primarily on carbohydrate rather than peptide determinants, based on the inhibitory potential of the different protein analogues. rP21 failed to inhibit the fucosylation of GP21-I, GP21-II, and pNP-LNB (Table VI), and in fact mildly stimulated the fucosylation of GP21-I and GP21-II. Since this stimulatory effect was not observed for pNP-LNB fucosylation, it may reflect substrate-substrate interactions, as 1) rP21 stimulated the fucosylation of GP21-I more than that of GP21-II, 2) GP21-I fucosylation was cooperative with respect to its own concentration (see above), and 3) GP21 and FP21 are known to aggregate in the concentration range examined (8). FP21-I was only a weak inhibitor (Table VI) compared to FP21-II (see below), showing an effect only at the highest inhibitor:substrate ratio tested against 0.028 M GP21-I (Table VI). The absence of inhibitory effects by both rP21 and FP21-I suggested that the enzyme does not recognize the polypeptide backbone of FP21.
In contrast, FP21-II was a good inhibitor of the fucosylation of all three substrates, pNP-LNB, GP21-I, and GP21-II, with Ͼ50% inhibition observed at 10 -100-fold concentration excess over the GP21-II and GP21-I substrate concentrations tested. The effect of FP21-II may represent product inhibition, as the reaction product, Fuc␣1,2Gal1,3GlcNAc-pNP, was found to be an inhibitor of pNP-LNB fucosylation, when tested over a similar range of inhibitor:substrate ratios (Table VI). The failure of FP21-I to exert inhibition, except at the highest concentrations, may be due to its extra mole of Gal (8) which, if applied following fucosylation, may mask recognition of the FP21-I oligosaccharide. If rP21 and the GP21 and FP21 isoforms differed only in their glycosylation, the simplest interpretation is that the specificity for fucosylation and inhibition of fucosylation resides in the sugar portion of the substrate.
Developmental Regulation-S100 extracts were prepared from proliferating, cell cycle synchronized, stationary, and developing cells (See "Experimental Procedures") and assayed in the presence of 1.0 M GDP-Fuc and 0.36 mM pNP-LNB. The specific activity of the cFTase was found to vary less than 50% in all samples tested, indicating that the enzyme is constitutively expressed.
FIG. 6. Co-chromatography of the pNP-LNB cFTase reaction product with Fuc␣1,2Gal1,3GlcNAc-pNP. pNP-LNB was fucosylated in the presence of GDP-[ 3 H]Fuc by preparation A of the cFTase and recovered on a C 18 Sep-Pak and by gel filtration. The 3 H-reaction product was combined with 10 g each of pNP-LNB and synthetic Fuc␣1,2Gal1,3GlcNAc-pNP, and the mixture was applied to a PA-1 column and eluted isocratically with 150 mM NaOH. Peak 1 is unknown material present in the reaction product, peak 2 is at the position of Fuc␣1,2Gal1,3GlcNAc-pNP, and peak 3 is at the elution position of pNP-LNB. No sugars or radioactivity were detected from 25-60 min. Ͼ95% of the radioactivity coeluted with Fuc␣1,2Gal1,3GlcNAc-pNP on this column, and also on a PA-100 column eluted with a gradient of NaAc in 0.1 N NaOH (not shown). (30 -33, 40). Separate ␣1,2-FTases appear to be encoded by the H and Secretor loci in humans (33,39), and DNAs encoding the H and Secretor enzymes have been cloned (40 -43). Like all other known Golgi glycosyltransferases (45,46), the Secretor and H enzymes are synthesized as type 2 membrane proteins consisting of a large C-terminal catalytic ectodomain, attached via a linker region to a transmembrane domain and a small N-terminal endodomain. The soluble, secretory forms of Golgi glycosyltransferases are apparently derived by proteolytic cleavage within the linker region (45). It is not clear whether the secretory form is physiologically meaningful. The K m values of Golgi and Golgi-derived FTases for GDP-Fuc are in the range of 10 -100 M, and apparent K m values for oligosaccharide acceptor substrates range from 0.1 to 20 mM (30 -33, 40). The order of magnitude of these K m values is consistent with the expected concentrations of these substrates in the Golgi apparatus.
The Dictyostelium cFTase, which is also capable of catalyzing the formation of a Fuc␣1,2Gal linkage, shows both similarities to and differences from the mammalian ␣1,2-FTases. The cFTase occurs in soluble form after gentle cell lysis that maintained the latency of Golgi enzymes, and its activity could not be detected in particulate extracts of the cell (7). Its polypeptide could be purified to apparent homogeneity using conventional and affinity chromatography and electrophoresis methods which have been successfully applied to secreted forms of mammalian enzymes. Although the cFTase appears to be fairly hydrophobic when chromatographed on phenyl-Sepharose columns and certain detergents stabilized its activity after purification, detergents were not used during purification. The apparent M r of the cFTase polypeptide (FT85), at 85 ϫ 10 3 , is approximately twice that of known Golgi enzyme cleavage products. The implication that the cFTase diverges from the Golgi type 2 membrane protein structural paradigm and resides in the cytosol must await confirmation from sequencing and cloning studies.
TABLE VI
Inhibition of cFTase Reactions were carried out using preparation A in the presence of 1.0 M GDP-[ 3 H]Fuc and the indicated concentration of acceptor substrate and inhibitors, for 1 h at 30°C. Fucosylation of pNP-LNB was assayed using a C 18 Sep-Pak cartridge, and fucosylation of GP21-I and GP21-II was assayed by SDS-PAGE and counting of gel bands. The Sep-Pak assay did not detect GP21 fucosylation, and the gel assay did not detect pNP-LNB fucosylation. rP21, FP21-I, and FP21-II were not fucosylated under the conditions of the assay. The K m of the purified cFTase for GDP-Fuc, at 0.34 M, is unusually low compared to Golgi FTases, but similar to the value of a cytosolic O-GlcNAc transferase for its donor substrate UDP-GlcNAc (34). The high affinity of the cFTase for GDP-Fuc is consistent with its proposed cytosolic localization, because in the cytosol the cFTase would be in competition with the Golgi GDP-Fuc transporter (2) for this substrate. Although the cFTase differs from mammalian Golgi ␣FTases in its high affinity for GDP-Fuc, its nucleotide binding is more similar to ␣1,2-FTases than ␣1,3-FTases in its preference for GDP-hexanolamine over GDP-adipate (31). The proportionately high sensitivity of the cFTase to inhibition by other guanine nucleotides (Table II) raises interesting regulatory questions about the effects of their intracellular pools.
The K m of the purified cFTase for the type 1 disaccharide conjugate pNP-LNB (Gal1,3GlcNAc-pNP) is similar to that of mammalian ␣1,2-FTases. The monosaccharide conjugate pNP-Gal, and the type 2 disaccharide conjugate Gal1,4Glc-NAc-pNP are only very poorly if at all fucosylated, and Gal1,6GlcNAc is at best a poor inhibitor. In contrast, the enzyme will fucosylate the type 3 disaccharide Gal1,3Gal-NAc␣-pNP, thus tolerating alternate sugars of opposite anomeric linkage penultimate to the terminal, fucosylated Gal. The acceptor specificity preferences of the cFTase are similar to that of highly purified porcine submaxillary ␣1,2-FTase (30,44) and the Secretor-type ␣1,2-FTase purified from human serum or plasma (31)(32)(33), and distinct from the H-type ␣1,2-FTase, which has a broader specificity range (31)(32)(33)40). Speculation that the Secretor-type gene preceded a gene duplication event leading to divergent evolution of the H-type gene (39) is reinforced by the present identification of a Secretor-type enzyme in the cellular slime molds. The specificity of the cFTase is compatible with what is known about the FP21 oligosaccharide, which contains Gal. However, the underlying sugar is not GlcNAc or GalNAc, as amino sugars are not detected in FP21 (8).
The cFTase exhibits varying V max and K m values for different aglycone moieties attached to the disaccharide. Of the three which have been tested, pNP has the highest activity, benzyl is intermediate, and the 8-methoxycarbonyloctyl moiety exhibits an order of magnitude lower activity compared to pNP when present at concentrations near or below their K m values. This suggests that acceptor substrate recognition involves more than the terminal disaccharide, which is consistent with the lower than expected inhibitory potential of free lacto-N-biose and the 3000-fold lower apparent K m of the native substrate GP21. Effects of alternate aglycone moieties have also been observed for mammalian FTases (31,33) and other Golgi glycosyltransferases (47), and polypeptide domains have been shown to be important determinants of recognition by certain glycosyltransferases (48). However, rP21 and FP21-I are not inhibitors of the cFTase, suggesting that the FP21 polypeptide is not an important determinant for cFTase recognition. The higher affinity of the enzyme for GP21-I compared to GP21-II, as determined by cross-inhibition studies (Table VI), in contrast to the higher inhibitory potential of FP21-II compared to FP21-I, may be most easily explained by glycosylation differences which have been described for the two FP21 isoforms (8). Why the different isoforms are differentially glycosylated remains to be determined. Since GP21 is the major acceptor of the reaction in crude extracts from the GDP-Fuc synthesis mutant (7), high affinity recognition of the FP21 carbohydrate implies that the carbohydrate structure is of limited distribution, and may be required because FP21 is not concentrated in a compartment. This is reminiscent of the relationship between UDP-Glc:glycoprotein Glc-1-phosphotransferase, another cyto-solic glycoprotein glycosyltransferase involved in peripheral modifications, and phosphoglucomutase, which appears to be its predominant acceptor substrate (5).
The V max of the purified cFTase with respect to pNP-LNB was 620 nmol/min/mg protein, at 1.0 M GDP-Fuc. The V max would be expected to extrapolate to 830 nmol/min/mg at saturating GDP-Fuc, or about 2.5 mol/min/mg with respect to the content of FT85 in the highly purified preparation A. This value is greater than the estimated specific activity of a highly purified Se-type FTase from serum (33), but less than that of a purified porcine Golgi ␣1,2-FTase (30). Projecting back to the cell, the specific activity suggests that there are on the order of 200 copies/cell of the cFTase in the cytosol. If the approximately 2 ϫ 10 5 copies/cell of the acceptor substrate FP21 2 were dissolved in the full volume of the cell, FP21 would be at a concentration of about an order of magnitude above its K m for fucosylation. If the cFTase were operating at its V max , there would be enough enzyme to be able to fucosylate all FP21 in about 1 h. | 2018-04-03T03:13:56.311Z | 1996-05-17T00:00:00.000 | {
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252163567 | pes2o/s2orc | v3-fos-license | The mechanism of colon tissue damage mediated by HIF-1α/NF-κB/STAT1 in high-altitude environment
The high-altitude environment damages the intestinal mucosal barrier, leading to a high incidence of intestinal diseases and seriously affects the working ability of people at high altitude. However, how high altitude induces intestinal mucosal barrier injury has not been well defined. The purpose of this study was to investigate the mechanism of colonic tissue injury induced by the influence of the high-altitude environment on the colonic microenvironment. Forty-eight SPF C57BL/6J mice were randomly divided into four groups: the control group and three other that were high-altitude exposure groups (Yushu, Qinghai; elevation: 4,010 m; 12 h, 24 h, 48 h). First, HE staining was used to observe the effect of the high-altitude environment on colon histomorphology of mice. The protein expression levels of claudin-1, occludin, and ZO-1 were analyzed by molecular biological methods. We found that altitude caused inflammatory damage to colon tissue. Intestinal hypoxia was measured with the hypoxic probe pimonidazole (PMDZ). Interestingly, we observed a decrease in the concentration of oxygen in the microenvironment in the colonic lumen. We sought to explore the mechanism of colonic mucosal barrier damage at different times when entering high altitude. The expression levels of hypoxia-inducible factors: HIF-1α, STAT1, and NF-κB and of inflammatory factors: IFN-γ, TNF-α, and IL-6 were significantly increased. This work highlights that the high-altitude environment leads to a reduction in the concentration of oxygen in the microenvironment of the colonic lumen, which disrupts the colonic mucosal barrier and ultimately induces and exacerbates intestinal injury.
Introduction
The intestinal mucosal barrier is an important part of the body's defense and is reinforced by tight junction proteins, which effectively prevent harmful substances from entering the body (Chelakkot et al., 2018). Hypoxic environments can lead to inflammation of the small intestine (Shah, 2016), and it is also the focus of inflammatory bowel disease. In the colitis mouse model, the intestinal mucosa is in a state of intense hypoxia, suggesting that hypoxia is involved in colitis (Shah et al., 2008). The main threat to human health at high altitudes is hypoxia. The high incidence of diseases of the digestive system seriously affects the physical functioning and working ability of populations living in plateaus (Frühauf, 2014) and also affects the pharmacokinetic parameters of drugs (Zhang and Wang, 2022) such that the drugs cannot achieve effective therapeutic effects. However, the mechanism of colon inflammatory injury caused by the high-altitude environment is still unclear.
We previously screened for cytokines that may be involved in inflammatory injuries in high-altitude environments through protein chip technology (Wang et al., 2016). The results showed that compared with the control group, there were significant changes in several inflammatory factors in the serum of the rats in the high-altitude groups. Hypoxiainducible factor 1α (HIF-1α), an O 2 -regulated protein, is a major transcription factor activated during hypoxia, and its stability is affected by the oxygen content. Studies have shown that hypoxic environment can lead to the activation of NF-κB in intestinal tissue (Nagpal and Yadav, 2017). NF-κB acts as a nuclear transcription factor and interferon regulator. After its activation, it enters the nucleus to promote the production of various inflammatory factors (Barnabei et al., 2021). NF-κB maintains the intestinal mucosal barrier. Signal transducer and activator of transcription 1 (STAT1) is an important member of a family of STAT proteins those act in a combinatorial fashion to help eliminate the expression of antibacterial and inflammatory response genes from foreign pathogens (Cohen Katsenelson et al., 2019). However, while inflammation is necessary to defend against foreign microbes, if not addressed in time, it can lead to cytokine overproduction, chronic inflammation, and even cancer (Fullerton and Gilroy, 2016). But the role of STAT1 in intestinal inflammation induced by high-altitude hypoxic environment has not been reported.
With the increase of high-altitude tourism and high-altitude workers, the incidence of intestinal-related diseases is high, and gastrointestinal reactions are one of the symptoms of acute altitude sickness (AMS). Gastrointestinal reactions caused by high-altitude environments mainly include nausea, vomiting, abdominal distension, diarrhea, etc. Among them, abdominal distention and diarrhea may be related to colon damage. It is urgent for us to further study the colonic inflammatory injury and related mechanisms caused by high-altitude environments. Since hypoxia plays an important role in regulating various physiological responses and pathological conditions, we hypothesized that decreased colonic tissue oxygen levels create hypoxic stress, which further exacerbates and accelerates inflammation and tissue damage. Here, we first need to clarify that the gut is a highly hypoxic environment, however, this "physiological hypoxia" has not been described in the highaltitude environment. In this study, relying on the established high-altitude field laboratory in Yushu, Qinghai (4,010 m), the morphological damage of the colon, colonic hypoxia, colonic barrier integrity, the expression of tight junction proteins, and the level of inflammatory signals in the colon were observed at different times of high-altitude hypoxia to evaluate inflammatory injury of colon tissue in the hypoxic environment. Molecular biology techniques were used to analyze the protein expression levels of HIF-1α, NF-κB, and STAT1 under high-altitude hypoxia environment. The mechanism of inflammatory injury of colon tissue provides a useful reference for the prevention and treatment of high-altitude intestinal diseases.
Animals
All experiments were performed on 48 SPF-grade 8-week-old male C57BL/6J mice (body weight 20 ± 2 g, license number: SCXK (Beijing) 2019-0010), which were purchased from Beijing Speifu Biotechnology Co., Ltd.). All protocols were approved by the 940th Hospital of Joint Logistics Support Force of the Chinese People's Liberation Army and were performed in accordance with relevant guidelines and regulations. The approval number is 2021KYLL175. The mice were placed under controlled temperature (20-25°C), humidity (50 ± 10%), and light (alternating 12-h light-dark cycle) conditions and fed following laboratory standards. Before the experiment started, they were adaptively reared in the SPF animal laboratory of the 940th Hospital of the Joint Logistics Support Force for 1 week. United States); Anti-NF-κB p65 (Abcam, United States); Anti-STAT1 (Proteintech).
Experimental method 2.3.1 Experimental animals and groups
We randomly divided healthy adult male SPF C57BL/6J mice into four groups, which were divided into the control group (C group, 1,500 m above sea level, Lanzhou), the high-altitude group for 12 h (H12 group, 4,010 m above sea level, Yushu, Qinghai), high-altitude group for 24 h (H24 group, 4010 m above sea level, Yushu, Qinghai), and high-altitude group for 48 h (H48 group, 4, 010 m above sea level, Yushu, Qinghai). The mice in the H24, H24, and H48 groups were transported by truck to the high altitude area and transported from Lanzhou to Qinghai within 12 h. After arriving at the destination, the experimental group were made to fast for 12 h before the experiment and the colon tissue collected at 12, 24, and 48 h. For the control group, the experiments were performed at Lanzhou.
Colon tissue hematoxylin and eosin staining and pimonidazole staining
Six mice from C group and H12, H24 and H48 groups were taken and fixed, three of which were used for PMDZ staining and the other three for HE staining. Pimonidazole (60 mg/kg) was administered intraperitoneally at the corresponding time. The mice were killed by dislocation half an hour later. A 2-cm mouse colon tissue was dissected and fixed, washed with precooled normal saline to remove blood stains, dried with filter paper, and immediately placed in 4% paraformaldehyde for fixation. Dehydration, embedding, slicing, and staining with HE were performed after adequate fixation. Pathological changes and immunohistochemical experiments were conducted to study the effect of the high-altitude environment on the intestinal barrier.
The fixed tissue requiring immunohistochemistry was dehydrated, trimmed, and then embedded in paraffin, sectioned, dewaxed to water, quenched using endogenous catalase in the tissue, washed twice with buffer, and placed in a target retrieval reagent which was incubated at 90°C for 20 min for antigen retrieval. After cooling to room temperature, it was washed twice with buffer again. A protein-blocking agent was added to block the background staining. Incubate in FITC conjugated to mouse IgG1 monoclonal antibody (FITC-MAb1) primary antibody for 60 minutes at room temperature, washed twice with buffer, then incubate set with Rabbit anti-FITC conjugated with horseradish peroxidase secondary antibody for 30 min at room temperature, washed twice with buffer, and stop color development with DAB, counterstained, dehydrated in alcohol, clear, and mounted. A scanner was used to scan the fluorescence images in a panoramic view, and the fluorescence images of the three mice were collected in each group of colon tissues.
Determination of inflammatory factors in colon tissue
Once the colon tissue was removed from the −80°C refrigerator, it was rinsed with pre-cooled PBS (0.01 M, pH = 7.4); the blood residue was removed, and 30-35 mg of colon tissue weighted. It was then placed in a corresponding volume of PBS (according to the ratio of 1:9). The colon tissue was ground in a prechilled homogenizer. Finally, the homogenate was centrifuged at 5,000 rpm for 10 min, and the supernatant was collected to detect the expression levels of IFN-γ, TNF-α, and IL-6 according to the instructions.
FITC-dextran for the detection of intestinal permeability
Four groups of mice were made to fast for 12 h before the experiment, and FITC-dextran was dissolved in sterile water for injection. During the experiment, the mice were given FITCdextran (500 mg/kg) by gavage, 4 h before the start of the experiment, and after 4 h of water deprivation, the blood samples were collected from the eye socket and the fluorescence value was detected by using a fluorescence microplate reader (excitation wavelength, 485 nm; emission wavelength, 520 nm), and quantitative analysis was performed according to the calibration curve of serum FITC-dextran concentration.
Western blotting
A total of 45-55 mg of frozen intestinal tissue was taken in 1.5 ml of the EP viewer, 450-550 μL of the pre-prepared tissue lysis solution (high-efficiency RIPA lysis solution:PMSF = 100:1, V:V) was taken; two steel balls were added to each tube; and the colon tissue was placed in a pre-chilled homogenizer and homogenized three times of 1 min for each time. After homogenization, the cells were lysed on ice for 30 min, vortexed every 10 min, then centrifuged in a cryogenic centrifuge at 12,000 rpm for 10 min, and the supernatant was aspirated to get the total protein. After measuring the protein concentration with BCA, according to the ratio of supernatant to 4× loading buffer = 3:1 (V:V), it was mixed well, boiled at 100°C for 10 min to denature the protein, and then stored in aliquots at −80°C refrigerator.
A total of 15-40 mg of the tissue was cut into small pieces and placed in a microcentrifuge tube. The tissue was washed with PBS, centrifuged at 500 × g for 5 min, and the supernatant aspirated. The tissue was then homogenized using a tissue grinder in the appropriate volume of CER I. The volume ratio of CER I:CER II:NER reagents was maintained at 200:11:100 µL. The tube was vortexed vigorously on the highest setting for 15 s to fully suspend the cell pellet. It was intubated on ice for 10 min. Ice-cold CER II was added to the tube. The tube was vortexed for 5 s on the highest setting. It was intubated on ice for 1 min. The tube was then vortexed for 5 s on the highest setting and centrifuged for 5 min at maximum speed in a microcentrifuge (~16,000 × g). The insoluble (pellet) fraction, which contains the nuclei, was suspended in ice-cold NER. The sample was placed Frontiers in Physiology frontiersin.org 03 on ice and vortexed for 15 s every 10 min, for a total of 40 min. The tube was centrifuged at maximum speed (~16,000 × g) in a microcentrifuge for 10 min. The supernatant fraction was immediately transferred to a clean prechilled tube to get the nucleoprotein. This was placed on ice, and the extracts were stored at −80°C until use.
Equal amounts of proteins was loaded by 7-12% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk, and then incubated overnight with the antibodies against HIF-1α, STAT1, NF-κB P65, claudin-1, occludin, and ZO-1. The next day, the membranes were incubated with secondary antibodies, and protein bands were visualized by using ChemiDoc MP and quantified using Image Lab software.
Statistical analysis
Data were processed using the SPSS 22.0 software. The data were expressed as mean ± standard deviation (SD). Multiple comparisons between the groups were conducted by one-way analysis of variance (ANOVA) followed by the LSD post hoc test. p < 0.05 was considered as significantly different.
Effects of high altitude environment on the morphology of colonic mucosa in mice
The pathological results of the colon tissue of mice with highaltitude exposure at different times are shown in Figure 1. It can be seen that the colon tissue of the mice in the C group is normal in shape; the cells are closely arranged; and there is no obvious pathological change. High-altitude environments can lead to increased spacing between crypts in colon tissue, distortion of crypts, thickening of crypt bases, obvious shedding of mucosal epithelium, and disordered arrangement of intestinal glands. The results have shown that the high-altitude environment can lead to changes in colon cell morphology, infiltration of inflammatory cells, and destruction of the lamina propria, leading to damage of the intestinal mucosal barrier.
Effects of high altitude environment on the expression of tight junction protein in mouse colon tissue
The experimental results of FITC-D4 and expression of tight junction proteins in mouse colon tissue is shown in Figure 2. The experimental results of FITC-D4 have shown that the high-altitude environment leads to an increase in intestinal permeability in the colon tissue of mice, indicating that the intestinal barrier is affected by the high-altitude environment. The expression of claudin-1 in mouse colon tissue was observed by hypoxia at different times, and the results have shown that the high-altitude environment leads to a decrease in claudin-1 expression in mouse colon tissue. Compared with the control group, the expression of claudin-1 protein in tissues was significantly downregulated by 9.1, 28.5, and 39.6%, respectively, after 12, 24, and 48 h of hypoxia at high altitude, while the tissue expression of occludin protein was significantly downregulated by 43.5, 69.9, and 39.3%, respectively. Tissue expression of ZO-1 protein showed a downward trend. The results of FITC-D4 and tight junction proteins have confirmed the results of HE staining from the level of the protein molecules that constitute the mechanical barrier, and the high-altitude exposure at different times destroyed the integrity of the colon tissue barrier in mice.
FIGURE 1
Changes in colonic barrier functions in mice from each group. (A) HE staining of colon tissue from mice in each group (×20). (B) HE staining of colon tissue from mice in each group (×40). (C) HE pathological scoring results. C, control group; H12 group, the high-altitude group for 12 h; H24 group, the high-altitude group for 24 h; H48, the high-altitude group for 48 h, n = 3. The data are presented as mean ± standard error of the mean. *p < 0.05 vs. N group, **p < 0.01 vs. C group.
Effects of high altitude environment on colonic tissue hypoxia in mice
The fluorescence signal intensity of the colon tissue hypoxia probe in each experimental group is shown in Figure 3. It can be seen from the data that when compared with the control group, the average fluorescence intensity of H12, H24, and H48 groups increased significantly by 43.19, 43.19, and 43.43%, respectively (p < 0.05). This shows that the hypoxic external environment such as high altitude can indeed lead to a decrease in oxygen concentration in the colon lumen of mice, which is likely to be the reason for the destruction of the integrity of the colonic mucosal barrier. H12 group, the high-altitude group for 12 h; H24 group, the high-altitude group for 24 h; H48, the high-altitude group for 48 h. The data are presented as the mean ± standard error of the mean. *p < 0.05 vs. N group, **p < 0.01 vs. C group.
FIGURE 3
Hypoxia of colon tissue in each experimental group. (A) Expression of hypoxia probe signal intensity in colon tissue of each experimental group (10×), n = 3. (B) Quantitative experimental map of colon tissue hypoxia probe signal intensities in each experimental group, n = 3. 1: C, control group; 2: H12 group, the high-altitude group for 12 h; 3: H24 group, high-altitude group for 24 h; H48, high-altitude group for 48 h. The data are presented as the mean ± standard error of the mean. *p < 0.05 vs. N group, **p < 0.01 vs. C group.
Effects of high altitude environment on inflammatory factors in mouse colon tissue
ELISA kits were used to measure the expression of IFN-γ, TNFα, and IL-6 in mouse colon tissue in order to observe inflammatory injury to mouse colon tissue caused by high-altitude environments. The results are shown in Figure 4. Entering the high-altitude environment results in varying degrees of increased inflammatory cytokines in mouse colon tissue. Compared with C group, H12 resulted in upregulation of IFN-γ, TNF-α, and IL-6 in mice colon tissue by 39.25, 41.36, and 53.78%, respectively. IFN-γ, TNF-α, and IL-6 were upregulated by 33.89, 7.68, and 13.14%, respectively, in the colon tissue of H24. IFN-γ, TNF-α, and IL-6 were upregulated by 21.55, 40.07, and 21.47%, respectively, in the colon tissue of H48. Compared with H12, inflammatory factors were downregulated at H24 and H48, but when compared with the C group, the inflammatory factors were still higher. Overall, the results of the elevated levels of the inflammatory factors indicated that the highaltitude environment caused some inflammatory damage to the colon tissue in mice.
Effects of high altitude environment on the expression of HIF-1α/STAT1/NF-κB protein in colon tissue of mice
The expression of various proteins in the colon tissue of mice on high-altitude exposure at different times was observed ( Figure 5). The results showed that the high-altitude hypoxia environment led to an increase in the expressions of NF-κB, HIF-1α, and STAT1 in the colon tissue of mice. Compared with the C group, NF-κB, HIF-1α, and STAT1 were upregulated by 29.72, 33.97, and 18.48%, respectively, in the H12 group. NF-κB, HIF-1α, and STAT1 were upregulated by 119.74, 98.66 and 76.72%, respectively, in the H24 group. NF-κB, HIF-1α, and STAT1 were upregulated by 156.39, 154.99 and 148.59%, respectively, in the H48 group. At the same time, the expression of NF-κB in nuclear protein was upregulated by 67.9, 148.6 and 320.8% at 12, 24, and 48 h, respectively. The above results show that HIF-1α, NF-κB, and STAT1 were all upregulated and time dependent. HIF-1α acts as an oxygen concentration sensor to reflect hypoxia in the colon. Meanwhile, NF-κB and STAT1 reflect the activation of inflammationrelated pathways in the colon. The results have indicated that colonic tissue damage may be mediated by the overexpression of HIF-1α/NF-κB/STAT1 under the high-altitude environment.
Discussion
AMS refers to high-altitude idiopathic disease at an altitude of more than 2,500 m. The most common clinical manifestations are headache, gastrointestinal symptoms, fatigue, dizziness, etc. AMS is caused by the hypoxic environment in the plateaus that seriously threatens the health and life safety of the people stationed in the plateaus, and significantly reduces the work efficiency of the working population. Among the symptoms of AMS, gastrointestinal (GI) reactions are the main symptoms (Zhang et al., 2018). Although it has been proved that the intestinal mucosal barrier is damaged at high altitudes, most of the research objects are small intestinal tissue. However, the mechanism of colonic tissue inflammatory injury mediated by low oxygen concentration in high-altitude environments is still unclear. The purpose of this study was to investigate the mechanism of inflammatory damage to colonic tissue caused by the oxygen concentration in the colonic lumen unraveling in the high-altitude environment. Our findings may provide effective measures for the search of prevention and treatment of intestinal diseases caused by high-altitude exposure.
In this study, we analyzed the pathological changes of the colon tissue in C57BL/6J mice after exposure to high-altitude hypoxia for 12, 24, and 48 h and found that the colon tissue in the high-altitude hypoxia group had obvious morphological changes, accompanied by inflammatory cell infiltration, indicating that high-altitude environments can lead to inflammatory damage in
FIGURE 4
Expression of inflammatory factors in colon tissues of each experimental group. 1: C, control group; 2: H12 group, the high-altitude group for 12 h; 3: H24 group, high-altitude group for 24 h; H48, high-altitude group for 48 h, n = 6. The data are presented as the mean ± standard error of the mean. *p < 0.05 vs. N group, **p < 0.01 vs. C group.
Frontiers in Physiology frontiersin.org 06 the colon tissue. The high-altitude environment induces increased colonic barrier permeability in mice at the overall animal level. Tight junctions between the intestinal epithelial cells are one of the important components of the intestinal mechanical barrier. Whereas, how high-altitude environments affect the tight junction proteins between colonic epithelial cells is unclear. Tight junction proteins are mainly composed of occludin-related proteins, claudin, and other proteins. The highaltitude environment reduces the protein expression levels of claudin-1, occludin, and ZO-1 in the colon tissue, suggesting that the colonic barrier function is impaired and colonic intestinal permeability has been increased. The gastrointestinal tract has unique oxygenation characteristics, and even in the normal physiological state, the intestinal mucosa epithelial cells are in a state of hypoxia, which is called "physiological hypoxia." It is well known that the most severe characteristic of high-altitude environments is the reduced oxygen concentration. We used the hypoxic probe pimonidazole (PMDZ) to detect colonic hypoxia and quantified PMDZ retention, and the mean fluorescence intensity reflected the hypoxia probe signal intensity. We observed a significant increase in the fluorescence intensity of the colon tissue signal in the high-altitude groups, indicating that the high-altitude environment leads to aggravated "physiological hypoxia" in colon tissue.
HIF-1α, as an oxygen sensor, plays a key regulatory role in hypoxia-related diseases. In a hypoxic environment, HIF-1α is activated and regulates cell survival, metabolism, and tumorigenesis. Studies have shown that the activation of HIF-1α contributes to the progression of inflammatory bowel disease (Kim et al., 2018), while the inhibition of HIF-1α activity helps to alleviate intestinal inflammation (Kerber et al., 2020). The present findings have shown that the expression level of HIF-1α in mouse colon significantly increased in a time-dependent manner. Our findings suggest that high-altitude exposure induces an anoxic microenvironment in the colonic lumen and promotes inflammatory responses in the colon of mice, and the maximal activation of HIF requires the activation of the other pathways. NF-κB is an important nuclear transcriptional regulator that plays an important role in regulating inflammation, immune response, cell proliferation, transformation, apoptosis, tumorigenesis, and other cellular processes (Kunnumakkara et al., 2020). In addition, NF-κB transcriptional dysregulation leads to chronic inflammation and cell death (Barnabei et al., 2021). Studies have shown that by downregulating the expression of TLR4 and NF-κB, the body's inflammatory response can be reduced (Ye et al., 2022), and it has a therapeutic effect on inflammatory bowel disease in the mouse model by inhibiting the over-activation of TLR4/NF-κB (Liu et al., 2017). Studies have shown that there is an NF-κB binding site in the HIF-1α gene promoter. This leads to an increase in the expression of HIF-1α mRNA (Bonello et al., 2007;van Uden et al., 2008). All the above studies have indicated that the activation of the NF-κB signaling is a key factor in causing intestinal inflammation. The current study shows that the expression of NF-κB in the colon of mice and the expression levels of inflammatory factors IFN-γ, TNFα, and IL-6 in the colon tissue were significantly increased. IFN-γ, IL-6, and TNF-α are associated with increased IEC death and the disruption of gut epithelial barrier function in IBD (Neurath, 2014). Expression of NF-κB P65 in nuclear proteins. 1: C, control group; 2, H12 group, the high-altitude group for 12 h; 3, H24 group, the high-altitude group for 24 h; H48, high altitude group for 48 h, n = 6. The data are presented as the mean ± standard error of the mean. *p < 0.05 vs. N group, **p < 0.01 vs. C group.
Frontiers in Physiology frontiersin.org 07 At the same time, studies have shown that TNF-α and IFN-γ act synergistically to kill IEC cells through the STAT1 module (Woznicki et al., 2021). IL-6R signaling is known to induce phosphorylation of both STAT1 and STAT3 in T cells (Barnstorf et al., 2019). STAT proteins are well known for their roles in transducing cytokine-mediated signals and specifying Th cell differentiation. Among them, STAT1 is responsible for the transduction of type 1 IFN signals (α and β) and type 2 signals (γ) through a JAK1/2-dependent mechanism. In particular, it is a major signal transmitter of IFN-γ, an important mediator of immune responses and inflammation . Accumulating evidence have indicated that STAT1 is also involved in the regulation of HIF-1α, and studies have shown that STAT1 can activate HIF-1α (Chen et al., 2019). At the same time, the more classic pathway between NF-κB and STAT1 is NF-κB/JAK/STAT1, and studies have shown that multiple inflammatory factors can play a role through the NF-κB-JAK/ STAT1 pathway, such as IL-6R, IL-3R, and IFN receptor (IFN-R) (Coskun et al., 2013). The current findings suggest that the expressions of STAT1, NF-κB, and HIF-1α proteins are hyperactivated with increasing high low-oxygen exposure time, which ultimately leads to inflammatory damage in the colon in mice. The relationship between HIF-1α/NF-κB/STAT1 has been reported in the literature, but the mechanism of colonic barrier damage caused by the high-altitude environment has not been reported, so we studied it. The abovementioned results further provide evidence for the conclusion that high low oxygen exposure induces hyperactivation of HIF-1α/NF-κB/STAT1 signaling in mouse colon tissue, resulting in colonic inflammatory damage. It has been suggested that high low-oxygen exposure causes intestinal inflammation, and the longer the exposure time, the more severe the damage to the colonic barrier in mice. The mechanism is shown in Figure 6.
In conclusion, high altitude significantly induces the reduction of oxygen concentration in the colonic microenvironment. This change in the microenvironment results in time-dependent damage to the colonic barrier in mice. At the same time, high-altitude exposure significantly induces the activation of inflammatory signaling in mice colon. However, this is an animal model study, and we will collect more evidence in the clinic in the future. In a word, our findings suggest that the HIF-1α/NF-κB/STAT1 pathway plays an important role in inflammatory colon injury. Frontiers in Physiology frontiersin.org 08 | 2022-09-10T13:06:00.064Z | 2022-09-09T00:00:00.000 | {
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1886452 | pes2o/s2orc | v3-fos-license | Isolation and Screening of Polyhydroxyalkanoates Producing Bacteria from Pulp, Paper, and Cardboard Industry Wastes
Background. Polyhydroxyalkanoates (PHAs) are storage materials that accumulate by various bacteria as energy and carbon reserve materials. They are biodegradable, environmentally friendly, and also biocompatible bioplastics. Unlike petrochemical-based plastics that take several decades to fully degrade, PHAs can be completely degraded within a year by variety of microorganisms into CO2 and water. In the present study, we aim to utilize pulp, paper, and cardboard industry sludge and waste water for the isolation and screening of polyhydroxyalkanoates (PHAs) accumulating bacteria and production of cost-effective PHB using cardboard industry waste water. Results. A total of 42 isolates showed black-blue coloration when stained with Sudan black B, a preliminary screening agent for lipophilic compounds, and a total of 15 isolates showed positive result with Nile blue A staining, a more specific dye for PHA granules. The isolates NAP11 and NAC1 showed maximum PHA production 79.27% and 77.63% with polymer concentration of 5.236 g/L and 4.042 g/L with cardboard industry waste water. Both of the selected isolates, NAP11 and NAC1, were classified up to genus level by studying their morphological and biochemical characteristics and were found to be Enterococcus sp., Brevundimonas sp. and, respectively. Conclusion. The isolates Enterococcus sp. NAP11 and Brevundimonas sp. NAC1 can be considered as good candidates for industrial production of PHB from cardboard industry waste water. We are reporting for the first time the use of cardboard industry waste water as a cultivation medium for the PHB production.
Introduction
Plastic materials that have been universally used in our daily lives are now causing serious environmental problems. Millions of tons of these nondegradable plastics accumulate in the environment per year. For efficient management of used-plastic materials, recycling is one solution. Another solution to reduce plastic residue is the use of biodegradable plastics [1,2] and among them polyhydroxyalkanoic acids (PHAs) are drawing much attention. Polyhydroxyalkanoic acids (PHAs) are common intracellular compounds found in bacteria, archaea, and in few eukaryotes such as yeasts and fungi. PHAs are carbon and energy reserve polymers produced in some microorganisms when carbon source is in plentiful and other nutrients such as nitrogen, phosphorus, oxygen or sulfur are limited. PHAs are found to accumulate in varieties of microorganisms as reserve food material for example, Alcaligenes latus, Ralstonia eutropha, Azotobacter beijerinckii, Bacillus megaterium, and Pseudomonas oleovorans, including some fungi and archaea. Among the members of PHA family, polyhydroxybutyrate (PHB) is the most common biodegradable polymer and promising alternative to synthetic nondegradable plastics. These polymers are accumulated intracellular membrane enclosed inclusion up to 90% of the cell dry weight under conditions of nutrient stress and act as energy reserve material. It has similar mechanical properties as those of the oil-derived conventional plastics like polypropylene or polyethylene which can be molded, made into films, spun into monofilaments, and used to make heteropolymers with other synthetic polymers and many more applications in agriculture, packaging, and medical field being biodegradable and also immunologically compatible with human tissue [3]. Recently, another application of PHAs is reported as biofuel [4].
In spite of these interesting properties, industrial production of PHAs is still not well established. In the 1950s, North-American Company W.R. Grace Co. made the first attempt to produce PHB at commercial level. However, this process was not successful due to low production efficiency and a lack of suitable purification method. Then, in the 1970s, Imperial Chemical Industries (ICI, UK) started producing PHAs by using a mutant stain Cupriavidus necator, NCIB 11599 from various carbon sources such as 1,4butanediol, 1,6-hexanediol, and butyrolactone. The commercial product was recognized as Biopol. The patents were later sold to Zeneca, then to Monsanto and are currently the property of Metabolix, Inc. (USA). Commercially, some other biopolyester products with different monomer composition are also produced with trade names such as poly(3HB-co-3HV) Nodax, poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) poly(3HB-co-3HA) as Biogreen, and poly(3HB) as Biomer [5]. But the large scale production was halted at commercial level due to the high production cost as compared with the oil-derived plastics [1,6]. With the aim of commercializing PHA, great efforts have been employed to reduce the production cost by the development of bacterial strains and more efficient fermentation/recovery process [7,8]. From the literature, it has been found that the major cost in this biopolymer production is the cost of the substrate [9,10] which accounts for more than 50% of production cost [5,11] and makes the difference in price of poly-3hydroxybutyrate (P3HB) from Biomer about 12 times the cost of polypropylene [12]. To solve this problem, inexpensive substrate, renewable substrates, waste material, and waste water are used as nutrient source for microorganisms for PHA production. Various types of waste products have been used for PHB production because it provides dual benefits of utilizing the waste and cost-effective production of biodegradable microbial bioplastic.
Cardboard industry is one of the parts of pulp, paper, and packaging industry. Cardboard industry includes two main industries corrugated cardboard industry and noncorrugated cardboard or paper-board industry. Waste water from chemical and mechanical pulping contains 12-25 kg of BOD/t of ADP, but the BOD discharges are 3 to 10 times higher in chemimechanical pulping as compared to mechanical pulping. Nitrogen and phosphorus are also present in waste waters and released from the pulping process of raw material such as wood, agricultural waste, and paper waste. Waste waters released from pulp and paperboard mills are typically rich in carbohydrates but poor in fixed nitrogen. If we consider the scenario of India, pulp and paperboard industry around 905.8 million m 3 of water is consumed and around 695.7 million m 3 of waste water is discharged annually. The largest part of the fresh water is used in sheet formation on the cardboard machine (200 m 3 h −1 ) and the smallest quantity is used in stock preparation (90 m 3 h −1 for thickening). Furthermore, the treatment of waste stream to purified effluent needs much effort and is very difficult, because the waste stream often contains various organic compounds. So instead of costly treatment, we can exploit the waste water directly for cultivation of PHB accumulating microorganisms. In this study, polyhydroxyalkanoic acids (PHAs) accumulating bacterial strains were isolated and screened using cardboard industry waste water as a sole carbon source with dual benefit of utilizing the waste and cost-effective production of biodegradable microbial bioplastic.
Isolation of Polyhydroxyalkanoic Acids (PHAs) Producing
Bacteria. For the isolation of PHA producing bacteriam activated sludge and waste water were collected from pulp, kraft, and cardboard manufacturing industry from Khanna pulp and paper mills at Amritsar and Topara Kurdh, Yamuna Nagar, India, respectively. The samples were stored at room temperature until analysis. In 99 mL sterilized water, 1 gm of sludge sample was dissolved. Then, the sample was serially diluted in sterile distilled water and followed by plating on the nutrient agar medium with 1% glucose. For isolation from waste water sample, 1 mL of water sample was added in 99 mL sterilized water. After serial dilution (10 −3 to 10 −7 ), 1 mL of each dilution was spread on carbon rich nutrient agar plate. For the rapid detection and isolation of PHB producing bacteria, 0.02% alcoholic solution of Sudan black B was applied to stain bacterial colonies and the plates were kept undisturbed for 30 min. The excess dye was then decanted and plates were rinsed gently by adding 100% ethanol. Colonies unable to incorporate the Sudan black B appeared white, while PHB producers appeared bluish black [13].
2.2.
Screening for PHA Producing Bacteria. Sudan black B positive isolates were checked for PHA production by Nile blue A staining, a more specific stain for Polyhydroxyalkanoic acids (PHAs) by a more rapid and sensitive, viable colony method [14]. This dye at concentrations of only 0.5 g/mL was directly included in carbon rich nutrient agar medium (glucose 1%, beef extract 0.3%, peptone 0.5%, sodium chloride 0.8%, and agar 1.5%) and growth of the cells occurred in the presence of the dye. This allowed an estimation of the presence of PHAs in viable colonies at any time during the growth experiment and a powerful discrimination between PHA-negative and PHA-positive strains. The PHA accumulating colonies, after Nile blue A staining, showed bright orange fluorescence on irradiation with UV light and their fluorescence intensity increased with the increase in PHA content of the bacterial cells. The isolates which showed bright orange fluorescence on irradiation with UV light after Nile blue A staining were selected as PHA accumulators.
Pretreatment of Cardboard Industry Waste
Water. Untreated cardboard industry effluent was collected from the cardboard industry, Topara Kurdh, Yamuna Nagar, Haryana, India, and stored at 4 ∘ C until used for analysis. The effluent was first filtered through the muslin cloth and then by rough filter paper to remove the undesired suspended solid materials from waste water. After this pretreatment step, cardboard industry waste water was used as quantification and production medium for PHA production by selected bacteria.
Extraction and Quantitative
Analysis of PHA. The PHB production was observed in 250 mL Erlenmeyer flask containing 50 mL of treated cardboard industry waste water, as production medium under stationary conditions of growth. After 72 h of incubation at 37 ∘ C, culture broth was centrifuged at 8000 rpm for 15 min. The pellet along with 10 mL sodium hypochlorite was incubated at 50 ∘ C for 1 h for lyses of cells. The cell extract obtained was centrifuged at 12000 rpm for 30 min and then washed sequentially with distilled water, acetone, and absolute ethanol. After washing, the pellet was dissolved in 10 mL chloroform (AR grade) and incubated overnight at 50 ∘ C and was evaporated at room temperature [15]. After evaporation, 10 mL of sulphuric acid was added to it and placed in water bath for 10 min at 100 ∘ C. This converts polyhydroxyalkanoic acids (PHAs) into crotonic acid, which gives maximum absorbance at 235 nm [16,17]. PHB (Sigma Aldrich) was used as standard for making standard curve. For quantitative analysis of PHA, cell culture was grown as described earlier and cell pellet was dried to estimate the dry cell weight (DCW) in units of g/L [18]. Residual biomass was estimated as the difference between dry cell weight and dry weight of extracted PHA [19]. This was calculated to determine the cellular weight and accumulation other than PHAs. The percentage of intracellular PHA accumulation is estimated as the percentage composition of PHA present in the dry cell weight: (1)
Morphological Characterization and Biochemical
Identification of PHA Producing Bacteria. Microscope Stereo Olympus was used to observe the morphology of bacterial colonies grown on nutrient agar. The growth characteristics such as structure, shape, color, margin, surface characteristics, surface upwards, smell, elevation, opacity, end of cells, cell's arrangement, and Gram-staining of the bacterial colonies were observed to characterize the bacterial colonies. Various biochemical tests were performed in selected PHB producing bacteria, namely, indole production test, methyl red and Voges-Proskauer, citrate utilization test, and H 2 S production for their biochemical characterization. The fermentative utilization of various carbohydrates was also followed for 48 hrs at 37 ∘ C by inoculating the isolates separately in the defined medium to which various sugars like xylose, mannose, maltose, sucrose, raffinose, dextrose, trehalose, fructose, glucose, ribose, lactose, rhamnose, esculin, inulin, mannitol, arabinose, sorbitol, and melibiose were added.
1 H-NMR Spectroscopy and Thermal Gravimetric Analysis (TGA).
The identity of individual monomer unit was confirmed by proton nuclear magnetic resonance ( 1 H-NMR) spectroscopy. 1 H-NMR spectra were acquired by dissolving the polymer in deuterochloroform (CDCl 3 ) at a concentration of 10 mg/mL and analyzed on a Bruker Avance II 500 spectrometer at 22 ∘ C with 7.4 ms pulse width (30 ∘ pulse angle), 1 s pulse repetition, 10,330 Hz spectral width, and 65,536 data points. Tetramethylsilane was used as an internal shift standard. Thermal gravimetric analysis (TGA) was performed using a TGA instrument (Mettler-Toledo, TGA/SDTA 851, USA) calibrated with indium. The temperature was ramped at a heating rate of 10 ∘ C/min under nitrogen to a temperature (700 ∘ C) well above the degradation temperature of the polymers.
FT-IR Analysis.
FT-IR analysis of the polymer sample was carried out on MB-3000, ABB FTIR spectrophotometer in the range 4000-600 cm −1 .
GC-MS Analysis.
Purified polymer, prepared as described before, was dissolved in 2 mL of chloroform and then 2 mL of methanol was added and acidified with 3% (v/v) H 2 SO 4 and heated at 100 ∘ C for 3.5 h for depolymerization and methanolysis of polyesters and 3 L was injected into GCMS-QP 2010 Plus model. The samples were injected in the splitless mode and the injection temperature was 260 ∘ C and column oven temperature was 100 ∘ C.
Isolation and Selection of PHA Producing Bacteria.
A wide variety of bacteria are known to accumulate PHA. Today, approximately 150 different hydroxyalkanoic acids are known to be incorporated into polyhydroxyalkanoates [20], with microbial species from over 90 genera being reported to accumulate these polyesters [21]. These bacteria have been reported from various environments, but only a few from the waste water and sludge ecosystems. For the rapid detection and isolation of PHB producing bacteria, 0.02% alcoholic solution of Sudan black B and Nile blue A staining viable colony method [14] was used. The isolation of PHA producing bacteria was done from cardboard manufacturing industry waste water and pulp cardboard and kraft industry sludge. A large proportion about 35% of isolated bacteria produced PHA as energy reserve material. A total of 42 isolates showed black-blue coloration when stained with Sudan black B, a preliminary screening agent for lipophilic compounds, and a total of 15 isolates showed positive result with Nile blue A staining (Figure 1), a specific dye for the of PHA granules. Both gram-positive and gram-negative bacteria showed PHA production, but gram-positive bacteria dominated the waste material microflora of pulp, kraft, and cardboard manufacturing industry. Teeka et al. [22] used this method to screen the potential PHA producing bacteria from soil, and Ramachandran and Abdullah [23] also observed the colonies formed on nutrient rich medium under ultraviolet light (UV) to screen for the pink fluorescence which indicated the presence of PHA producers. Kitamura and Doi [24] first demonstrated this viable colony method on agar plates; they induced the isolates to accumulate PHA by culturing in E 2 medium containing 2% (w/v) glucose before Nile blue A staining. The PHA accumulating colonies, after Nile blue A staining, showed bright orange fluorescence on irradiation with UV-light and their fluorescence intensity increased with increase in PHA content of the bacterial cells.
Production, Extraction, and Estimation of PHA with Cardboard Industry Waste Water as a Sole Carbon Source.
The PHA-positive isolates selected after Nile blue A staining were grown in 50 mL cardboard industry waste water in Erlenmeyer flasks and were employed to extract PHA after 2 days of incubation under stationary conditions of growth. The PHA from the isolates was extracted by the hypochlorite and chloroform method [15] as described earlier. The isolates NAP11 and NAC1 showed maximum PHA production 79.27% and 77.63% (Table 1) with cardboard industry waste water and were selected for further biochemical identification and chemical characterization.
Organic matter from wastes and waste waters has high BOD and COD values, and hence microorganisms can grow, utilizing the nutrient present in waste water, and can convert them into valuable compounds and polymers. Based on this idea, many researchers reported the PHA production from various industrial waste materials. PHA production by A. vinelandii from swine waste liquor was studied by [25]. The raw liquor supported the production of only 0.43 g/L PHA, at a polymer content of 37% w/w, whereas twofold dilution and supplementation with 30 g glucose/L allowed a PHA concentration of 5.5 g/L at a 58.3% w/w polymer content. Few researchers have proposed coupling PHA production to biological waste water treatment [26][27][28]. Ceyhan and Ozdemir [29] reported polyhydroxybutyrate (PHB) production from domestic waste water using Enterobacter aerogenes 12Bi strain with good yield ranging from 16.66 to 96.25% (w/w). The use of pure C. necator cultures to produce PHAs from waste waters has been explored by Ganzeveld et al. [30]. They used a supernatant, obtained by centrifuging fermented organic waste, as the sole carbon source for the production of P(3HBco-3HV), and obtained a maximum polymer concentration of 1.13 g/L at a polymer content of 40.8% in 45 h. Cardboard industry waste water is typically rich in carbohydrates but poor in fixed nitrogen, due to the high C/N ratio. This high carbon-nitrogen ratio favors the growth of PHA producing bacteria. It is the first time that cardboard industry waste water is used for the isolation, screening, and production of polyhydroxyalkanoates. This waste has high BOD and COD values 680-1250 mg/L and 3400-5780 mg/L and COD/BOD ratio between 3.9 and 5 [31], which is suitable for microbial growth.
Extracted PHA of selected isolates was quantified and its efficiencies were compared with the standard. Standard pure culture of Ralstonia eutrophus MTCC no. 1473 was used for PHA production with cardboard waste water producing a polymer concentration of 2.974 g/L and PHB content up to 41.30% with cardboard industry waste water. The selected isolates NAP11 from pulp sludge have produced 79.27% w/w PHA with polymer concentration of 5.236 g/L using cardboard waste water which are 37% higher as compared to standard stain of Ralstonia eutrophus (Figure 2). The other NAC1 isolates showed PHA production up to 77.63% with polymer concentration of 4.042 g/L under stationary conditions of growth.
Morphological and Biochemical Characterization of Selected Isolates. By using Bergey's Manual of Determinative
Bacteriology [32] and by ABIS Online-Advanced Bacterial Identification Software, bothisolates were classified up to genus level using the morphological and biochemical characteristics (Table 2). NAP11 and NAC1 were found to be Enterococcus sp., and Brevundimonas sp., respectively. Other researchers also reported these genuses from the waste effluents. Silva et al. [33] studied the ecology of Enterococci and related bacteria in raw and treated waste water from a treatment plant receiving domestic and pretreated industrial effluents. The predominant species found in the raw waste water were Enterococcus hirae, Enterococcus faecium, and Enterococcus faecalis. Jiang et al. [34] isolated 3,851 in total Enterococci isolates from eight individual source categories including feces from animals and birds, soil, and sewage water samples to establish antibiotic resistance analysis (ARA). Reddy and Mohan [35] also reported the Enterococcus italicus sp. in mixed consortia in waste water treatment and produced PHA up to 71.4%. During their study of influence of substrate load and nutrient concentration (nitrogen and phosphorous) on PHA production using waste water as substrate and mixed culture as biocatalyst, they found that PHA accumulation was high at higher substrate load (40.3% of dry cell weight (DCW)), low nitrogen (45.1% DCW), and low phosphorous (54.2% DCW) conditions by mixed consortia containg in Enterococcus sp. this paper confirms that Rani et al. [36] reported Brevundimonas with other bacteria as the dominant cultured bacteria in microbial diversity in functional pesticide effluent treatment plants (ETPs). Brevundimonas aveniformis sp.nov. A stalked species, was isolated from activated sludge by Ryu et al. [37]. Brevundimonas sp. MIFC and Brevundimonas diminuta was isolated from refinery active sludge and olive mill waste water, respectively, [38] and a Brevundimonas sp. were isolated from tannery waste treatment plant [39]. PHA production also reported up to 64% from the acid hydrolyzed saw dust (hydrolyzed wood) by Brevundimonas vesicularis. They also optimized the C : N ratio for PHA production in Brevundimonas vesicularis sp. and found that C : N proportion of 100 : 3.5 yielded maximum PHA up to 64% of cell dry weight. Thus, they concluded that acid hydrolyzed saw dust can be used as substrate by Brevundimonas vesicularis sp. for PHA production [40].
Polymer Analysis by 1 H-NMR Spectroscopy.
Based on the characterization of the PHA produced by NAP11 and NAC1 through NMR comparison with the standard PHB (Sigma), it was observed that the PHA obtained from NAP11 and NAC1 is having properties similar to that of the standard PHB (Sigma) (Figure 3(a)), so the PHA produced by both bacteria is polyhydroxybutyrate (PHB (Figure 3(b)) of the PHAs extracted from Brevundimonas sp. NAC1 (Figure 3(c)) show the following resonance signals: HC=CH bond at 5.30 ppm, CH 2 O-COOH bond at 2.574 ppm, a high signal at 1.30 ppm that belongs to the hydrogen of methylene in the saturated lateral chain, and a terminal -CH3 group at 0.857 ppm [15]. The 1 H NMR spectra of the samples and the standard are almost identical, conferring that extracted intracellular compounds are polyhydroxybutyrates (PHBs).
Fourier Transform Infrared Spectroscopy (FTIR).
Polymer extracted from NAP11 and NAC1 was used for recording IR spectra in the range 4000-600 cm −1 . IR spectra (Figure 4) showed two intense absorption bands at 1720 and 1281 cm −1 of NAP11 and at 1720 and 1273 of NAC1 specific for C=O and C-O stretching vibrations, respectively. The absorption bands at 2932 and 2954 cm −1 are due to C-H stretching vibrations of methyl, methylene groups. These prominent absorption bands confirm the structure of poly--hydroxybutyrate.
3.6. Thermogravimetric Analysis (TGA). TGA results of NAM5 showed that the is 171.33 ∘ C and the enthalpy of PHA fusion is 85.56 J/g. The result showed similarity with the data obtained from standard PHB (176.29 ∘ C and 86.49 J/g) [41] and from other studies from the literature also [42,43].
GC-MS Analysis of Extracted PHA.
In this study, the PHB was methanolysed in the presence of sulphuric acid and methanol, and the methanolysed 3HB was then analyzed International Journal of Biomaterials 7
Conclusion
In this study, inexpensive cardboard industry waste water was tried as a carbon source to produce PHA. Different bacterial strains were isolated from cardboard industry waste water and pulp, kraft, and cardboard manufacturing industry sludge and screened for polyhydroxyalkanoate production using cardboard manufacturing industry waste water as a carbon source. The bacterial isolates NAP11 and NAC1 can be regarded as potential strains for the conversion of cardboard industry waste water into PHB. Both of the selected isolates utilized cardboard industry waste water as sole carbon source for growth and PHB biosynthesis, accumulating PHB up to 79.27% and 77.63% of the cell dry mass, respectively. As a conclusion, isolates NAP11 and NAC1 can be considered as good candidates for industrial production of PHB from cardboard industry waste water. Based on the morphological and biochemical characterization, NAP11 and NAC1 were identified up to genus level as Enterococcus sp. and Brevundimonas sp., respectively. Currently, these bacterial strains are further investigated to increase the productivity of PHB by the optimization of the process parameters and making the whole process more cost-effective.
Conflict of Interests
The authors declare that they have no competing interests. | 2016-05-12T22:15:10.714Z | 2013-10-29T00:00:00.000 | {
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13027843 | pes2o/s2orc | v3-fos-license | Whole grain for whom and why?
A definition of whole grain is a critical first step in investigating health claims for whole grain and its products. Today, there is no internationally accepted definition of whole grain. Some existing definitions are broad and commodity-based, including grains with similar end uses, while others are more restricted. Scientific knowledge must be the basis for inclusion of certain grains. It is better to start with a restricted list of grains (a precautionary principle) and extend this as more knowledge becomes available. An exact definition of the raw materials (milled, cracked, crushed, rolled, or flaked) and knowledge of the components providing health effects would appear to be crucial issues for the European authorities when approving health claims. It is important that health claims are evidence-based, sustainable, and officially validated.
F or a long time there has been a strong international trend to market whole grain foods as healthy. In many cases it has been difficult for consumers to obtain reliable information about food items and their health effects, and thus select the most desirable products from a nutritional point of view. Consumers, at least in Scandinavia, are well aware that whole grain is healthy, but are less well informed about what whole grain is and its relationships to health. Several population studies have shown correlations between increased intake of whole grain and decreased risk of developing diet-related diseases, such as cardiovascular disease (CVD), diabetes, certain cancers, and obesity (1,2). However, these studies have mainly been conducted in Western populations and have included different definitions of whole grain. In Western populations, whole grain intake typically comes from wheat, oats, rye, barley, and sometimes popcorn, and therefore the estimated risk reductions are most likely associated with intake of these cereals. Results from intervention studies have been more variable. Some studies have shown no effects of whole grain consumption on intermediate endpoints for CVD and diabetes, while others have shown effects (3). A number of these intervention studies have evaluated the effects of whole grain from oats, wheat, rye, and barley. In order to provide consumers with appropriate dietary advice and to give the industry the opportunity to develop and market innovative and healthy foods, a definition of whole grain based on compelling evidence from a totality of population and intervention studies is needed.
Cereals: a good source for nutrients Whole grain cereals are well known as a major source of dietary carbohydrate and protein, as well as containing high amounts of a variety of dietary fibers and co-passengers, i.e. minerals, vitamins, and other bioactive components. These latter components are mainly present in the outer parts of the grain or in the germ, and are removed with the bran during extraction of sifted flour. As a result, health authorities around the world are recommending increased intake of whole grain cereals. However, evidence-based recommendations on the exact amounts and types of whole grains to be consumed are rare.
Consumers are well aware that whole grain is healthy and that consumption of whole grain foods should be increased. However, there is a mismatch between whole grain recommendations and the actual behavior of consumers. Knowledge among consumers of the nutritional content and health effects of grain seems to be limited. Questions that ought to be raised in this context are therefore: Do consumers know what is meant by whole grain? Do they know which grains are included in this group? Are all whole grains or whole grain foods the same when it comes to nutritional content and health effects?
Whole grain definitions
With the current interest in whole grain, not only among health authorities and consumers, but also within the food industry, a definition of whole grain is urgently needed. Most of the target groups Á scientists, industries, authorities, and consumers Á are interested in the health benefits in whole grain. Even depending on different aims (e.g. dietary recommendation, nutrition claims, or health claims), the definition of whole grain needs to be strict. A generally higher content of vitamins, minerals, and dietary fiber is not enough for dietary recommendations on whole grain even if this might be enough regarding nutrient content behind nutritional claims for selected nutrients. Knowledge about the exact content of nutrients in whole grains (milled, cracked, crushed, rolled, or flaked) of different cereals is, however, needed, both for dietary recommendation and nutrient claims. Only those whole grains, which have a similar content of nutrients should be included. To demonstrate the beneficial effects and/or disease risk reduction for whole grain, an exact and strict definition must be available.
To date, however, there has been no internationally accepted definition of whole grain. Currently, the scientific community is actively involved in discussions regarding an official definition, including issues such as the types and parts of grains that should be included in the definition. An important aspect in finding an applicable definition is the target group: individual consumers, to facilitate their choice of healthy food alternatives; the scientific community, to establish a uniform dietary factor to study in relation to health; the authorities, to allow them to make recommendations; or the food industry, to produce new whole grain products. A definition of whole grain would almost certainly benefit all these interests. However, for a definition to be useful in a long-term perspective, it must be grounded on evidence-based nutritional aspects rather than simply classification of food items.
In the scientific literature and popular press, much of the discussion concerns the seeds that should be included in the definition 'whole grain' (4). It is not clear whether such a definition should be based solely on whole grain cereals or whether the so-called pseudocereals and other starch-rich seeds should also be included, and the rationale behind their inclusion has to be defined.
Cereals are members of the grass family (Poaceae or Gramineae) and produce dry one-seeded fruits (caryopsis) which are commonly called a kernel or grain (5). All cereals consist of a fruit coat (pericarp) surrounding the seed. The seed contains an embryo (germ) and an endosperm surrounded by a nucellar epidermis and a seed coat (testa). In general, all cereals have broadly similar proportions of these botanical structures. However, it is not possible to specify a standard ratio for the different structures, since these vary within and between cereals. In addition, some cereals, such as rice, oats, and barley retain their husk during threshing and this must be removed to produce acceptable foods for humans. Bran is a technical fraction from the milling industry. It generally comprises the fruit wall, seed wall, aleurone layer, and small amounts of the starchy endosperm and germ. The composition of a bran fraction is highly dependent on the milling technology and type of grain used.
Pseudocereals, such as amaranth, quinoa, and buckwheat are not members of the grass family, but because of the high starch content in their seeds and their use in cereal-like products, it has been suggested that they be classified as whole grain together with the cereals in the grass family. These seeds contain no gluten and are therefore suitable alternatives for people with celiac disease.
Pulses or grain legumes are other seeds with a high content of starch that can be used in different types of cereal-like products. Because of these characteristics, they may fall into the definition of whole grain although they are generally not regarded as such.
American Association of Cereal Chemists (AACC) International and American Whole Grain Council (WGC) definition
Whole grain was defined back in 1999 by American Association of Cereal Chemists (AACC) International (6): 'Whole grain shall consist of the intact, ground, cracked or flaked caryopsis whose principal anatomical components Á the starchy endosperm, germ and bran Á are present in the same relative proportion as they exist in the intact caryopsis.' Inclusion or noninclusion in this definition is not based on fiber content, and although nuts and legumes are regarded as healthy plant foods, they are not included in the definition. Pseudocereals (buckwheat, amaranth, and quinoa), on the other hand, are included since they are considered to have similar macronutrient composition to whole grain cereals and are eaten in the same way.
The AACC International definition of whole grain was adopted by the US Food and Drug Administration (FDA) in the document 'Whole Grain Label Statements' in 2006, to provide guidance for the industry (7).
The American Whole Grain Council (WGC) defines whole grain in a similar way to AACC International, using the following wording in their definition in 2004 (8): 'Whole grains or foods made from them contain all the essential parts and naturally occurring nutrients of the entire grain seed. If the grain has been processed (e.g. cracked, crushed, rolled, extruded, and/or cooked), the food product should deliver approximately the same rich balance of nutrients that are found in the original grain seed. ' This definition includes the following cereals and pseudocereals (and forms of these): amaranth, barley, buckwheat, corn, including whole cornmeal and popcorn, millet, oats, including oatmeal, quinoa, rice, both brown rice and colored rice, rye, sorghum (also called milo), teff, triticale, wheat, including varieties such as spelt, emmer, farro, einkorn, Kamut † , durum and forms, such as bulgur, cracked wheat and wheatberries, and wild rice. It also includes more unusual cereals belonging to the grass family such as canary seed, Job's tears, montina, and fonio when consumed with all of their bran, germ, and endosperm.
Oilseeds and legumes (such as flax, chia, sunflower seeds, soy, chickpeas, etc.) are not considered whole grains by the WGC, AACC International, or the FDA.
Definition in Denmark, Sweden, and the Scandinavian keyhole A Danish Task Force (2) from 2008 defines whole grain as intact, ground, cracked, or flaked kernels after removal of the husks. In this definition the nine main cereals within the grass family (barley, oats, wheat, rye, rice, millet, maize, sorghum, and triticale) are included. It is permissible to combine different milling fractions, but the relative proportions of bran, starchy endosperm, and germ must be the same as in the intact kernels. Only dry flour of whole maize is included, but not fresh maize and popcorn. Pseudocereals are not included.
In Sweden, the National Food Administration uses a similar definition of whole grain to that of the Danish Task Force, but does not include triticale, since this cereal is not used in Swedish human foods.
The three Scandinavian countries, Denmark, Sweden, and Norway, have agreed on common rules for declaration of healthy foods in a system entitled 'The Scandinavian Keyhole' (9). These rules, accepted by the health authorities in the three countries, include a definition of whole grain. 'Whole grain is defined as intact and processed (dehulled, ground, cracked, flaked, or the like) products where endosperm, germ, and bran are present in the same proportions as in the intact grain. If these fractions are separated under processing, they should be added back so that the final product has approximately the same relative proportions of the three parts as in the intact grain. The whole grain definition includes the following whole grain cereals: wheat, rye, oats, barley, maize (dry seeds), rice, millet, and sorghum. Wild rice, quinoa, amaranth, and buckwheat are not included.' Health claims in the USA and Europe A whole grain health claim was the first to be allowed by the FDA in 1999: 'Diet rich in whole grain foods and other plant foods and low in fat, saturated fat and cholesterol may reduce the risk of heart disease and some cancers' (10). The product should contain 51% whole grain cereals or more per reference amount. This is easily achieved for dry foods such as breakfast cereals, but not for products with higher moisture contents such as bread. The FDA has produced a draft guidance to industry about what they consider to be whole grain and to assist the manufacturers in labeling their products (11). Three health claims have also been approved for grain products (not for whole grains) relating to coronary heart disease and certain cancers (3,12).
A health claim was subsequently accepted by the Joint Health Claim Initiative in UK in 2002: 'People with a healthy heart tend to eat more whole grain foods as part of a healthy lifestyle' (13). The food should contain 51% or more whole grain ingredients by weight per serving. The term 'whole grain' refers to the major cereal grains, such as wheat, rice, maize, and oats.
A Swedish Code of practice entitled 'Health Claims in the Labeling and Marketing of Food Products: The Food industry's Rules' (14) has been developed in close co-operation with relevant authorities. In 2003, a whole grain claim was adopted: 'A healthy lifestyle and a balanced diet rich in whole grain products reduce the risk of heart disease (http://www. snf.ideon.se). Product X is a good source of whole grain.' The food should contain at least 50% whole grain from wheat, rye, oats, or barley on a dry matter basis.
Awaiting European Union regulations, health claims in Europe primarily have been country-specific. Scientific substantiation of claims is one of the most important aspects of providing truthful information to consumers, satisfying regulatory requirements, and allowing fair market competition.
Basis for health claims for grains Currently, accepted health claims for grains have mostly been substantiated by observational/epidemiological studies. Unfortunately, different definitions for whole grain have been used. While AACC International has used the broadest inclusion criteria for grains (both cereals and pseudocereals), the Swedish and UK definitions have included only the most commonly eaten grains. Another problem may arise in that some studies include not only whole grain, but also products containing extra added bran and germ.
In population studies no cause and effect of the whole grain can be demonstrated. Assessment of intake is usually based on self-reporting, which is prone to systematic and random measurement errors (15). Furthermore, the lack of a general definition for whole grain foods also raises questions about the accuracy of reporting about the amount of wholegrain products consumed by the study participants. There have only been a few intervention studies that relate whole grain intake as defined by FDA/AACC International to intermediate markers of effect for CVD and diabetes. A recent review (12) concluded that there was insufficient scientific evidence for claims that whole grain reduces the risk of CVD. The evidence for whole grain consumption and lower risk of diabetes is also only suggestive and inconclusive. However, it is important to bear in mind that many large epidemiological studies of long duration are supportive when it comes to relationships between whole grain intake and a reduced risk of several Western diseases (1,2).
Many components in whole grain, such as certain fibers (e.g. b-glucan), vitamins (e.g. folate and tocols), minerals (e.g. magnesium), and bioactive components (e.g. phytoestrogens and plant sterols), can act as biomarkers or influence biomarkers for the abovementioned diseases. At present it is not possible to verify the specific components in whole grain responsible for reduced risk of disease. There is most likely a combination of different components providing such an effect. The effects of the individual components are impossible to separate, as they are often correlated in different ways to each other. In addition, the effects of consumption of one type of whole grain do not necessarily reflect the magnitude of benefits for other whole grains due to the diversity of whole grains in terms of macronutrients, micronutrients, and bioactive components.
For soluble fibers, i.e. b-glucan, in certain whole grain foods of oats and barley, health claims based on effect studies have been allowed by FDA from 2006: 'A soluble fiber from food such as (name of food) as part of a diet low in saturated fat and cholesterol may reduce the risk of heart disease. A serving (name of food) supplies (x) grams of the soluble fiber per day to have this effect' (16). The Swedish Code also includes a claim regarding b-glucan in oats and barley to blood cholesterol level and risk of CVD (14).
Why is there a need for a definition of whole grain? From the existing literature it is evident that there is no internationally accepted definition of whole grain. Some existing definitions are broad and commodity-based, including grains with similar end uses. These definitions include not only cereals (the grass family), but also pseudocereals and will perhaps be extended to other seeds. Other definitions are narrower and include only the most used and studied cereals in the respective population. It is important that international consensus can be reached on this point.
The chemical composition (macronutrients, micronutrients, and bioactive components) of some of the grains suggested for inclusion in the definition is well known, while others are much less well characterized. It is evident that little is known about the composition and health effects of rarely used cereals, such as teff, Job's tears, and fonio, and to some extent the pseudocereals.
For consumers, health authorities, and the relevant industries, the nutritional aspects are the main concern. It is therefore important that relevant scientific knowledge be made available, regarding both composition and health aspects, before including any grain in a definition. Simply considering the starch content, as has been suggested, would not be sufficient. Scientific documentation of health effects must form the basis for inclusion, and it is better to start with a restricted list of grains (a precautionary principle) and extend this as more knowledge becomes available.
It is also important to restrict grains included in the definition to true cereals belonging to the grass family (Poaceae) with similar botanical and chemical composition. Inclusion of all grains, such as pseudocereals and perhaps pulses and other seeds, due to high starch content and the same end uses and regardless of family membership, could be confusing for all those wishing to use the definition.
Two steps are necessary for definition
In agreeing a definition of whole grain and whole grain food there are two essential steps: definition of the ingredients of whole grain and definition of whole grain foods. It is important to begin by agreeing a definition of whole grain in terms of the raw dry materials (milled, cracked, crushed, rolled, or flaked). A definition of whole grain foods prepared by thermal, enzymatic, and chemical treatments, such as baking, malting, and fermentation, should then only be agreed once evidence on their composition and health effects becomes available.
However, the definition of whole grain food urgently needs to contain information about the effects of processing. An example of this is b-glucan from oats and barley, which will generally not have the same nutritional effects in a fermented product such as bread as in the minimally processed grains. This is also true for sensitive vitamins such as folate. This is highly relevant information, which must be considered by the food industry when producing foods with desired health effects.
Conclusions
We believe that an internationally accepted definition of whole grain is the first essential step in establishing health claims for whole grain and whole grain foods. An exact definition of the raw materials and knowledge about the components providing the health effects would appear to be crucial issues for the authorities when approving health claims.
Conflict of interest and funding
The authors have not received any funding or benefits from industry to conduct this study. | 2014-10-01T00:00:00.000Z | 2010-03-12T00:00:00.000 | {
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245838759 | pes2o/s2orc | v3-fos-license | Platelet, Plasma, Urinary Tryptophan-Serotonin-Kynurenine Axis Markers in Hyperacute Brain Ischemia Patients: A Prospective Study
Background and Purpose: Ischemic stroke is one of the most common causes of morbidity and mortality and has numerous clinical mimics. Previous studies have suggested a potential role of the tryptophan-serotonin (5-HT)-kynurenine (TSK) axis in ischemic stroke. Studies assessing this axis in the hyperacute phase of ischemic stroke (<4.5 h) are lacking. This prospective study thus evaluates the TSK axis in transient ischemic attack (TIA) and hyperacute ischemic stroke (AIS) patients. Methods: This study included 28 patients (24 AIS and 4 TIA) and 29 controls. The blood and urine samples of patient were collected within 4.5 h of symptoms onset (day 0, D0), then at 24 h and 3 months. Control blood and urine samples were collected once (D0). The TSK axis markers measured were platelet serotonin transporter (SERT) and 5-HT2A receptor (5-HT2AR) densities and platelet, plasma, and urinary 5-HT, plasma and urinary 5-hydroxyindole acetic acid (5-HIAA), and plasma kynurenine and tryptophan (TRP) levels. Results: At D0, patients exhibited a lower (p = 10−5) platelet SERT density, higher (p < 10−6) platelet 5-HT2AR density, higher (p = 10−5) plasma kynurenine/tryptophan (K/T) ratio, and higher urinary 5-HT (p = 0.011) and 5-HIAA (p = 0.003) levels than controls. Conclusions: We observed, for the first time, a hyperacute dysregulation of the serotonergic axis, and hyperacute and long-lasting activation of the tryptophan-kynurenine pathway in brain ischemia.
INTRODUCTION
Stroke is the second most common cause of disability-adjusted life-years worldwide, affecting 15 million people annually (1). Approximately 85% of strokes are ischemic but we still lack biomarkers that can help differentiate brain ischemia from its many mimics (2). Pivotal players in this process are endothelial dysfunction and platelet aggregation. Although activation of the tryptophan (TRP)-kynurenine axis (the main route of TRP catabolism) may play a role in ischemic stroke, a recent systematic review highlighted the absence of clinical studies on the topic (3). TRP-kynurenine activation in acute ischemic stroke may possibly be a consequence of increased activity of the initiating enzyme of this pathway (i.e., indoleamine 2,3dioxygenase, IDO), which is upregulated by inflammatory stimuli (3)(4)(5). TRP is internalized by small intestine enterochromaffin cells, which then hydroxylate and decarboxylate it to 5-HT. 5-HT is released into the bloodstream and stored in vesicles in platelets through a serotonin transporter (SERT), and, when released from platelets, mainly catabolized into inactive 5-hydroxyindole acetic acid (5-HIAA) via the monoamine oxidase A (MAO-A), contained in various tissues, such as liver, adipocytes, and vascular endothelium. Certain 5-HT receptors, such as the 5-HT 2A R present on platelet membranes, have platelet pro-aggregating and vasoconstrictive effects (6). 5-HT and pro-serotonergic drugs, such as selective serotonin reuptake inhibitors (SSRIs), have platelet pro-aggregating actions and vasoconstrictive effects, both may play a role in hyperacute ischemic stroke (AIS) (7). Indeed, SERT polymorphisms have been shown to be associated with a higher ischemic stroke risk (8). Nevertheless, only a few previous studies have explored certain markers of the tryptophan-serotonin (5-HT)-kynurenine (TSK) axis 24 h after brain ischemia (3,5), and there are no prospective studies that have measured TSK axis markers in blood and urine in the hyperacute phase (<4.5 h) of brain ischemia.
We thus aimed at evaluating TSK axis markers in blood and urine, such as, for the first time, platelet SERT and 5-HT 2A R, during the hyperacute phase (<4.5 h) of brain ischemia, as well as with repeated measurements at day 1 (D1) and 3 months (M3).
METHODS
Briefly, blood and urine samples of patients with acute brain ischemia were collected within 4.5 h of symptoms onset (day 0, D0) and then at 24 h (D1) and M3 from symptoms onset. The blood and urine samples of controls were collected once (D0). TSK axis markers were measured and compared between patients and controls and between different time points, as detailed in the following paragraphs. Detailed inclusion and exclusion criteria, clinical evaluation and demographic data of the participants, and a flowchart of the study design are provided in the Supplementary Material.
Study Population-Recruitment
In this prospective, monocentric, observational study, 53 consecutive adults (>18 years of age) patients with suspected AIS and candidates for intravenous thrombolysis were included from September 10, 2012 to November 27, 2014 at the Stroke center of Versailles Hospital (France), as detailed in Supplementary Figure 1. After screening of 53 patients, 12 patients were excluded due to an alternative diagnosis and negative MRI, 13 due to informed consent problems. In total 28 patients were included in this study, 24 diagnosed with ischemic stroke and 4 with transient ischemic attack (TIA). The hospital was pre-notified of all patients and they underwent brain imaging upon hospital arrival <4.5 h from symptoms onset. In addition, 60 controls without clinical evidence of cerebral ischemia were screened and 29 enrolled, including healthy volunteers or historical controls, i.e., inpatients that had been examined before orthopedic surgery for other clinical studies. Further details are provided in the Supplementary Material.
Statistical Analyses
Non-identifying data from clinical and biological measurements were analyzed using Statistical Package for the Social Sciences (SPSS) and Statistical Analysis System (SAS) software. The Shapiro-Wilk normality test was used to evaluate the data distribution. Student's T-test was used for comparisons between patients and controls for normally distributed data and Pearson's correlation coefficient was used to evaluate relationships between variables. Qualitative data were compared using Fisher's exact test and quantitative non-normally distributed data using Wilcoxon's non-parametric test for paired samples when appropriate. Spearman's non-parametric correlation coefficient was used to evaluate relationships between non-normally distributed variables. All tests were two-sided and a p < 0.05 was considered significant. Age and tobacco exposure (to quantify smoking and exposure to environmental tobacco smoke) were included as co-variables when making comparisons. Drug therapies that could interfere with the biological parameters being evaluated, especially serotonin, were recorded. In particular, subjects were divided into subgroups based on SSRI usage at the time of inclusion.
Biological Sampling and Parameters
All controls and patients were studied in the Versailles Hospital and the same biological parameters were measured using the same techniques and equipment (9)(10)(11). The blood and urine samples at 3 months (M3) from symptoms onset were collected during the neurological follow-up consultation.
The TSK axis markers measured were platelet SERT and 5-HT 2A R densities and platelet, plasma, and urinary 5-HT, plasma and urinary 5-HIAA, plasma kynurenine, and total (free + bound) tryptophan (TRP) levels. Besides, platelet aggregation and plasma homocysteine levels were measured. Sample collection and biochemical determinations were performed according to the procedures previously described by our group (9-11) for plasma TRP and kynurenine, plasma and platelet 5-HT, and plasma 5-HIAA using various standardized specific and sensitive high-performance liquid chromatography (HPLC) with coulometric detection methods, except for platelet 5-HT, for which blood was used instead of serum. The urinary 5-HT and 5-HIAA determinations used were derived from plasma 5-HT and 5-HIAA methods (10,11). The results of the urinary 5-HT and 5-HIAA assays were adjusted for renal function. The molar ratio between urinary 5-HIAA and 5-HT levels was calculated and used as an indirect index of monoamine oxidase-A (MAO-A, the enzyme catabolizing 5-HT to 5-HIAA) activity, and the molar ratio between plasma kynurenine and total TRP levels x100 was calculated and used as an indirect index of indole amine 2,3-dioxygenase (IDO) activity. Platelet SERT and 5-HT 2A R densities were determined through radioligand bindings. They were carried out in duplicate tubes containing 50 mM Tris buffer (pH 7.7), [ 125 I]DOI 2.5 nM (5-HT 2A R) or [ 3 H]paroxetine 1 nM (SERT), and 40 µl of platelet membrane suspension with (nonspecific bindings) or without (total bindings) 1 µM ketanserin (5-HT 2A R) or 10 µM fluoxetine (SERT) in a total volume of 100 µl. After incubation for 2 h at 37 • C, filtration (GF/B filters) and washing (3 × 5.0 ml cold buffer + 0.01% BSA) filters were counted.
The blood and urine collection of patients and controls were carried out at the same times and in the same manner, minimizing any potential bias from these sources on the results. In vitro addition of 5-HT 10 µM to ADP to platelets samples, even at minimal concentration of ADP (0.5 µM), significantly increased platelet aggregation intensity and speed for both patients and controls (p < 10 −5 ), with no significant differences in platelet aggregability between the two groups (Supplementary Tables 7, 8).
Results of Platelet Analyses
There were no significant differences in these parameters (i.e., platelet SERT and 5-HT 2A R densities, plasma, urinary, and platelet 5-HT) between male (n = 14) and female (n = 14) patients, as detailed in Supplementary Table 4, nor between participants taking SSRI and those who were not (p > 0.05 for each comparison). Further details are provided in the Supplementary Material.
There was no significant difference between patients and controls or between patients at D0, D1, or M3 for platelet 5-HT.
Further details are provided in the Supplementary Material and Supplementary Table 3.
The D0 urinary 5-HT levels of patients were significantly higher than those at D1 (p = 0.016) and M3 (p = 0.007), whereas there was no difference between the D1 and M3 urinary 5-HT values (p = 0.461).
There was no significant difference in D0 MAO-A activity index between patients and controls, nor between D0, D1, or M3 for the other measured urinary parameters in patients Supplementary Figure 3. There was no significant difference in urinary 5-HT and urinary 5-HIAA between male and female subjects (Supplementary Table 4).
(urinary 5-HIAA and MAO-A activity index), as detailed in
Further details are provided in the Supplementary Material and Supplementary Table 3.
There were no significant differences in these and others plasma parameters (i.e., plasma 5-HT, plasma 5-HIAA, and plasma K/T ratio) between male (n = 14) and female (n = 14) patients, as detailed in Supplementary Table 4, nor between participants taking SSRI and those who were not (p > 0.05 for each comparison).
There was no significant difference between patients and controls or among patients at D0, D1, or M3 for the other measured blood biological parameters, i.e., plasma 5-HT, plasma 5-HIAA, and plasma homocysteine. There was no significant difference between patients and controls or between patients at D0, D1, or M3 for the other measured plasma biological parameters.
In addition, we found no significant correlation between plasma homocysteine levels and those of other D0 TSK markers.
Further details are provided in the Supplementary Material and Supplementary Table 3.
Sub-Analyses
We performed a sub-analysis excluding patients with TIA (n = 4). At D0, platelet SERT density remained significantly lower (p < 10 −5 ) in patients than controls. Platelet 5-HT 2A R density remained significantly higher (p < 10 −6 ) in patients than controls, as were urinary 5-HT (p = 0.03) and urinary 5-HIAA (p = 0.008). The D0 plasma K/T ratio of patients remained higher than controls (p < 10 −4 ). The plasma K/T ratio of patients at D0 and D1 remained lower than at M3 (p < 0.05 for all comparisons; Supplementary Material and Supplementary Table 5).
Differences in blood markers (i.e., platelet SERT density, platelet 5-HT 2A R density, and plasma K/T ratio) and urinary markers (i.e., urinary 5-HT and urinary 5-HIAA) remained significant (p < 0.0001 and p < 0.022, respectively) after excluding the six patients taking SSRI from the analyses.
Globally, patients were slightly older than controls (p = 0.0483). Further details are provided in the Supplementary Material.
Non-Significant Results
There were no significant differences between patients and controls for sex or tobacco exposure (Supplementary Table 1), two important confounders for the interpretation of serotonergic parameters data. However, it ought to be noted that the study was not powered enough for detecting sex differences in these parameters. Among all participants, four patients (three women and one man) were tobacco smokers, and six female patients were taking SSRI. There was no significant correlation between any of the measured TSK axis markers and the NIHSS score of patients at the emergency department or in the stroke unit of hospital.
Further details are provided in the Supplementary Material and Supplementary Table 3.
DISCUSSION
Overall, as recapitulated in the graphical abstract, this study provides the first evidence of abnormalities of the TSK axis (i) for patients in the hyperacute phase (<4.5 h) of cerebral infarction relative to controls and (ii) for intra-subject measurements obtained from patients after 1 or 90 days from symptoms onset.
The 5-HT levels are mainly regulated by SERT and MAO-A. The marked decrease in platelet SERT and increase in platelet 5-HT 2A R densities observed in patients may be possible mechanisms that increase the ischemic risk or reflect the presence of a thrombotic process, as such alterations could promote platelet aggregation, local platelet release of 5HT, and local proischemic vasoconstriction. Although, this is the first time that this clinical finding has been reported, it is in accordance with results from a recent study on 834 patients with AIS and TIA showing that genetic polymorphisms associated with increased SERT expression are linked to a lower risk of cerebral ischemia (12). Additionally, it is consistent with the results of studies showing a decrease in platelet SERT densities in drug-free depressed patients, further reduced by SSRIs (11), and an increased AIS risk associated with SSRI treatment (8,12). Furthermore, an increase in platelet 5-HT 2A R density and aggregation response has been observed with certain antidepressant treatments, e.g., clomipramine (11). The increased ex vivo platelet aggregability found both in patients and controls after the addition of 5-HT confirm the role of this neurotransmitter in platelet aggregation. On the other hand, the absence of difference between patients and controls in this test ex vivo cannot be interpreted as a reflection of in vivo processes.
The fact that alterations were found in the levels of urinary serotonergic markers but not in plasma is consistent with the very short half-life of 5-HT in plasma. Furthermore, patients showed increased urinary 5-HT levels only in the first hours following brain ischemia, further supporting the hypothesis of a transient serotonergic storm in the setting of brain ischemia.
The higher plasma K/T ratio (IDO activity index) observed in patients with TIA and AIS is in accordance with the results of previous AIS studies, that reported lower TRP levels and higher kynurenine levels in patients with AIS than in controls (4,5,13). At biochemical level, kynurenine and serotonin pathways are linked by their common precursor tryptophan, which influences both serotonergic and kynurenine pathways. We found the TRP-kynurenine pathway to be already activated in the hyperacute phase (<4.5 h) of brain ischemia, not just within 24 h of symptoms onset, as previously reported (13,14), and it was still activated 3 months later. The K/T ratio has been found to correlate with brain infarction volumes in patients with stroke (14). Although the exact effects of kynurenine and its catabolites are still debated (15), as mentioned in the introduction, the TRP-kynurenine pathway appears to be activated in acute ischemic stroke, possibly as a consequence of increased activity of the initiating enzyme of this pathway, i.e., indoleamine 2,3-dioxygenase (IDO), which is upregulated by inflammatory stimuli (3)(4)(5). This pathway generates neurotoxic and pro-apoptotic catabolites (3).
This study thus further confirms the activation of the TRPkynurenine catabolic pathway in acute brain ischemia and shows that such an alteration is already present a few hours after symptoms onset and is long-lasting.
Such findings may have important clinical implications in the quest for biomarkers specific for the hyperacute phase of brain ischemia [differentiating it from its numerous mimics (2)].
The present findings are of interest also considering that proserotonergic, anti-depressant drugs, such as SSRI, have platelet pro-aggregating actions and vasoconstrictive effects, and may play a role in AIS. Thus, these results add to previous knowledge on the relationship between stroke and depression, with the dysregulation of the serotonergic system being one link between these two conditions. However, both technological advancements and further studies are needed to translate the above results to real-life prehospital evaluation of patients with suspected AIS or TIA.
LIMITATIONS
This study has some limitations. Due to its preliminary nature, the sample size is small, in particular, the numerosity of patients with TIA is low. Thus, the study is underpowered to allow comparisons between patients with TIA and AIS, who were analyzed together, as they all suffered a cerebral ischemic insult. While exclusion criteria were different between controls and patients, patients were not taking other antidepressants apart from SSRI, nor monoamine oxidase inhibitors (MAOIs), opioids, triptans, and valproate. Only one patient was treated with L-dopa. The therapies of patients included antihypertensive drugs, anticoagulants, antiplatelets, statins, bronchodilators, antidiabetics, and antiulcer drugs. We did not collect information on herbal medicine. Furthermore, patients were slightly older than controls, but this weakly significant difference could hardly explain, alone, the very significant difference we observed in serotoninergic parameters. Control patients did not undergo MRI to exclude silent ischemic strokes but the low likelihood of such events and the differences observed between the groups suggest that this limitation did not compromise the soundness of the study.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of Ile de France XI of Saint Germain en Laye ("Comité de protection des personnes, CPP, " reference number: 11040; 2011) and by the AFSSAPS ("Agence française de sécurité sanitaire et des produits de santé" reference number: B111102-90; 2011). The patients/participants provided their written informed consent to participate in this study. | 2022-01-11T14:21:22.320Z | 2022-01-11T00:00:00.000 | {
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260994243 | pes2o/s2orc | v3-fos-license | Engaging with communities and precarity theory to bring new perspectives to public mental health
ABSTRACT In this paper we explore the role of precarity theory in bringing new perspectives to public mental health. The paper draws on a qualitative, participatory research study carried out in Glasgow that illuminates the entangled and complex relations between the social, bio-political, cultural, economic, and environmental conditions that produce mental health and ill health. Through the accounts of Sassy Queen, Tony, Simba, John, and Rhianna, we explore the attrition of everyday lives that are lived in constant states of crisis, the forms of precarity that it precipitates, and how this plays out for them concerning how they talk about and experience ‘mental health’. Their experience provides an alternative vantage point to understanding mental distress to those which are made possible through recourse to prevalent social determinant and behavioural models of public health. The paper concludes that the operations of the political as everyday biopolitics must become more visible within public health, and this requires interdisciplinary and narrative approaches to fully understand the border zone between macro-level politics and the biopolitics of everyday life.
Introduction
As the acute stage of the COVID-19 pandemic recedes and the cost-of-living situation intensifies in the United Kingdom what has been called a crisis of mental health has come to the fore (Broadbent et al., 2023).This 'crisis' occupies a longstanding fault line in public health situated between the medical and the political.Major public health bodies premise a social determinants of health model (SDH) that claims the major causal factors of poor mental health are social, economic, and environmental (Bonnar, 2017;Herrick & Bell, 2022;WHO, 2014).This proposition sits in tension with health policy that is often preoccupied with individual bodies, minds, and ailments (Goldberg, 2017).Articulating the complex relations between the social, political, cultural, environmental, and individual factors that shape our mental health experiences is a challenge for public health.Several critical mental health scholars (Prozorov, 2021;Rau, 2013;Stiegler, 2008;Thomas, 2016) argue that both the tendency to medicalise social problems and the associated data-driven technologies of measurement are a facet of both biopolitical and psychopolitical governance of life.They bemoan the displacement of economic and political failures as individual pathology of mental ill health and argue that it is a new politics that is required to address these issues to improve population mental health.But what forms of political understanding can offer insight into the relations between systems of governance and individual anxieties/worries/troubles?Despite the political nature of CONTACT Heather Lynch heather.lynch@gcu.ac.uk health being recognised in behavioural and social determinant models of public mental health, it is often enacted through a medicalised lens of objectivity.These enactments include public health programmes that focus on screening and prevention which are underpinned by the belief system that 'mental health' is individually experienced and is identifiable, knowable, and preventable.Further, although these behavioural and social determinant models often identify 'political' factors as determinants of health and as spheres of influence they tend to do so in a realm beyond the individual.They are therefore ill-equipped to understand how the political is implicated through life as it is lived.
In this article we look at the biopolitical theorisation of precarity as it articulates a relationship between the current 'politics of crisis ' (Gentili, 2021) and individual suffering (Butler, 2004(Butler, , 2009;;Vij, 2019).On a practical level, precarity theory is useful for understanding 'mental health' and has much to offer public mental health as it brings a biopolitical lens to a complex phenomenon and through doing so offers an understanding of the 'uncertainties' of 'life worlds' (Kelly et al., 2009).Thus, it provides public mental health with new perspectives not necessarily visible from more deterministic viewpoints.As Kelly et al. (2009, p. 18) state 'it is not possible to predict individual health outcomes' based on population models, as individual lives are complex, dynamic, and unique.In contrast, precarity theory implicates the 'political' as it exists in everyday life rather than as a set of political responses to human suffering encapsulated in a set of pathological categories of mental health.In turn, it shifts the emphasis from signs, symptoms, and manifestations of mental ill-health which might be experienced by individuals to seeing these as expressions of a system at work.
Using contemporary theory of precarity, we make sense of the complex ways in which political, social, economic, and embodied experiences of a group of people who live in the North of Glasgow are expressed as struggles with mental health.We offer insight into how the everyday precarity expressed by Sassy Queen, Simba, John, Rhianna, and Tony relates to the politics of crisis encapsulated in precarity theory.Following an outline of our use of precarity theory, we summarise methods adopted for this study.
Precarity: a new lens for public mental health
The term 'precariat' (Standing, 2011;Lorey, 2015) is increasingly used to define the gig economy workers whose precarious economic conditions are the result of insecure employment.However, Butler's (2004Butler's ( , 2009) ) precarity theory has much deeper and wider implications, that connect contemporary models of governance and their perpetual crisis, with the crisis experienced by individuals, foremost those living at the harshest front of unequal societies.For Butler, precariousness is a facet of life.She states: 'precariousness is coextensive with birth itself' and that 'precariousness implies living socially, that is, the fact that one's life is always in some sense in the hands of the other ' (2009, p. 14).Precarity may be unequal in distribution but does not belong to a particular group of insecure people but to all lives and modes of governance.The route to understanding the distribution of precarity and the politics of crisis runs through Foucauldian biopolitics (Berlant, 2011;Bird & Lynch, 2019;Butler, 2004Butler, , 2009;;Povinelli, 2011;Vij, 2019).In this section, we summarise how the precariousness of all life, relates to biopolitical governance and how this, in turn, relates to public mental health.Foucault's (2008) biopolitical theory extends from his theory of distributed power, where power does not belong to a sovereign but circulates through 'state and non-state actors and discourses' (Butler, 2004, p. XV).Unlike sovereign power that operates through law, biopower operates in the governance of the everyday.In this scheme, biopolitical governance is comprised of the meshwork of practices that operate in and through societal norms.Foucault (1978) argues that biopower ascended with modernity, where science and technology afforded the means of measuring, classifying, and intervening on populations, literally to 'foster life'.
There are clear implications for public health in these theoretical perspectives and Foucault (2003) noted the 'medical gaze' as a mechanism of biopolitical governance.However, Foucault's writing theorises the operations of biopolitics at population level while more recent biopolitical scholarship explores the operations of biopolitics through subjective experience (Butler, 2004(Butler, , 2009;;Tarizzo, 2011;Gentili, 2021).This work offers insight into the relationship between precarity and mental health in its widest sense.Tarizzo (2011) proposes that biopolitical attempts to 'optimise' life affect mental health.He argues that 'mood disorder' may be an act of resistance to biopolitical management, or conversely may 'pre-suppose' this field of management.In other words, this is the subjective manifestation of a 'mental health' problem that lends itself to intervention.Through the critique of liberalism, Gentili (2021) connects biopolitics with precarity and mental health through his theorisation of 'crisis' as a mode of governance that reaches into everyday life.He argues that this crisis circularity locks governments and governed into a mutually produced endless state of uncertainty and its associated distress.This predisposition to constant change generates a much deeper sense of precarity than can be explained by employment conditions and one where crisis 'activates those subjective states of mind such as trust, credit, belief, guilt, expectation, fear, and sacrifice' (2021, p. 95).From this position precariousness and its associated problem moods 'is rather a form of life' (Gentili, 2021, p. 175) that has emerged from modes of governance that are biopolitical.
The biopolitics of precarity presents a stark reality of the benefits and costs of any intervention in life.Public health has a strong history of achieving more equitable distribution of good health.A biopolitical view on precarity however might allow us to understand why despite extensive public health efforts, interventions focused on improving mental health and increasing equality have had some, yet limited success to date (Alegría et al., 2018).
Glasgow, the location of our study, is a place where health inequalities endure despite the best efforts of public health institutions.Despite attempts to reconfigure Glasgow in positive frames as a cultural capital, its high mortality, healthy life expectancy, drugs deaths, and poverty mean that it is frequently held up as an example of failure (Schofield et al., 2021;Walsh et al., 2017Walsh et al., , 2021)).It has been subject to numerous interventions that seek to improve the population health of Glasgow (Scottish Government, 2008Government, , 2018)).Individual Glaswegians who are seen as a part of this failure are required to live differently and improve their own health and, in doing so, that of the population.Our study offers insight into how this is experienced and what everyday precarity looks and feels like for them.In this next section, we describe the setting and methods of the study.
Methods
The qualitative, participatory research methods used in this study contrast with many of the datadriven and quantitative measures traditionally used in public health.In recent years, there has been a turn towards 'lived experience' as being vital for good policy-making and public health action (Bramley et al., 2020).As Case and Deaton (2021) indicate, the complexities of circumstance that generate despair do not lend themselves to causal analysis.Rose (2019Rose ( , 2020) ) argues that there is a need for sociological and ethnographic investigations capable of mapping experiences of 'stress, poverty, exclusion, isolation and violence' and the ways this is 'shaped by cultural narratives and beliefs' (Rose, 2020, p. 47).The study reported in this paper takes such an approach.
In this study, we worked with 9 service users involved with a North Glasgow community organisation.They participated in the process by documenting their daily lives, as diaries offering access to participants' experience in its 'natural, spontaneous context' (Bolger et al., 2003, p. 581).Participants varied in terms of their literacy and confidence and so they were encouraged to find a method of documenting how they felt each day that best suited them.In practice, this meant keeping a traditional written diary, audio recording, memos, mood boards, and photographs over a 4-week period between March and April 2021.A downside of diaries is that participants may not remember, be too tired, or think that they have nothing to say.To mitigate this our participants were supported by social work students on placement from Glasgow Caledonian University (GCU).The students facilitated participants' involvement by explaining the process, discussing options to document the experience, and providing participants with materials.Participants chose a pseudonym, and their material has been anonymised.Recruited through a family support organisation, all the participants were parents.Caring and mental health featured heavily in their logs; as well as in their day-to-day decision-making.It was a challenge to find methods of documentation that were achievable in the context of their demanding lives.Many responses were a somewhat staccato, direct 'unstoried' (Connelly & Clandinin, 1990) response to the request to document their daily lives.This extract from Tony exemplifies this: 'Nervous because I don't know what will happen today'.
Despite this, participation in its many forms still happened allowing a unique documentation of lives rarely seen in the research literature.The longitudinal, albeit short, method of data collection affords insight into the rhythms, rise, and fall of feelings and how these relate to other life events across the weeks.After the completion of the four-week period and an initial scan of the data, we undertook in-depth interviews with each student and the support worker.This process allowed us to ask follow-up questions and clarify any ambiguities in the diaries.The interviews with the students and support worker provided important data concerning the context of the participants' lives.In a bid to keep the lens of the exploration on the lives of the participants, we have chosen for the most part not to draw directly on the accounts of the students or support worker.They were, however, invaluable in providing the context which allows for the in-depth explorations of this paper.
The study received ethical approval from GCU School of Health and Life Sciences Ethics Committee (Approval Number: HLS/PSWAHS/20/086).Data analysis was carried out by the authors through an abductive (Timmermans & Tavory, 2012) process framed by precarity theory.Abduction is a process whereby the research brings theory into conversation with empirically derived data.It depends 'on a theoretically sensitized observer who recognizes their potential relevance' (Timmermans & Tavory, 2012, p. 173).An abductive approach involves thinking simultaneously with empirical and theoretical knowledge.Each individual expressed their daily experience in different ways, some through written diaries, others through single words, and others in conversations that were recorded or noted by the interviewer.To honour the uniqueness of each participant we have not looked for cross-cutting themes but instead explored the resonance between precarity and their self-reported mental health.So that we could 'bring to life' the lives of our participants we have chosen to focus on reporting the findings about 5 of our participants rather than all 9.The contribution of all the participants was equally valued and informed our overall analysis.In the illustrative speech used in the findings section, we have chosen to keep the Scots dialect spoken by the participants to respect them and their local identities.Our use of precarity theory brings the biopolitical lens of precarity into conversation with everyday life and affords insight into this relationship with practical implications for the field of public mental health.
Findings
In this next section, we consider the lives of the participants from the vantage point of precarity theory and in relation to several emergent overarching themes: precarity of everyday life, precarious care, precarious services, and precarity subverted.We focus on the lives of Rhianna, Tony, Simba, John and Sassy Queen.
Precarity of everyday life
Rhianna's days oscillate between recurrent worries and small wins that are recited with humour and a sense of hope.She lives with chronic pain as she awaits news of surgery; financial concerns are ever present as she frets about paying for food and household bills, and who she can borrow from to achieve this.She worries about the events in her children's lives; exams, health, and relationships.She is also anxious about what might seem to be less important occurrences such as disastrous selfadministered hair dye.She narrates how these specific concerns are addressed; she borrows money, receives a benefits payment, makes the hospital appointment and her child survives the day at school.Any moments of joy are short-lived, always marked by a turn to the next worry.Perhaps the most harrowing aspect of her account is that behind these specific concerns, she expresses an undercurrent of general worries about her 'kids not coping', her enduring pain, and her selfidentified capacity to overthink.There are moments of respite.She can 'take time out' but knows that this continuous mental strain will 'start all over again'.She is acutely aware of her lack of control and yet she absorbs the social expectations of her children's performance and managing household finances as a measure of her value.
Tony's diary entries are similarly underpinned by pervasive uncertainty.He has lived in Glasgow for 15 years, yet he feels the absence of his family and home in Germany.His written diary entries are succinct, often not much more than a sentence but each statement communicates emotional depth that is not evident in conversation.The student who facilitated his participation was surprised to read his diary entries.'He says his mental health is good, then you read this' she mused.His record makes bleak reading.His choice of words betrays hopelessness.He is 'stressed', 'nervous', 'tired' and 'worried', where 'the same thing every day is doing my [his] head in'.Tony certainly bears many of the markers of the type of economic precarity discussed by Standing (2011) and Foti (2017).He is a migrant whose work status is unclear.He 'picks up casual work, sometimes'.However, his daily account of life offers little on issues linked to precarious employment, instead he offers glimmers of insight into unpredictable and perpetually strained family relationships.These are not presented as a roller coaster of dramatic events but of life in a perpetual low-simmer crisis.A life conditioned by the acute awareness that nothing is certain, but the refrain of uncertainty.Like Rhianna, Tony's daily reflections offer insight into the subjective experience of inhabiting the precarity of 'endless crisis' (Gentili, 2021).Bonnar's (2017) neuroscientific approach might encourage us to conclude that this sense of endless crisis is a result of complex trauma, while Marmot et al. (2010) promote interventions that allow them to relieve their sense of crisis by taking control.However, this would evade the glaring reality that Rhianna and Tony are dealing with tangible problems, that precipitate reasons for concern daily.Their endless crisis is not a symptom of neuropathology but the real circumstances of their lives, and the challenges in fulfilling their roles as parents and carers.They report a constant struggle to fulfil these roles, through the relentless daily occurrences that they take responsibility for.These daily concerns are not significant through order of magnitude but volume.This is what Povinelli (2011) terms the precarity of the quasi event.In her critique of 'late liberalism' Povinelli distinguishes the 'event' and the 'quasi event'.The event is the remarkable, the terror attack, the company collapse, or in the significant individual loss, while the quasi event is the slow attrition of everyday life.A 'decomposition' that happens below the 'threshold of awareness and theorisation' (Povinelli, 2011, p. 133).It is the relentless and persistent number of 'quasi events' reported by Rhianna, Tony and other participants that is notable, coupled with the belief that they must rise to each occasion.This internalisation of value is a mechanism that produces a perpetual cliff edge of failure and in turn, impacts mood and sense of control.
Precarious care, another cruel optimism
Simba also lives with pervasive uncertainty.She articulates a map of experience that navigates one caring responsibility to the next.Simba cares for her three children, one of whom is diagnosed with autism, her partner who is unwell and awaiting surgery, her dad who is also seriously unwell with illness associated with COVID-19, and two cats.Alongside daily domestic tasks of shopping, washing, cooking, cleaning, and entertaining her children she has additional tasks of going to the chemist for her father's medication, helping him to get a shower, and encouraging him to do his exercises as part of his recovery from COVID-19.The pressure of these tasks rachets and each daily expression progresses her feelings of despair.Frequently she talks about her mood, and the struggle to hold onto her 'sanity'.
Simba's care labour has a different set of characteristics and rewards than that of Standing's (2011) gig economy worker.Unlike the Deliveroo rider, she knows that she will work each day.She talks about 'hating Fridays' due to the need to go to the shops, the chemist, and help her dad shower, as she has only just completed these tasks when her children return home demanding her attention.Plans frequently get disrupted by sickness or by events such as her son getting a fractured finger after being hit by another child at school.Montori (2021) promotes care for others as a social determinant of health, that has been eroded by contemporary lifestyles.However, it is hard to imagine that Simba or indeed any of our participants could care more.Indeed, much of their persistent sense of crisis is a result of their caring responsibilities.Cubellis (2018, p. 638) provides fascinating insight into care work that relies on the precarity of the giver.Her study involves peer support for people with mental illness, however, there are similarities.She notes that care 'can wound when that exposure is seized on as a means to mitigate the fissures in a larger broken system', a system in perpetual crisis.Each day Simba attempts to mitigate the suffering of her father, ease the anxieties of her children and occupy the space where there could be money for bills, food, and clothing with the fullness of devotion to their wellbeing.To achieve this, she needs to be present and malleable to whatever challenges each day throws up.She writes, 'Constantly worry about everyone else.I've let myself go in every way'.The terms of her care provision require her to lose herself, in order that she can bridge the gaps that appear for her family.
She is caught between a belief that her problem is a lack of time for herself, 'just feel like screaming, it's getting too much, no time for myself', and that peace lies in the successful fulfilment of her caring responsibilities, 'I'm still exhausted but I will get there as long as they (kids) are ok'.She is in an impossible situation, where the two appear mutually exclusive and the belief that time for herself will make things better is a cruel optimism (Berlant, 2011).Berlant defines cruel optimism as 'a relation of attachment to compromised conditions of possibility whose realization is discovered either to be impossible, sheer fantasy, or too possible, and toxic' (Berlant, 2011, p. 23).Simba looks to the moments of relief from her care role as a means of wellness.Yet, these are moments of respite, not an answer to problems that lie deep within a beleaguered health system, an eroded benefits system, and overwhelmed child and adolescent mental health services.That she takes the burden of her inability to care for herself, on herself, is another twist.She states, 'I feel like a failure.It's [her son's] birthday tomorrow.I haven't been able to get him what I would normally'.Simba's precarious care labour is found in the gap between structures incapable of providing sufficient care and the people she cares for.She is always on call, and the moments of respite, are just that.
Precarious services
John lives with his partner and baby son who, at the time of the fieldwork, was eight months old.His baby was taken into care at birth and his delight in being able to care for him again is clear.For John, precarity involves managing 'life' through multiple agencies and support put in place to help him and his partner care for their son.John does not explicitly discuss feeling under surveillance, but it is implicitly expressed in the way that he communicated with the interviewer.He describes the presence of services as, at times, overwhelming and can get in the way of the routine that they are trying to establish for their son.He does not offer the direct expressions of being at the end of his tether that we find in Simba and Rhianna's diaries, but he does express this through his body.He states, 'Sometimes it's hard to actually speak about how you are feeling' (John, Interview 2).
By his account health and social care services have not helped him with this.Quite the contrary, and reading between the lines of his account, he does not want to report anything negative about services or say anything against them for fear that this will impact his relationship with his son.This suggests that health and social care services can be a part of the engine of precarity.Morgan (2014) discusses the multiple and complex problems associated with everyday surveillance by health and social care services alongside forms of self-surveillance that this propagates.She argues that more research is needed on the impacts of these everyday curtailments of freedom and privacy.Understanding John's relationship with services requires longer and more in-depth study, but it is apparent that services designed to give support are also an engine of uncertainty.
Precarity subverted
Sassy Queen offers a distinct view of her precarious life.Her bold proclamation, 'ah come weh drama' speaks to precarious living in ways that confound Gentili's (2021) negation of 'crisis'.Sassy Queen gives an account of challenges, historical and present, that would increase the anxiety levels of most people.Her choice of pseudonym reflects her larger-than-life personality that radiates through her storytelling.She comes 'weh drama' and in this statement, she owns her precarity, she puts it to work as a feature that distinguishes her as uniquely immersed in a life of unpredictable and stressful events.Sassy Queen's first interview began with a succession of worries, her recent MRI scan, ongoing tremors, the emotional and financial pressure of family birthdays, Mother's Day, COVID-19 restrictions, worry over her elderly mother's health, waiting for results, feeling 'rotten for goin aff the handle at the slightest wee thing', the need to furnish her children with technology and activities that she believes they cannot do without, worries over accumulating debt, and the risk of blood clots associated with the COVID-19 vaccine.In this succession of stressors, the significant health issue that necessitated her MRI scan gets rolled into worries over birthday presents and Mother's Day planning.Her free-flowing conversation is structured around anxieties that she presents with equivalent intensity and peppered with statements that reinforce her life as one that is characterised by uncertainty.
Like other participants, Sassy Queen's challenges evolve from fundamental relations of family, material needs, and health.Her eldest son has a diagnosis of autism.She claims that he suffers from anxiety 'through the roof' and alongside his brother is an open case with Child and Adolescent Mental Health Services (CAMHS).Sassy Queen in her words has had 'mental health' since her teens, the origins of which she relates to an experience of sexual abuse by a GP.She claims that her first son 'saved my life': and that she would be dead through alcohol if it were not for the greater need to care for him and his siblings.Her children are a huge source of stress and yet they are also what keeps her focused.She navigates each day from one source of anxiety to the next.She said 'stress follows me.Ah, jist get wan thing sorted and another thing comes alang'.These simultaneous and competing worries are associated with her constant awareness of her struggle to find the money to pay for what she views as necessities, that her children's mental health is unpredictable and that her own mental and physical health hover at breaking point.Like other participants, Sassy Queen endures endless crises however, her story differs from theirs as she presents her endurance of challenges as a strength.She states that her children are both a big source of worry and what 'saved' her.Sassy Queen appears to use the discourse of mental health as a means of making sense of her life and how she feels in a way that generates certainty from uncertainty.
Sassy Queen has found what Marmot et al. (2010) might call a 'sense of belonging' in the realm of quasi evental perpetual crisis.Sassy Queen finds value in her precariousness, it affirms her.This capability is not to diminish the very real problems that she deals with, but positioning her as, in need, or pathologising what she calls her 'mental health' would mischaracterise her approach to life.She is not a pitiable excluded character but someone whose liveliness cannot be disentangled from the challenging conditions through which she expresses subjectivity.Like other participants, Sassy Queen is not a one-dimensional victim of an unequal world.
Discussion
Our participants express the daily concerns of their lives and how these affect their mood in different ways.However, each of their accounts is characterised by a pervasive sense of anxious uncertainty, that is expressed in the register of mental health and the struggle not to be subsumed by this.Their stories evidence that the precariat does not just include an exclusive category of gig economy workers but also people who cannot work and are reliant on services.Precarity is not tied to a particular problem or role but is the architecture of their everyday lives.
It is the volume and pervasiveness of events that need to be managed that leads them to feel that they are always on the brink of losing control.All the participants are carers and this responsibility and the associated concern for their loved ones dominates the everyday events that generate uncertainty.Moments of respite are short-lived, and for some cruel, as they do not change the circumstance of uncertainty.The resources that they might draw on such as family and services are also mired in precarity and therefore rather than offering relief can compound the sense of losing control.These insights present a set of challenges to public mental health that we set out below.
Our contributors live with a relentless awareness of the misfortunes each day may bring to those they care for and to them.Their expectation is that they should manage these events yet, as much of it links to the behaviour of others and the responses of public services, they are plagued with the uncertainty of events that are out of their hands.Stress and mental health are words that are used to describe these feelings.Tarizzo (2011) draws a line between biopolitical governance that seeks to optimise population behaviour through the identification and management of risk, and the ways that this is internalised by individual subjects.What our participants most often express is a striving to fulfil social norms around care and parenting and a pervasive sense of living on the brink of failure.None of their reports seeks to blame policy or structures.However much the discourse of stress, anxiety and despair provides a frame for these feelings, this framing continues to place the onus on them rather than the social conditions that produce these expectations.This quandary raises a question about the role of public health.At population level, our contributors would be situated at the harsh end of the health inequality spectrum, where the multifaceted pressures of poverty are difficult to disentangle from mental health risks.However, at the individual level, it is difficult to make the case that the burden of responsibility they carry and the substantive everyday issues they deal with are 'mental health' issues.Challenges with benefits, housing, education, and care responsibilities are linked to the politics of societal choices and aspirations.And yet our participants reach for the register of mental health to express their challenges rather than that of the political.It seems the link between the political and the everyday is imperceptible to them.This finding poses a question for public health on how it might engage with the politics of everyday life in a way that does not further individualise societal problems.We have some suggestions on how this might be achieved.
First, there is a need within public health strategy to better understand how population-level identification of problems is expressed through subjective experience.This measure involves understanding not just how categories such as 'economically disadvantaged' may position those classified in deficit, but to develop an understanding of how macro-level societal expectations are configured through the subjective experience of the everyday.Second, to understand the politics of everyday life is to get to grips with what people and families value, how this informs their choices and intersects with wider community and societal values.This labour is public health work that takes place in the messy border zone between population and individual.It involves public health practitioners occupying positions of not knowing, and following the indeterminate uncertainties of the everyday, as opposed to imposing frameworks developed through classification systems generated at population level.This value-driven work requires modes of listening which are relational where the solutions do not lie with any one person or organisation but are generated through dialogue.This dialogue has the scope to go beyond current narratives on individuals' lived experience as a form of policy knowledge.Dialogue that creates meaningful engagement has the scope to shift the relations of power that keep health inequalities firmly entrenched.To bear witness to experience in relational ways that do not objectify people, nor individualise their distress is an important role for public health organisations.This is not a rejection of knowledge generated at population level but an understanding of its limits, and acknowledgement that life's uncertainties cannot be managed out of existence but must be worked with.Finally, achieving this will require narrative approaches and methodologies capable of holding the multiple facets at play in everyday life. | 2023-08-19T15:02:27.968Z | 2023-08-17T00:00:00.000 | {
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119429702 | pes2o/s2orc | v3-fos-license | submitted to THE ASTROPHYSICAL JOURNAL LETTERS, 05/04/99 EFFICIENCY AND SPECTRUM OF INTERNAL γ-RAY BURST SHOCKS
We present an analysis of the Internal Shock Model of GRBs, where gamma-rays are produced by internal shocks within a relativistic wind. We show that observed GRB characteristics impose stringent constraints on wind and source parameters. We find that a significant fraction, of order 20 %, of the wind kinetic energy can be converted to radiation, provided the distribution of Lorentz factors within the wind has a large variance and provided the minimum Lorentz factor is higher than 10^(2.5)L_(52)^(2/9), where L=10^(52)L_(52)erg/s is the wind luminosity. For a high,>10 %, efficiency wind, spectral energy breaks in the 0.1 to 1 MeV range are obtained for sources with dynamical time R/c<1 ms, suggesting a possible explanation for the observed clustering of spectral break energies in this range. The lower limit to wind Lorenz factor and the upper limit, around (R/10^7 cm)^(-5/6) MeV to observed break energies are set by Thomson optical depth due to electron positron pairs produced by synchrotron photons. Natural consequences of the model are absence of bursts with peak emission energy significantly exceeding 1 MeV, and existence of low luminosity bursts with low, 1 keV to 10 keV, break energies.
Introduction
The widely accepted interpretation of the phenomenology of γ-ray bursts (GRBs) is that the observable effects are due to the dissipation of the kinetic energy of a relativistically expanding wind, a "fireball" (see Mészáros 1995 andPiran 1996 for reviews). The discovery of GRB afterglow emission in X-ray (Costa et al. 1997), optical (van Paradijs et al. 1997) and radio wavelengths (Frail et al. 1997) confirmed (Waxman 1997, Wijers, Rees & Mészáros 1997, standard model predictions of afterglow (Paczyński & Rhoads 1993, Kats 1994, Vietri 1997) that results from the collision of the expanding fireball with surrounding medium.
The steep low energy spectra has led several authors to consider inverse-Compton emission as the source of γ radiation (e.g. Lazzati, et al 2000). However, steep low energy spectra may also be accounted for within the context of the synchrotron emission hypothesis by the effects of inverse-Compton suppression at low energies (Derishev et al. 2000) and by the contribution to observed γ-ray radiation of photospheric fireball emission (e.g. Mészáros & Rees 2000). As we show here (see discussion in §4), the contribution of latter effect is expected to be significant.
The main goal of the present paper is to determine the constraints imposed by GRB observations on the relativistic fireball wind parameters, and hence on the underlying source producing the wind, under the assumption that the observed radiation is due to synchrotron emission produced in internal wind shocks. In particular, we address the questions of whether, and under what conditions, high radiative efficiency can be obtained, and whether the clustering of peak emission energies can be naturally explained by the model.
It has been demonstrated in earlier work (e.g. Kobayashi et al. 1997;Beloborodov 2000), that high radiative efficiency may be obtained in the internal shock model provided the variance of the wind Lorentz factors distribution is large. Kobayashi et al. (1997) found ∼ 10% efficiency for uniform Lorentz factor distribution with maximum to minimum Lorentz factor ratio ≈ 10, and Beloborodov (2000) demonstrated that the efficiency may approach 100% for non-uniform distributions with wide range of Lorentz factors 3 . In the analysis presented here we consider several issues which have not been addressed in earlier work: The constraints imposed on the model by the observed peak emission energy distribution, the effect of optical depth due to e ± pairs produced within the fireball wind, and the upper limit imposed on the maximum wind Lorentz factor by the acceleration process. The analysis presented here is also more general, as we study the dependence of radiative efficiency on a wider set of models parameters. We find that the radiative efficiency is limited to values well below the 80%-90% derived by Beloborodov. The inclusion of e ± pair optical depth effects is essential in determining both the efficiency and the peak emission energy.
High radiative efficiency clearly requires that a large fraction of the internal energy generated by internal shocks be carried by shock accelerated electrons, i.e. that the electron and proton energy densities be close to equipartition. Moreover, high radiative efficiency requires the magnetic field energy density to be not far below equipartition as well (e.g. Derishev et al. 2000). Thus, we assume in most our calculations that electrons, protons, and magnetic field energy densities are close to equipartition, and focus on the study of the dependence of observed γ-ray flux and spectrum on wind luminosity and on wind dynamical parameters (source size, variability time, Lorenz factor and mass distribution of shells within the wind). For completeness, we demonstarte using our calculations (in §3.2) that near equipartition is required in order for model predictions to be consistent with the observed peak emission energy distribution.
The model outline is presented in §2. The results of a numerical analysis of the model are presented and discussed in §3. The main conclusions are summarized in §4.
Outline of the model
In the fireball model of GRBs, a compact source, of linear scale R 0 ∼ 10 6 cm, produces a wind characterized by an average luminosity L w ∼ 10 52 erg s −1 and mass loss rateṀ = L w /ηc 2 (R 0 ∼ 10 6 cm corresponds to three times the Schwarzschild radius of a non-rotating, solar mass black hole, and L w ∼ 10 52 erg s −1 is the characteristic luminosity inferred from observations). At small radius, the wind bulk Lorentz factor, Γ, grows linearly with radius, until most of the wind energy is converted to kinetic energy and Γ saturates at Γ ∼ η ∼ 300. η ∼ > 300 is required to reduce the wind pair-production optical depth for observed high energy, > 100 MeV, photons to less than unity. If η > η * ≈ (σ T L w /4πm p c 3 R 0 ) 1/4 = 2 × 10 3 (L w,52 /R 0,6 ) 1/4 , where L w = 10 52 L w,52 erg s −1 and R 0 = 10 6 R 0,6 cm, the wind becomes optically thin at Γ ≈ η * < η, and hence acceleration saturates at Γ ≈ η * and the remaining wind internal energy escapes as thermal radiation at ∼ 1 MeV temperature. Variability of the source on time scale t v , resulting in fluctuations in the wind bulk Lorentz factor Γ on similar time scale, then leads to internal shocks in the expanding fireball at a radius where Γ = 10 x Γ x , t v = 10 −3 t v,−3 s. If the Lorentz factor variability within the wind is significant, internal shocks reconvert a substantial part of the kinetic energy to internal energy. It is assumed that this energy is then radiated as γ-rays by synchrotron (and inverse-Compton) emission of shock-accelerated electrons.
In this work, we use an approximate model of the unsteady wind described in the preceding paragraph [following Kobayashi et al. 1997;Panaitescu, Spada, & Mészáros 1999;Spada, Panaitescu & Mészáros 2000 (SPM00)]. The wind evolution is followed starting at radii larger than the saturation radius, i.e. after the shells have already reached their final Lorenz factor following the acceleration phase, and the GRB photon flux and spectrum resulting from a series of internal shocks that occur within the wind at larger radii are calculated. We consider both synchrotron and the inverse-Compton emission, and take into account the effect of e ± pair production.
We approximate the wind flow as a set of discrete shells. Each shell is characterized by four parameters: ejection time t j , where the subscript j denotes the j-th shell, Lorentz factor Γ j , mass M j , and width ∆ j . Since the wind duration, t w ∼ 10 s, is much larger than the dynamical time of the source, t d ∼ R 0 /c, variability of the wind on a wide range of time scales, t d < t v < t w , is possible. For simplicity, we consider a case where the wind variability is characterized by a single time scale t w > t v > t d , in addition to the dynamical time scale of the source t d and to the wind duration t w . Thus, we consider shells of initial thickness ∆ j = ct d = R 0 , ejected from the source at an average rate t −1 v . The shell thickness may increase with time, due to variations in the expansion Lorenz factor across the shell. The Lorenz factor distribution across the shell depends on the entropy distribution, which is determined by the details of the ejection from the source. We therefore consider two extreme cases: uniform Lorenz factor across the shell, in which case the shell thickness is time independent, and order unity variation of LF across the shell, ∆Γ/Γ ∼ 1, in which case the shell thickness is given by ∆ j = max(R 0 , R/Γ 2 j ).
We assume that the Lorenz factor (LF) of a given shell is independent of those of preceding shells, and consider 4 different LF distributions: uniform, modulated, bimodal, and lognormal. In the uniform distribution case, the shells' LFs are randomly drawn from a uniform distribution over the range Γ m to Γ M , where Γ m < Γ M are time independent. In the modulated case, Γ M varies as sin 2 (2πt j /t w ). In the bimodal case, the LFs are drawn from a bimodal distribution, Γ j = Γ m or Γ j = Γ M with equal probability. Finally in the lognormal case, shell LFs are drawn from a lognormal distribution with an average Γ = 1000 and Γ 2 1/2 / Γ = 4, truncated at Γ > η * .
The time intervals t j+1 − t j are drawn randomly from a uniform distribution with an average value of t v . The shell masses M j are chosen to be either independent of j, or inversely proportional to Γ j . That is, we consider the two qualitatively different possibilities: equal shell mass and equal shell energy. The total mass is determined by the constraint N j=1 M j Γ j c 2 = L w t w , where Once shell parameters are determined, we calculate the radii where collisions occur and determine the emission from each collision. We assume that following each collision the two colliding shells merge and continue to expand as a single shell. Under this assumption, the internal energy carried by the merged shell following the collision is where is the expansion LF of the merged shell and M 1,2 , Γ 1,2 are the masses and the LFs of the colliding shells. The dynamical efficiency ǫ d , defined as the fraction of total kinetic energy converted to internal energy, increases with the ratio of the fast and slow shell LFs, and, for a given ratio, is maximized for equal masses. The first collisions, for which the differences in the shell LFs are largest, are the most efficient.
In each collision a forward (FS) and a reverse (RS) shock are formed, propagating into forward and backward shells respectively. The plasma parameters behind each shock are determined by the Taub adiabat, requiring continuous energy density and velocity across the contact discontinuity separating the two shells (Panaitescu & Mészáros 1999). Since the energy density in the downstream regions of both shocks is similar, we divide the internal energy between the reverse and forward shock according to the ratio E f /E r = ∆ f /k∆ r , where ∆ r,f are the shell thicknesses prior to the collision and k is a factor of the order of few that takes into account the compression ratios of the two colliding shells.
The energy released in each shock is distributed among electrons, magnetic field and protons with fractions ǫ e , ǫ B and 1 − (ǫ e + ǫ B ) respectively. We assume that electrons are accelerated by the shocks to a a power-law distribution, dn e /dγ e ∝ γ −p e for particle Lorentz factors γ min < γ e < γ max . γ max is the electron Lorenz factor at which the synchrotron cooling time equals the acceleration time, estimated as the electron Larmor radius divided by c, while γ min is determined by the requirement that the energy carried by electrons be equal to ǫ e E in .
The GRB energy spectrum and flux is derived by summing the contributions of individual shell collisions. For each collision, synchrotron and inverse-Compton emission by the shock-accelerated electrons is calculated. In order to achieve high radiative efficiency, electrons must radiatively loose most of their energy on a time scale shorter than shock shell crossing time. Under this condition, the synchrotron spectrum may be approximated as where ν sy is the characteristic synchrotron emission frequency of electrons with γ ≃ γ min . The inverse Compton spectrum has a similar shape but is shifted to higher energy as described in SPM00. In determining the inverse-Compton flux, we take into account the Klein-Nishina suppression of the inverse-Compton cross section.
The photons radiated can be scattered by the electrons within the shell. The Thomson optical depth τ T for such scattering is evaluated by taking into account the electrons that were accelerated but have cooled radiatively while the shock crossed the shell, and those within the yet un-shocked part of the shell. The optical depth to Thomson scattering may be increased significantly beyond the value derived taking into account cooled shell electrons, due to the production of e ± pairs. This contribution has not been taken into account in previous analysis of the internal shock model, since for a uniform distribution of Lorentz factors, and under the hypothesis of equipartition, the break energy, hν sy , in the shell co-moving frame is well below the pair production threshold. However, since the photon spectrum extends to high energy as a power law with spectral index −p/2 ≈ −1, there is an equal number of photons per logarithmic energy intervals, and there may exist a large number of photons beyond the pair production threshold. The pairs produced by these photons may contribute significantly to the Thomson optical depth.
In order to take the effect of pair production into account, we determine for each collision the photon energy ǫ pairs for which the pair production optical depth τ γγ equals unity. All the photons that have energies higher than max(m e c 2 , ǫ pairs ) (measured in the shell frame) are assumed to form pairs, leading to a suppression of the high energy photon tail. We assume that the energy of photons that undergo pair production is converted to sub-relativistic pairs, which are taken into account in determining the Thomson optical depth.
We assume that a fraction (1 − e −τ r,f )/τ r,f , where τ r,f are the optical depths of the RS and FS, of the photons produced by each shock escapes and reaches the observer (The RS flux is also absorbed by the forward shell, and is thus further reduced by a factor e −τ f ). The energy of the absorbed photons, as well as the internal energy which has not been converted to radiation, are converted back to ordered kinetic energy by adiabatic losses during the shell expansion. During this process, we take into account the increase in shell thickness by assuming this thickness grows as the shell's (comoving frame) speed of sound.
Our assumption, that following a collision two shells merger into one shell which expands with a single (uniform) Lorenz factor, leads to an overestimate of the reduction due to collisions of shell Lorenz factor variance. Clearly, the expansion of the shocked shells due to the pressure produced by the shocks results in the leading edge of the two shells moving faster (in the observer frame) than the trailing edge. It is therefore clear that the variance in Lorenz factor distribution following the collision is larger than obtained under our assumption of a "complete" merger. This, in turn, implies that a more detailed calculation of the shell merger process, which may require dividing the merged shells into several distinct shells following the collision, will enhance the overall efficiency of kinetic to thermal energy conversion. The magnitude of such enhancement depends on the density and Lorenz factor distributions within the shells prior to the collision, and is expected to be of order unity. We therefore do not consider this effect in the approximate analysis presented in this paper.
Results and discussion
In this section we determine the dependence of the wind radiative efficiency ǫ γ , defined as the fraction of wind energy converted to radiation, and peak emission energy ε p , the photon energy at which the maximum of νf ν is obtained, on wind model parameters. As explained in the introduction, we adopt electron and magnetic field energy fractions close to equipartition and analyze the dependence of ǫ γ and ε p on wind luminosity, variability time, source size, and on the distribution of wind Lorentz factors and shell masses. We use ǫ e =0.45, ǫ B = 0.1, and p = 2 throughout the paper, except in the calculations presented in Fig. 4, where smaller ǫ e and ǫ B values are assumed in order to demonstarte that near equipartitrion is required by observations. We first discuss in §3.1 the various Lorenz factor distributions described in §2. We then present a detailed discussion of the bimodal case, which best reproduces observed GRB characteristics, in §3.2. Fig. 1 shows the results of numerical simulations of the model described in the previous section. Each panel shows ǫ γ versus ε p for one of the LF distributions described in §2. Different points within each panel correspond to different choices of Γ m , Γ M and t v . Results are shown for 10 < Γ m < Γ M < 2500 and 10 −4 s < t v < 1 s. We have assumed equal mass shells, R 0 = 10 6 cm, L w = 10 51 erg s −1 and source redshift z = 1 for all calculations. Results are presented for the two extreme assumptions on shell width evolution with radius described in §2, i.e. for both constant width ∆ = R 0 and maximal expansion, ∆ = max(R 0 , R/Γ 2 ).
Comparison of various Lorenz factor distributions
Based on Fig.1, a uniform LF distribution can be ruled out since the radiative efficiency is small, ǫ γ < 5%. The low radiative efficiency in this case is due to low dynamical efficiency, i.e. to the fact that only a small fraction of the kinetic energy is converted to internal energy in collisions. In order to increase the dynamical efficiency it is necessary to either enhance the variance of the LF distribution of colliding shells at the first set of collisions (as is the case for bimodal or lognormal distributions) or to keep the variance moderate over a wind range of collision radii (as is the case for the modulated case).
In the modulated case the maximum dynamical efficiency is 40%. The first collisions remove the initial random differences and the merged shells have LFs near the ejection average value Γ = (Γ m + Γ M )/2. If the wind is homogeneous Γ is similar for all shells, resulting in a steady decrease of ǫ d during the wind expansion. If the range of LFs is varying on time scale of order of t w , Γ reflects the initial modulation of Γ M and larger radii collisions that are dynamically efficient are still possible. Thus, a modulation in the wind can solve the efficiency problem (ǫ γ ≈ 10%). However, the peak emission energy in this case is well below the BATSE range, since most of the emission originates at large radii, much larger then the radius of the photosphere R ± , the radius at which the wind becomes optically thin to Thomson scattering by e ± pairs [see the discussion preceding Eq. (5) in §3.2].
The dynamical efficiency in the case of a bimodal LF distribution can reach 50%, since the Lorentz factors of the colliding shells are very different. Contrary to the case of modulated ejection, the collisions at the photospheric radius R ± dominate wind emission, and a region of the parameter space exists, where model radiative efficiency and peak emission energy are consistent with observations, ǫ γ > 10% and ε p ∼ 100 keV.
The case of a lognormal LF distribution can be considered as an intermediate case between the random and bimodal ones. In this case the maximal radiative efficiency expected is of the order 10-15%, much lower than the 80-90% value derived in Beloborodov (2000). This is due to the pair optical depth, which sets a lower limit of Γ m ∼ > 100 for an optically thin wind (see §3.2 below), and to the constraint imposed on the maximum LF by finite source size, Γ M < η * ∼ 10 3.5 . These constraints limit the variance of the LF distribution, and hence the dynamical efficiency.
Considering shells of equal energy, rather than of equal mass, the main results do not change. Uniform LF distribution is still characterized by low efficiency and modulation leads to low peak emission energy. In the case of a bimodal distribution, the efficiency is lower for shells of equal energies than for shells of equal mass, due to an increase in wind optical depth. The LF of the merged shell, given by Eq.(3), is reduced in the equal energy case by a factor Γ M /Γ m with respect to the equal mass case. Thus, the average wind LF is lower, implying smaller collision radii and higher opacity. ¿From the analysis of Fig. 1 we conclude that a bimodal distribution of shell LFs, that is, LF distribution with large variance, and equal shell mass are favored by observations. Note, that this conclusion is independent of the assumption of near equipartition, using which the above calculations were performed, since for electron and magnetic field energy fractions below equipartition the peak emission energy ǫ p would be lower than obtained above [see also §3.2, Fig. 4 and Eq. (9), below]. In what follows we study the bimodal LF case in more detail, in order to examine the dependence of observable characteristics on wind parameters.
Bimodal LF distribution
Figures 2 and 3 present the dependence on Γ m and on t v of peak emission energy, ε p , and radiative efficiency, ǫ γ , respectively, for Γ M = 2500, L w = 10 52 erg s −1 , R 0 = 10 6 cm and equal shell masses. Results are presented in Fig. 2 for the two extreme assumptions on shell width evolution with radius, ∆ = R 0 and ∆ = max(R 0 , R/Γ 2 ). The efficiency contour plot, shown in Fig. 3, is similar for both cases.
The dependence of ε p on parameters can be understood considering the dependence of the dynamical efficiency and of the optical depth on Γ m and t v . For high values of Γ m (> 300), both reverse and forward shocks are optically thin. Decreasing Γ m leads to a decrease in the initial radius of the collisions, increasing the efficiency up to 15% and the peak emission energy up to 0.1-1 MeV. At lower values of Γ m , the efficiency decreases and steep breaks are observed in the ε p contour plot. These breaks can be understood considering the difference between the FS and RS efficiencies, due to different comoving densities of the two colliding shells (E f /E r ∼ ρ c,r /ρ c,f ). In the case of maximal shell expansion, ∆ = R/Γ 2 , the ratio E f /E r ∼ Γ r /Γ f , and the FS dominates the emission. Thus, the break in the ǫ p contour plot (left panel of Fig. 2) corresponds to the region of Γ m and t v values where the FS, and consequently also the RS, become optically thick due to pair production (Γ m = 50 to Γ m = 130 for t v varying between 1 s and 10 −4 s respectively). A small decrease in Γ m results in collisions below the photosphere, leading to a steep decrease in peak emission energy, to a value corresponding to the second set of collisions, which occur at larger radii. These collisions dominate the emission and are characterized by a lower efficiency and a lower peak emission energy. In the case of a constant shell width ∆ = R 0 , the ratio E f /E r ∼ Γ f /Γ r , and the RS dominates the emission. Thus, there are two breaks in the peak energy contour plot (right panel of fig 2). The first corresponds to the values of Γ m and t v where the RS becomes optically thick due to pair production and the FS starts to dominate the emission. This break represents the shift of the peak energy from the RS value to the FS one. The other occurs in the parameter region where the FS becomes optically thick, and corresponds to the shift of the peak energy from the first to the second set of collisions.
The contour plot of the radiative efficiency in Fig. 3 shows that values of ǫ γ higher than 10% and peak emission energy between 0.1 and 1MeV are reached at similar regions in the Γ m -t v plane, in accordance with the correlation between the peak emission energy and radiative efficiency shown in Fig. 1. High efficiency, > 10%, is obtained only for large Γ M , Γ M ≈ η * ≈ 2 × 10 3 , i.e. for values close to the maximum allowed by the finite source size. For lower values of Γ M , the dynamical efficiency decreases leading to lower ε p and ǫ γ .
In Fig. 4 we present results of calculations similar to those presented in Fig. 2, for ǫ e and ǫ B values below equipartition. The results demonstarte that the electron and magnetic field energy fractions can not be far below equipartition, ǫ e ∼ > 10 −1 and ǫ B ∼ > 10 −2 are required in order to obtain peak emission energies consistent with observations. These results are consisitent with our analytic estimates, Eqs. (8) and (9) below.
The contour plot shown in Fig. 2 demonstrates that values of ε p larger than ∼ 1 MeV can not be obtained. This result can be understood using the following arguments. For a collision of shells of thickness ∆ at radius R, we have -10 - The maximal value of ε p is obtained for collisions at the smallest radius R for which the wind is optically thin. The radius of the Thomson photosphere due to the electrons present in the original fireball is given by where M is the shell mass, and we have approximated M = L w t v /c 2 (Γ m + Γ M ). R ± , the Thomson photosphere radius due to e ± pairs resulting from pair production interaction of synchrotron photons, can be estimated by assuming that a significant fraction, ∼ 1/2, of the radiative energy is converted to pairs, as typically is the case. The number density of e ± is given in this case by n ± ≈ ǫ e E in /(8πm e c 2 R 2 Γ 2 ∆) and Comparing R ± and R T , we conclude that the optical depth is typically dominated by the pairs.
For fixed t v and Γ M , the maximal peak energy is obtained for Γ m = Γ m± for which the collision radius R i ≈ Γ 2 m ct v equals the photospheric radius R ± . Solving for Γ m± , and substituting in Eq. (5) we find where we have used γ min = (Γ M /Γ m )ǫ e m p /m e log(γ max /γ min ) with γ max = 100 γ min . The dependence of ε p on wind luminosity is demonstrated in figures 5 and 6. Under the assumption of fixed shell width, ∆ = R 0 , ε max p is weakly dependent on L, as indicated by Eq. (8). If the maximum Lorenz factor scales as Γ M ≈ η * ∝ L 1/4 , rather than being independent of L, the slow decrease with L of ε max p at fixed t v , ε max p ∝ L −1/6 , is replaced by a slow increase with L ε max The dependence on luminosity becomes stronger under the assumption of maximal expansion. Substituting ∆ = R/Γ 2 in Eq. (8), we find ε max p ∝ L −5/18 . The stronger dependence is also apparent in figures 5 and 6. However, allowing for a scaling Γ M ≈ η * ∝ L 1/4 , the peak emission energy dependence on L becomes ε max p ∝ L 7/36 , similar to the dependence shown in Eq. (9).
Conclusions
We have analyzed a model of GRBs, in which a compact source of linear scale R 0 produces a wind characterized by an average luminosity L w and mass loss rateṀ = L w /ηc 2 . At small radius, the wind bulk Lorentz factor, Γ, grows linearly with radius, until most of the wind energy is converted to kinetic energy and Γ saturates at Γ ∼ η. Variability of the source results in fluctuations in the wind saturation Lorentz factor Γ, leading to internal shocks in the expanding wind. These shocks reconvert a fraction of the kinetic energy back to internal energy, which is assumed to be radiated as γ-rays by synchrotron (and inverse-Compton) emission of shock-accelerated electrons. Since the wind duration, t w ∼ 10 s, is much larger than the dynamical time of the source, t d ∼ R 0 /c, variability of the wind on a wide range of time scales, t d < t v < t w , is possible. For simplicity, we have assumed that in addition to t d , which determines the initial shell thickness ∆ = ct d ∼ R 0 , and t w , the wind is characterized by a single time scale t v > t d , which determines the shell ejection rate t −1 v . We have addressed the questions of whether, and under what conditions, high radiative efficiency consistent with observations can be obtained, and whether the observed clustering of peak emission energies can be naturally explained by the model.
We have shown that a significant fraction, ∼ 15%, of the fireball energy can be converted to radiation. As pointed out at the end of §2, our simplified treatment of post-collision shell evolution, assuming complete merger of shells, leads to an overestimate of the reduction of the variance of colliding shells' Lorenz factors with wind evolution. A more detailed calculation of the merger process will lead to an enhancement of the radiative efficiency by a factor of order unity. The exact value of the enhancement factor will depend, however, on the unknown internal shell structure. Adopting simplifying assumptions regarding the shell structure and post-collision evolution, Kobayashi & Sari (2001) have recently pointed out that including post-collsion evolution effects may lead to radiative efficiency exceeding ǫ e (note, that this may be the case also when post-collision evolution is neglected). Although this effect may relax somewhat the constraint imposed on ǫ e by the requirement of high efficiency, we have shown here (see following paragraph), that ǫ e is constrained to be of order unity by the observed γ-ray spcetra.
In order to obtain high radiative efficiency and peak emission energy ∼ 1 MeV, the minimum radius R i at which internal collisions in the expanding wind occur is required to be similar to R ± , the radius where the Thomson optical depth due to e ± pairs produced by shock synchrotron emission equals unity [see Eq. (7)], and a large variance (compared to the mean) of the colliding shells' Lorenz factor (LF) distribution is required. In addition, the electron and magnetic field energy fractions should be close to equipartition, with ǫ e ∼ > 0.1 and ǫ B ∼ > 0.01, in order for model predictions to be consistent with the observed peak emission energy distribution [see Fig. 4 and Eq.(9)]. The constraint R i ∼ R ± is equivalent to a constraint on the minimum LF, Γ m , of expanding shells, Γ m ∼ Γ m± ≈ 10 2.5 (L w,52 /t v,−3 ) 2/9 , where L w = 10 52 L w,52 erg s −1 and t v = 10 −3 t v,−3 s [see Eq. (7)]. Large variance in the LF distribution of colliding shells than requires a non-uniform LF distribution (e.g. truncated log-normal or bimodal distributions, see Fig. 1) with Γ M , the maximum LF of wind shells, close to the upper limit set by the shell acceleration process, Γ < η * ≈ (σ T L w /4πm p c 3 R 0 ) 1/4 = 2 × 10 3 (L w,52 /R 0,6 ) 1/4 , where R 0 = 10 6 R 0,6 cm.
Our results do not agree with the very high, ∼ 80%, radiative efficiency values, obtained in the calculations of Beloborodov (2000) and Kobayashi & Sari (2001) by assuming wind Lorentz factor distributions extending from Γ m ∼ 10 to Γ M ∼ > 10 4 . In our opinion the minimum wind Lorentz factor must be significantly higher than 10 (as otherwise the wind becomes optically thick) while the maximum Lorentz factor can not significantly exceed 10 3 (due to the acceleration process limitations).
We have shown that there is an upper limit to the observed energy of photons, ε p , at which the γ-ray flux peaks, ε p ∼ < ε max p ≈ 0.5R −5/6 0,7 MeV (where R 0 = 10 7 R 0,7 cm), with very weak dependence on L w and on t v [see discussion preceding Eq. (9), and figures 2, 5 and 6]. Thus, the source dynamical time t d ∼ R 0 /c must satisfy t d ∼ < 1 ms in order to allow ε p ∼ 1 MeV. ε p ≈ ε max p is obtained for Γ m ≈ Γ m± , for which the radiative efficiency is largest, and ε p values in the range of 0.1 MeV to 1 MeV are obtained for wind parameters for which the radiative efficiency is high, ∼ > 10% (see figure 3).
High radiative efficiency and peak emission energy consistent with observations are therefore obtained for Γ m ∼ Γ m± . This does not necessarily imply that fine tuning of this model parameter is required. For Γ m < Γ m± , most efficient collisions occur at radii where the optical depth is high, leading to low efficiency, and hence low luminosity, bursts with peak emission energy ∼ 1 keV (see figures 2 and 3), which would not have been detected by BATSE. For Γ m significantly higher than Γ m± , LF variance is small, leading to low efficiency, low luminosity bursts with peak emission energy ∼ 10 keV, which may have been difficult to detect with BATSE. Natural consequences of the model considered here are therefore absence of bursts with peak emission energy significantly exceeding ∼ 1 MeV, and existence of low luminosity bursts with low, ∼ 1 keV to ∼ 10 keV, peak emission energy. The frequency of such bursts depends on the distribution of Γ m in different winds.
It should be pointed out in this context that while the deficit, among bursts detected by BATSE, of bursts with peak emission energy below ∼ 50 keV clearly reflects a real deficit of such bursts, the deficit in detected bursts with peak emission energy > 0.5 MeV may be partly due to a selection effect (e.g. Llyod & Petrosian 1999). BATSE data are consistent with equal number of bursts per logarithimic peak energy interval beyond ε p ∼ 0.5 MeV (with luminosity comparable to that of lower peak energy bursts). Our model prediction, that bursts with ε p ≫ 1 MeV should be absent, should therefore be tested with future GRB detectors, which are able to better constrain the high energy end of the ε p distribution.
A note should be made here regarding the low energy spectral slope. The upper limit η * on Γ M is a consequence of the fact that for η > η * shell acceleration saturates at Γ ∼ η * < η, as the wind becomes optically thin to Thomson scattering, and the internal energy left in the shell escapes as thermal radiation rather than being converted to kinetic energy. The requirement Γ ∼ η * therefore implies that a significant fraction of the wind energy may escape as thermal radiation, leading to low energy spectral slopes steeper than those expected for pure synchrotron emission. This may account, at list partially, for observed steep low energy spectra.
Finally we note that the lower limit imposed on Γ m , Γ m ∼ > Γ ± , is not derived from the requirement that the pair production optical depth for high energy, > 100 MeV, photons be smaller than unity. While this requirement leads to a similar constraint, Γ m ∼ > 10 2 , high energy photons have been detected in a small number of cases only. The constraint Γ m > Γ m± is imposed in the present analysis by the requirement that the wind Thomson optical depth due to e ± pairs produced by synchrotron photons be smaller than unity at the internal shocks stage. Results are shown for 10 < Γ m < Γ M < 2500 and 10 −4 s < t v < 1 s, for two extreme assumptions on shell width expansion, maximal expansion ∆ = max(R 0 , R/Γ 2 ) and constant width ∆ = R 0 . We have assumed equal mass shells, R 0 = 10 6 cm, L w = 10 51 erg s −1 and source redshift z = 1 for all calculations. | 2014-10-01T00:00:00.000Z | 2000-01-01T00:00:00.000 | {
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258781387 | pes2o/s2orc | v3-fos-license | Fine-tuning mesoporous silica properties by a dual-template ratio as TiO2 support for dye photodegradation booster
Titanium dioxide (TiO2) has been integrated into the surface of mesoporous silica (SMG) synthesized via the hydrothermal approach and a dual template CTAB-Gelatin. XRD, nitrogen adsorption, FTIR, SEM-EDX, and UV–Vis DR spectroscopy were performed to evaluate a 1 wt% TiO2/SMG material. After titania incorporation, the addition of gelatin during the synthesis of SMG increases the pore volume to 0.76 cc/g. The expansion of the silica pores is caused by the development of TiO2 crystal grains on the mesoporous silica-gelatin. An increase in the gelatin-CTAB to mesoporous silica weight ratio modifies the surface area, pore size, and particle size without compromising the meso-structure. In this research, the TiO2/SMG composite demonstrated much greater photodegradability for methylene blue (MB) than the TiO2/mesoporous silica sample without gelatin. The experimental results indicate that the photocatalytic activity of methylene blue from SMG titania/silica samples is reliant on the adsorption ability of the composite and the photocatalytic activity of titania, with optimal activity from samples with the highest surface area and pore volume, which directly increase the Ti: Si ratio and decrease the photodegradability of the composite when the ratio is too high or too low.
Introduction
The presence of waste containing methylene blue is one of the most significant concerns in the world's oceans today [1]. Diverse efforts have been made to eliminate this waste because its adverse impacts on the health and growth of aquatic biota jeopardize the future availability of clean water [2]. Numerous attempts were undertaken, including adsorption techniques and photocatalysts. Photocatalyst is regarded as a promising clean approach for reducing methyl blue waste without accumulating residual compounds [2,3]. TiO 2 is a commonly used metal in photocatalysts. However, when applied, pure TiO 2 readily agglomerates, lowering the photodegradation efficiency by up to 80% [4]. Therefore, the good support material is required to ensure that TiO 2 is properly distributed and effective for photodegradation [5]. Mesoporous silica is among the most preferred support materials [6][7][8].
Mesoporous silica has drawn the interest of researchers in recent years due to its multiple features, including inertness, controllability, large pore size, high surface area, high pore volume, and uniform morphology [9][10][11][12]. All of these benefits of mesoporous silica are advantageous for numerous applications in adsorption, photocatalysis, separation, and energy storage [9,13,14]. Considering the high cost of hard templates and the complexity of the synthesis procedure, several researchers have selected the soft template technique, which utilizes synthetic surfactants as the major structure-directing agent in the synthesis of mesoporous silica [15][16][17]. The extensive use of synthetic surfactants such as P123, P127, CTAB, TMOH, and CTAC. Recently, CTAB was widely used in the synthesis process due to their self-assembly method into cylindrical-shaped, positively charged micellar then reacts with the negatively charged silica source, exchanging, releasing the counter anions of the surfactant and permits the modulation of silica structure [18]. Haynes et al. [19], proposed the Pluronic F127/P123 and CTAB as neutral and ionic soft templates to synthesize mesoporous silica. As a cosurfactant in the P123 system, CTAB directed the creation of large-pore mesoporous silica microspheres with tunable pore diameters. Moreover, the introduction of CTAB into the SBA-15 gel solution may result in the production of secondary micelles within the primary SBA-15 framework [19]. Another study, Nguyen et al. [20] also used CTAB as positively charged surfactant with various non-ionic surfactants i.e., Tween 20, Tween 80 and Brij S10 as co-template in the preparation of mesoporous silica nanoparticles (HMSN).
The utilization of synthetic surfactant: silica source ratio of approximately 1:2 (w/w) has an impact on the high costs required for large-scale silica synthesis, despite the fact that these surfactants or soft templates are removed through decomposition at the end of the stage [17,[21][22][23][24]. The usage of synthetic templates in large quantities is expensive, limiting the long-term production of large-scale mesoporous silica. Consequently, numerous attempts have been made to resolve this issue, including the natural template approach. Natural templates such as starch, gelatin, natural rubber, and gum Arabic are notable advances in the effort to limit the use of synthetic templates [25][26][27][28][29][30][31][32][33]. Gelatin has been discovered to be an effective structure-directing agent when combined with synthetic templates in a dual template strategy. Numpilai et al. [34] uses gelatin with ratio to silica source of 0-1.8 in the hydrothermal process to produce hollow porous silica spheres with a controlled shell thickness of 80-160 nm. The recent studies revealed that P123 and F127, when combined with gelatin, generated regular mesoporous silica and carbon with good application performance [12,35,36] with regular hexagonal pores of 104-320 Å and high surface area of 69-104 m 2 . However, no specific investigation has been conducted on the impacts of CTAB-gelatin dual template on mesoporous silica formation. Therefore, this study will employ a soft template of CTAB with gelatin to synthesize large pore and adjustable pore diameters of mesoporous silica subsequent TiO 2 doping for methylene blue photodegradation.
Material
The materials used in this research are HCl (37%, Sigma-Aldrich Merck KGaA, Mr 36.
Synthesis mesoporous silica doped with titania
The limitation of this study is the mesoporous silica synthesis as support material from dual templated CTAB-gelatin in a specific ratio (Table 1) in 480 ml of 0.015 M NaOH at 80 • C with stirring. The resultant mixture was added to 5 ml of TEOS placed in an autoclave at 80 • C for hydrothermal reaction for 24 h. To remove residual surfactant, the product was filtered, washed with distilled water, dried at 70 • C for 24 h, and then calcined at 550 • C for 5 h at a rate of 2 • C/min. SMG x -H is the outcome obtained, where x is the gelatin to CTAB ratio and H is the hydrothermal procedure. 0.024 ml of titanium isopropoxide dissolved in 20 ml of n-hexane was added to 0.99 g of SMG x -H while stirring at 45 • C for 16 h, followed by drying at 160 • C for 2 h. The mixture was then dissolved in hexane, followed by 45 min of drying at 80 • C and 5 h of calcination at 550 • C to produce TiO 2 /SMG-x-H.
The instruments used to analyze the samples included the X-Ray Diffraction (Pananalytical XRD, PW3050/60, copper anode, and 0.02 • /s scanning rate), Fourier Transform Infrared (Shimadzu FTIR spectrophotometer) with a resolution of 0.5 cm − 1 , Scanning Electron Microscopy-Energy Dispersive X-Ray (SEM-EDX, JEOL JSM-700 microscope) at a voltage speed of 15.0 kV, and Brunauer-Emmett-Teller (BET, Quantachrome Nova 1200e. The crystal size of mesoporous silica nanoparticles with the Debye Scherrer formula is as in Eq. (1) below: 0.9 λ B cos θ Eq. 1 D is the crystal size in Ǻ, λ is the wavelength used in the XRD test, which is 1.54056 Å, and B is the width of half the peak in radians. θ is the angular position of the peak formation. XRD results also show FWHM to be able to determine the value of B (rad) and cyrstallinity (Eq. (2)).
Photodegradation of methylene blue
TiO 2 /SMG x -H photocatalyst with mass of 50 mg was added to 10 ml of 5 ppm methylene blue, that had followed by a closed shaker procedure (dark adsorption) for 30 min in triplicate and then UV irradiation. Using a UV-Vis spectrophotometer with a wavelength range of 670 nm, absorbance readings were taken every 10 min. Analysis of the percent degradation of methylene blue using a UV-Vis spectrophotometer by observing the absorbance curve and determining its value using the %ED formula (Eq. 3). The kinetic study also is carried out by using first order (Eq. (4)) and second order (Eq. (5)) reaction.
Degradation efficiency of MB
Where C o = initial concentration of MB dan C t = concentration of MB at t minutes.
Result and discussion
The XRD profiles of each sample at 2θ = 10-70 • are shown in Fig. 1. The diffraction peaks centered at 2θ of 25.3 • correspond to (002) planes of SiO 2 and the hump is confirming the amorphous nature of SiO 2 . The peak is in accordance with the previous literature that amorphous silica has a diffraction peak of 2θ = 21-25 • (JCPDS No. 29-0085) [37]Utilization of gelatin or CTAB as a single template results in less crystalline silica than using dual template gelatin-CTAB. Dual template CTAB-gelatin in silica formation increases crystallinity by approximately 7% and does not significantly alter the crystallite size, as indicated in Table 2. However, due to the limited proportion of Ti in silica, no sample exhibited a significant peak of anatase phase on TiO 2 (JCPDS No. 2101272) [5,38,39]. The presence TiO 2 on the mesoporous silica was confirmed by the FTIR and EDS data.
The FTIR of SMG x -H/TiO 2 with different gelatin concentrations was depicted in Fig. 2. All samples exhibited wide absorption band at wavenumber of 3369-3454 cm − 1 as the hydroxyl (-OH). The peak at 1050, 800, and 949-963 cm − 1 revealed the Si-O-Si symmetric, asymmetric bending vibrations, and Si-OH symmetric vibration, respectively [3]. The samples with high gelatin concentrations have slightly higher intensity of hydroxyl and Si-O-Si absorption peak due to the high involvement of gelatin in the structure formation process. Moreover, the titania presence in the silica mesoporous was evidence by the peak at wavenumber of 1627-1633 cm − 1 as Ti-OH bending [40] and at 803-806 cm − 1 as Ti-O-Ti bond [41]. The wavenumber peak at this research are consistent with those of earlier research [3,6,7,21,[42][43][44]. Not only FTIR (Fig. 2), the presence TiO 2 on the mesoporous silica was confirmed by EDS data ( Table 2). The EDX spectra of all sample demonstrated the presence of TiO 2 and SiO 2 as the minority and majority component, respectively. The presence of TiO 2 is stable at low concentration from 0.25 to 0.51% w/w which show the succesfully the incorporation process. Increasing gelatin content in the samples led to the decreasing Si content due to the replacing Si by C which may be ascribed to the particle decomposition of carbon. By comparing the carbon contents, we found that not only a higher C content corresponded to larger aggregates on the surface but also led to the decreasing surface area which confirmed by nitrogen adsorption desorption analysis ( Fig. 3 and Table 3).
The analysis of N 2 adsorption-desorption (Fig. 3 a-e) depicts that all samples have isotherm type-IV with H-1 hysteresis, in which the hysteresis has a narrow loop. The type-IV isotherm exhibited the adsorption and desorption branches are almost vertical and almost parallel with the inflection position which lies at the relative pressure (P/P o ) in the range of 0.4-0.9 as indicative of mesoporous samples. Based on Table 3 as the summarized of the textural properties, the surface area of TiO 2 -impregnated silica produced from 100% gelatin (SMG 100 -H/TiO 2 ) and 100% CTAB (SMC 100 -H/TiO 2 ) was 577 m 2 /g and 763 m 2 /g, respectively. When dual template gelatin-CTAB technique with 1-20% w/w gelatin was used, the surface area increased as much as 5% compared to SMC 100 -H/TiO 2 and 22% compared to SMG 100 -H/TiO 2 to 799.89% for SMG 10 -H/TiO 2 . The pore volume of TiO 2 impregnated on mesoporous silica as supported synthesized from CTAB-gelatin increased by roughly 12%, 0.67 cc/g to 0.76 cc/g for SMG 10 -H/TiO 2 . The increment of surface area and pore volume was the synergetic effect of CTAB-gelatin which occurs when the polar groups of both molecules engaged strongly with Si in the precursor thus controlled the pore formation. Fig. 4(a-e) displayed the SEM analysis and particle size distribution of mesoporous silica at varying concentrations of gelatin-CTAB as support of TiO 2 . The particle size of silica reduced with increasing gelatin concentration up to 10%, from 0.11 to 0.095 μm, before increasing to 0.12 with the addition of 20% gelatin. The inclusion of gelatin of 1-20% alters the morphology of silica, which, in the absence of gelatin, tends to form dense granule aggregates in the shape of big aggregates. The negatively charged silica causes aggregation of positively charged amine gelatin to produce a gel network that controls the particle formation. The mesoporous silica as support for TiO 2 was used as photocatalyst to degrade methylene blue. According to Fig. 5, the degradation of methylene blue accelerates significantly with increasing UV exposure time at 5-40 min of irradiation and remains steady at 40-90 min. Exposure time affects the methylene blue degradation as the color of the solution fades, the UV light is easier to reach the photocatalyst. When irradiation time extends, the photon energy absorbed by the TiO 2 /mesoporous silica photocatalyst on the surface rises, more methylene blue will be degraded. In addition to photocatalytic, the dark adsorption process was conducted in the dark for 30 min prior to the irradiation process to homogenize the solution and reach equilibrium. Therefore, the methylene blue solution degrades more quickly when irradiated with UV since some of the methylene blue molecules have been trapped on the surface of the mesoporous silica as booster material. Due to adsorption in the dark, the concentration of methylene blue decreased by 0 min of irradiation time. The findings is consent with investigations that employed silica as a supporting material for titania in the photodegradation of methylene blue [45][46][47][48]. The SMG 10 H/TiO 2 would degrade the methylene blue at the highest rate, 95.81%, whereas TiO 2 degraded the least, at a rate of 94.80%. Increasing the concentration of gelatin to 10% improves the degradation of methylene blue, which occurs due to the participation of gelatin-CTAB to generate a structure with a large surface area and an even particle size distribution. The high degradation rate also influenced by highest surface area of 799.89 m 2 /g with a particle size of 0.095 μm of the SMG 10 H/TiO 2 . The degradation efficiency of TiO 2 /mesoporous silica photocatalysts by single template (SMG 100 H/TiO 2 and SMC 100 H/TiO 2 ) is significantly lower than that of TiO 2 /mesoporous silica photocatalysts from dual template. Without the synergetic effect CTAB-gelatin, TiO 2 lacks mesoporous silica as support material to aid in the dispersion of particles, causing agglomeration that limits photodegradation. The correlation between surface area and the degradation efficiency of material was displayed in Fig. 6. The degradation efficiency is also compared to other studies (Table 4). Among graphene oxide and other silica material, the SMG 10 H/TiO 2 demonstrated the high efficiency due to their high surface area and pore volume.
The schematic illustration of CTAB-gelatin role for mesoporous silica formation as support in TiO 2 displayed in Fig. 7. CTAB has a polar group CTA + while gelatin has an NH + group; both have a high affinity for Si because to the electronegativity difference, which exceeds 1.2. When the polar groups of gelatin and CTAB fight to interact with Si, the non-polar groups of gelatin and CTAB repel Si. Due to the carbon chains from the non-polar CTAB and gelatin groups, this causes the space framework of the silica particles to lengthen. Because gelatin with a molecular weight of 400 KDa [60] has a substantially longer chain than CTAB with a molecular weight of [61][62][63], the TiO 2 -impregnated silica generated by including gelatin-CTAB has a greater pore capacity than that synthesized from CTAB or gelatin alone. Gelatin can induce the condensation of silica precursors via hydrogen bonding or electrostatic interactions between -NH 3 + and silanol species, as well as CTA + from CTAB interacting with silanol from Si precursors.
Gelatin also contributes to the expansion of the silica core and the aggregation of silica particles, a chain aggregation process that is crucial to the generation of the morphology of mesoporous materials. The alteration of pore volume and surface area affect the methylene blue photodegradation. The reaction mechanism for the degradation of methylene blue on TiO 2 and TiO 2 /mesoporous silica photocatalysts when exposed to UV light is as follows (Eqs. (4)-(8)) [64]: Eq. 4 [51]. These two substances can migrate to the surface of TiO 2 and TiO 2 /mesoporous silica, where they will undergo redox reactions with organic contaminants, in this case methylene blue. Hole electrons will react with OH − to form hydroxyl radicals (•OH). This radical is an extremely powerful oxidizing agent and the primary oxidizer in the photocatalytic oxidation of methylene blue to carbon dioxide, water, and other mineralization byproducts. Meanwhile, electrons (e − ) will react with methylene blue to form CO 2 and H 2 O as reduction products [35,[65][66][67]. TiO 2 content also influenced the band gap energy of each silica sample. The band gap energy increases as the particle size of semiconductor materials decreases, as predicted by the model theory of the cohesive energy of the confinement effect [68]. The band gap of the samples was estimated by analyzing diffuse reflectance spectra (DRS) in the UV-vis range (Fig. 8). The plots of the band gap energy of silica following titania impregnation and the calculated band gap energy were shown in Fig. 9. The material band energy gap fell as its gelatin content increased, reaching a maximum of 3.23 eV for samples containing 20% gelatin. The values did not vary linearly with gelatin concentration, therefore a smaller crystallite size at 10% gelatin (4.8 nm) represents a decrease in band gap energy. The non-uniform dispersion of TiO 2 particles on the silica surface may account for this inconsistency. Fig. 10 depicts the pseudo zero, first order, and second order kinetics models of methylene blue decomposition. During this investigation, these models were applied to each of the catalyst samples that were analyzed. The coefficients of determination (R 2 ) that were obtained based on the various models utilized to fit the kinetic data are presented in Table 5. Due to the assumption that the ratelimiting step is the chemical sorption of MB as target molecules and that the oxidation occurs via photo-induced electron transfer between the reactants and photoactive particles, the photodegradation of the entire sample is more accurately described by the pseudo first order model with R close to 0.99. Aforementioned assumption that the rate-limiting step is chemical sorption of MB as target molecules [55,69,70].
The measured values of k as the kinetic constant degradation process by pseudo first order model are the best fit for all samples. SMG 10 /TiO 2 -H has the highest degradation rate constant from the decomposition of methylene blue among all catalysts based on kinetic constant and initial rate value. Rate constant is almost 2.7 times higher than pure TiO 2 . The increase in photocatalytic activity for SMG 10 /TiO 2 -H (Table 4) is due to the enhanced surface plasmonic resonance effect of dispersed TiO 2 on the surface of mesoporous silica under visible light as well as an electron storage space for degradation of methylene blue which allows the separation of charge carriers. These result which was in good agreement with the previous photogenerated carrier migration rate of the catalysts research [51,[71][72][73]. Comparative photocatalytic degradation revealed the superiority of TiO 2 compared to catalysts reported elsewhere.
Conclusion
Combining hydrothermal and physical techniques, dual template gelatin-CTAB was used to produce TiO 2 /mesoporous silica as an accelerator material for methylene blue photodegradation using TiO 2 /mesoporous silica. Prior to the inclusion of 20% gelatin, the particle size of silica decreased from 0.11 to 0.095 m with increasing gelatin concentrations up to 10%. In addition to particle size, the gelatin-CTAB ratio had a negligible impact on the sample's surface area, pore volume, and pore diameter. The mesostructured anatase TiO 2 was relatively dispersed in accordance with the 799.89 m 2 /g silica surface area. Titania on mesoporous silica without gelatin addition (SMC) exhibited significantly lower MB photodegradability (88.2%) than titania on mesoporous silica with gelatin addition (SMG) (95.81%).
Author contribution statement
Maria Ulfa: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper. Cindy Nur Anggreani: Performed the experiments; Wrote the paper. Novia Amalia Sholeha: Analyzed and interpreted the data; Wrote the paper.
Data availability statement
Data will be made available on request.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper | 2023-05-19T15:18:56.254Z | 2023-05-01T00:00:00.000 | {
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9596651 | pes2o/s2orc | v3-fos-license | Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8
The cytochrome P450, CYP2C8, metabolizes more than 60 clinically used drugs as well as endogenous substances including retinoic acid and arachidonic acid. However, predictive factors for interindividual variability in the efficacy and toxicity of CYP2C8 drug substrates are essentially lacking. Recently we demonstrated that peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor primarily involved in control of lipid and energy homeostasis directly regulates the transcription of CYP3A4. Here we investigated the potential regulation of CYP2C8 by PPARα. Two linked intronic SNPs in PPARα (rs4253728, rs4823613) previously associated with hepatic CYP3A4 status showed significant association with CYP2C8 protein level in human liver samples (N = 150). Furthermore, siRNA-mediated knock-down of PPARα in HepaRG human hepatocyte cells resulted in up to ∼60 and ∼50% downregulation of CYP2C8 mRNA and activity, while treatment with the PPARα agonist WY14,643 lead to an induction by >150 and >100%, respectively. Using chromatin immunoprecipitation scanning assay we identified a specific upstream gene region that is occupied in vivo by PPARα. Electromobility shift assay demonstrated direct binding of PPARα to a DR-1 motif located at positions –2762/–2775 bp upstream of the CYP2C8 transcription start site. We further validated the functional activity of this element using luciferase reporter gene assays in HuH7 cells. Moreover, based on our previous studies we demonstrated that WNT/β-catenin acts as a functional inhibitor of PPARα-mediated inducibility of CYP2C8 expression. In conclusion, our data suggest direct involvement of PPARα in both constitutive and inducible regulation of CYP2C8 expression in human liver, which is further modulated by WNT/β-catenin pathway. PPARA gene polymorphism could have a modest influence on CYP2C8 phenotype.
The cytochrome P450, CYP2C8, metabolizes more than 60 clinically used drugs as well as endogenous substances including retinoic acid and arachidonic acid. However, predictive factors for interindividual variability in the efficacy and toxicity of CYP2C8 drug substrates are essentially lacking. Recently we demonstrated that peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor primarily involved in control of lipid and energy homeostasis directly regulates the transcription of CYP3A4. Here we investigated the potential regulation of CYP2C8 by PPARα. Two linked intronic SNPs in PPARα (rs4253728, rs4823613) previously associated with hepatic CYP3A4 status showed significant association with CYP2C8 protein level in human liver samples (N = 150). Furthermore, siRNA-mediated knock-down of PPARα in HepaRG human hepatocyte cells resulted in up to ∼60 and ∼50% downregulation of CYP2C8 mRNA and activity, while treatment with the PPARα agonist WY14,643 lead to an induction by >150 and >100%, respectively. Using chromatin immunoprecipitation scanning assay we identified a specific upstream gene region that is occupied in vivo by PPARα. Electromobility shift assay demonstrated direct binding of PPARα to a DR-1 motif located at positions -2762/-2775 bp upstream of the CYP2C8 transcription start site. We further validated the functional activity of this element using luciferase reporter gene assays in HuH7 cells. Moreover, based on our previous studies we demonstrated that WNT/β-catenin acts as a functional inhibitor of PPARα-mediated inducibility of CYP2C8 expression. In conclusion, our data suggest direct involvement of PPARα in both constitutive and inducible regulation of CYP2C8 expression in human liver, which is further modulated by WNT/β-catenin pathway. PPARA gene polymorphism could have a modest influence on CYP2C8 phenotype.
INTRODUCTION
The human CYP2C subfamily consists of four highly homologous genes, CYP2C18, CYP2C19, CYP2C9, and CYP2C8, which are localized in this order in a ∼390 kb gene cluster on chromosome 10q23.3. The CYP2C8 is responsible for the oxidative metabolism of many clinically available drugs from a diverse number of drug classes, including thiazolidinedione and meglitinide antidiabetics, non-steroidal anti-inflammatory agents (NSAIDs), antimalarials (e.g., amodiaquine, chloroquine), chemotherapeutics (e.g., taxanes, imatinib), and numerous other drugs (Daily and Aquilante, 2009;Xiaoping et al., 2013;Zanger and Schwab, 2013). CYP2C8 is also involved in the endogenous metabolism of arachidonic acid and all-transretinoic acid to epoxyeicosatrienoic acids and 4-hydroxy-alltrans-retinoic acid, respectively (Zeldin et al., 1996;McSorley and Daly, 2000). CYP2C8 shares more common substrates with CYP3A4 than it does with CYP2C9, despite its closer sequence homology to CYP2C9 (Totah and Rettie, 2005). All CYP2C enzymes are primarily expressed in liver although lower levels of functional CYP2C enzymes are also expressed in extrahepatic tissues, e.g., in human small intestine and in cardiovascular tissues (DeLozier et al., 2007;Achour et al., 2014). While CYP2C9 is the major CYP2C subfamily isoform in human liver, CYP2C8 has been suggested to be the major fetal CYP2C form (Achour et al., 2014;Johansson et al., 2014). Interestingly, hepatic expression of CYP2C8 is rather strongly correlated to CYP3A4 (Naraharisetti et al., 2010;Achour et al., 2014).
Although the CYP2C subfamily members CYP2C9 and CYP2C19 show clinically relevant genetic polymorphism, there is conflicting data regarding polymorphic effects on CYP2C8. While it has been reported that clearance of CYP2C8 substrates repaglinide, rosiglitazone, and pioglitazone is increased in homozygous and heterozygous carriers of CYP2C8 * 3 (Niemi et al., 2005;Kirchheiner et al., 2006;Tornio et al., 2008), other in vitro and in vivo showed contradictory results (Bahadur et al., 2002;Dai et al., 2001;Daily and Aquilante, 2009). Moreover, the CYP2C8 * 4 allele did not influence the pharmacokinetics of repaglinide (Niemi et al., 2003).
Thus, compared to other CYP2C genes, CYP2C8 appears to be less strongly affected by genetic variation and consequently regulatory events may have a more significant impact on variability. The transcriptional regulation of CYP2C genes has been thoroughly studied implying constitutive regulation by involving the liver-enriched receptor HNF4 (Jover et al., 2001;Ferguson et al., 2005;Rana et al., 2010;Yue et al., 2010) as well as inducible regulation with xenobiotic-sensing receptors CAR, PXR, and glucocorticoid receptor (GR) playing major roles (Pascussi et al., 2000;Ferguson et al., 2005;Chen and Goldstein, 2009;Rana et al., 2010). Interestingly, Prueksaritanont et al. (2005) observed pronounced induction of CYP3A4 and CYP2C8 in human hepatocytes by a series of fibrates including clofibric and fenofibric acids and gemfibrozil, but failed to link this to the fibrate receptor, PPARα. The finding was confirmed by other studies and appeared to be human-specific (Richert et al., 2008;Rakhshandehroo et al., 2009). While PPARα had been shown to transcriptionally activate some Phase II conjugating enzymes (e.g., EPHX2, GSTA, and UGT1A9; Barbier et al., 2009), direct regulation of cytochrome P450s was only recently shown by our group (Klein et al., 2012;Thomas et al., 2013). Elucidation of the molecular mechanism of PPARα-mediated regulation of CYP3A4 revealed direct transcriptional activation of the CYP3A4 promoter via at least three functional PPARα-binding regions (PBR-I, -II, and -III) within ∼12 kb of the CYP3A4 upstream gene region (Thomas et al., 2013). More recently, we found that the PPARα-mediated effects on CYP expression were additionally modulated by the WNT/β-catenin pathway (Thomas et al., 2015b).
In this context of CYP2C8 pharmacogenetics and expression regulation, the aims of this study were: (a) to characterize hepatic CYP2C8 expression variability in 150 liver samples from white individuals; (b) to assess the impact of two PPARA polymorphisms, previously shown to correlate with CYP3A4, on the expression and activity of CYP2C8; (c) to investigate the potential direct regulation of CYP2C8 by PPARα in human hepatocytes; and (d) to further elucidate the molecular basis for the modulation of PPARα-mediated effects on CYP2C8 by the WNT/β-catenin pathway. We demonstrate that PPARα directly binds and regulates CYP2C8 via specific binding elements within the CYP2C8 promoter. We also find a moderate influence of PPARA gene polymorphisms on hepatic CYP2C8 phenotype. These novel findings may help to better understand the interindividual variability in the response to various CYP2C8 drug substrates.
Cell Culture and Treatments
Detailed description of culturing HepaRG cells can be found elsewhere (Klein et al., 2015). Briefly, HepaRG cells (batch HPR101007) were obtained from Biopredic International (Rennes, France) and expanded according to the provider's instructions. The cells were cultivated for the first 14 days in HepaRG growth medium based on William's E Medium with supplements. At the final stage, HepaRG cells reached a differentiated hepatocyte-like morphology and showed liverspecific functions. The cells were further maintained in HepaRG differentiation medium for the duration of the experiments with exchange of medium every 2 days. All cells were maintained at 37 • C and 5% CO 2 in a humidified atmosphere throughout the experiment.
Transfections with siRNAs
For the RNA interference experiments, HepaRG cells were transfected with 20 nM siRNAs using 10 pmol Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) in 12well plates with serum-free medium. The siRNA targeting PPARα (Thomas et al., 2014(Thomas et al., , 2015a, β-catenin (Thomas et al., 2015b), and a non-targeting siRNA as a negative control (Lo GC Duplex 2) were obtained from Life Technologies. Onehundred microliters of the transfection cocktail was added per well to the cells containing 100 μl culture medium. Upon 20 min of complex formation, the liposomes were given to the cells. Twenty-four hours after the transfection cells were treated for an additional 48 h with 100 μM of PPARa agonist, WY14,643 (Sigma-Aldrich) or solvent control, DMSO (Sigma-Aldrich).
Human Liver Cohort
Liver tissues and corresponding blood samples were previously collected from 150 patients of Caucasian ethnicity (71 males and 79 females; average age of the subjects 58 ± 14 years). Patients who suffered from hepatitis, cirrhosis, or alcohol abuse were excluded. All tissue samples had been examined by a pathologist and only histologically non-tumorous tissue was used (Schröder et al., 2013). The study was approved by the ethics committees of the medical faculties of the Charité, Humboldt University, and of the University of Tuebingen and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient.
Quantitative Real-time RT-PCR Gene Expression Analysis
For the determination of the absolute amounts of CYP2C8 mRNA expresion in the cohort of the liver samples, high quality total RNA was isolated from liver tissue using Trizol/Qiagen RNeasy protocol as described previously (Gomes et al., 2009). Synthesis of cDNA was performed with 1 μg RNA using the TaqMan Reverse Transcription Kit (Applied Biosystems) according to the supplier's instructions. Expression of CYP2C8 mRNA in liver tissue was performed using a commercially purchased gene expression assay (Applied Biosystems, Hs_00946140_g1) with a TaqMan 7500 system (Applied Biosystems). A standard curve was obtained using CYP2C8 cDNA-containing linearized plasmid DNA purchased from OriGene (SC107944). Raw data were normalized to RPLP0 (60S large ribosomal protein P0) expression which was shown to be the most suitable reference gene in the liver tissue cohort using geNorm analysis (Vandesompele et al., 2002). RPLP0 was determined in the same samples using the endogenous control assay (4326314E) from Applied Biosystems. Normalized values were adjusted to the median value of all samples.
For qRT-PCR analysis of treated HepaRG cells, total RNA was isolated from HepaRG cells using the RNeasy Mini Kit, including on-column genomic DNA digestion with RNase free DNase Set (Qiagen). RNA was reverse transcribed to cDNA with TaqMan Reverse Transcription Reagents (Applera GmbH). Quantification of CYP2C8 expression was performed using ABI Applied Biosystems R 7500 Real-Time PCR System following the manufacturer's instructions using Life Technologies Assays (Hs_00946140_g1 for CYP2C8 and 4326314E for housekeeping gene, RPLP0). The mRNA expression levels were normalized to the RPLP0 mRNA expression. Relative gene expression changes were calculated using the delta delta Ct ( Ct)-method (Livak and Schmittgen, 2001;Feuer et al., 2015).
Western Blot Detection of Protein Expression
CYP2C8 protein expression in the liver microsomes of the liver samples cohort or following HepaRG cell treatments was analyzed by Western blot. Ten micrograms of protein were separated by electrophoresis on a 10% SDS-polyacrylamide gel and blotted to nitrocellulose membranes. Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor). For absolute quantification, a standard curve was generated by coanalyzing 250-4000 fmol of recombinantly expressed CYP2C8 (Becton Dickinson Gentest 455112) in each experiment.
Transfections and Luciferase Reporter Analyses
Cells were transfected with the Firefly luciferase reporter constructs using standard methods as recently described (Thomas et al., 2013(Thomas et al., , 2015a. The plasmid pRL-CMV, encoding Renilla luciferase under the control of a constitutively active viral promoter, was co-transfected for normalization purposes. 24 h after seeding of the cells, 800 ng of plasmid DNA (750 ng of the respective Firefly luciferase reporter plasmid, 50 ng pRL-CMV) were transfected per well of a 24-well plate using Lipofectamine 2000 (Invitrogen). Firefly luciferase reporter plasmids used in the study were: a pGL3-based 4xPPRE-driven reporter for luciferase expression under the control of 4 rat PPRE sites responsive to activation by the PPARα ("pGL3-PPRE pos"; Thomas et al., 2013) and a pGL3-based reporter plasmids driven by approximately 1000 bp of the human CYP2C8 promoter region between -2500/-3500 bp ("pGL3-CYP2C8-Site C") and -8500/-9500 bp ("pGL3-CYP2C8-Site A") to test the functional activation of the predicted sites. For the confirmations of the functional activity of the binding motifs following mutations were introduced (shown in bold): TCCCATTTGATGCTTC ["pGL3-CYP2C8-Site A * (mut)"]; ATCTCGAAGTCTAC ["pGL3-CYP2C8-Site C * (mut)"]. The plasmids were generated by GenScript sequence Synthesis Company. Transfection experiments with the pGL3Basic empty vector were conducted as controls. Cells were incubated with 100 μM WY14,643 or v/v DMSO control for 48 h prior to lysis with 1x Passive Lysis Buffer (Promega) and luciferase activity determination as previously described (Thomas et al., 2013).
Assessment of CYP Metabolic Activities
Cytochrome P450 enzyme activities were determined in the cohort of human liver samples and HepaRG cell culture supernatants using a liquid chromatography with tandem mass spectrometry-based substrate cocktail assay, as previously described (Feidt et al., 2010). The CYP substrate mix was added to cell cultures after 45 h of incubation with the enzyme inducers as detailed above. The following substrates were used: 50 μM phenacetin (CYP1A2), 25 μM bupropion (CYP2B6), 5 μM amodiaquin (CYP2C8), 100 μM tolbutamide (CYP2C9), 5 μM propafenone (CYP2D6), 100 μM atorvastatin (CYP3A4). Aliquots of the supernatant were taken after 3 h of incubation at 37 • C. Metabolite formation was normalized to cellular protein content.
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation was performed using MAGnify Chromatin Immunoprecipitation Kit (Invitrogen) according to the manufacturer's description and as previously described (Thomas et al., 2013). Briefly, DNA was sheared by sonication to an average length of ∼800 bp using Bioruptor (Diagenode) and incubated with 10 μg of anti-PPARα antibody (PP-H0723-00; R&D Systems), previously bound to 10 μl of magnetic beads at 4 • C for 2 h, and DNA was purified using DNA purification beads and eluted in 150 μl of elution buffer. Promoter occupation was analyzed with 5 μl of immunoprecipitated DNA by Sybr-Green polymerase chain reaction (PCR). The results were normalized to HMGCR (3-hydroxy-3-methyl-glutaryl-CoA reductase) promoter region (-21970/-22200 bp) used as positive control (van der Meer et al., 2010) and the untranscribed region Untr-5 as negative control (Hariparsad et al., 2009).
Statistical Analyses
Statistical analyses were performed using software R-3.2.0 1 with additional packages coin_1.0-24 (Hothorn et al., 2006), quantreg_5.11 2 , and RVAideMemoire_0.9-50 3 . Spearman's rank correlation coefficient was used to assess associations between CYP2C8 phenotypes (amodiaquine N-demethylation, CYP2C8 protein and mRNA expression) and of PPARα protein and mRNA expression. The effect of each PPARA-SNP on each of the three CYP2C8 phenotypes was studied in three genetic models: dominant, recessive, and additive model. For the first two genetic models, Wilcoxon-Mann-Whitney tests were applied for univariate association analyses, whereas the last model was investigated by Spearman correlation tests. In addition, multivariate analyses were performed considering 10 non-genetic factors (age, gender, nicotine and alcohol intake, exposure to P450 inducers, total bilirubin, GGT, CRP, cholestasis, diagnosis; Klein et al., 2010). To be more precise, for each of the CYP2C8 phenotypes, each SNP, and each genetic model, two median regression fits were compared with function anova.rq of library quantreg (using a rank test with Wilcoxon scores): (a) with the SNP in the respective genetic model plus the ten non-genetic factors and (b) only with the ten non-genetic factors. Reported p-values were adjusted for multiple testing (Bonferroni) where appropriate. For all calculations, all data were used, including all outlier data presented in various graphics. All statistical tests were two-sided and statistical significance was defined as p < 0.05; 95% confidence intervals (95% CI) were reported where appropriate.
Population Variability of Hepatic CYP2C8
We quantitated CYP2C8 hepatic phenotypes, i.e., mRNA, protein, and enzyme activity in RNA and microsomes of a cohort of 150 human liver samples, and examined the effect of non-genetic parameters. CYP2C8 expression varied considerably at the different phenotype levels and was not normally distributed (Table 1, Figure 1). Fold-variation was highest (65.5-fold) for protein and lowest (15.7-fold) for enzyme activity (Table 1), while the coefficient of variation (cv) as a normalized measure of variability was more comparable between the different phenotypes. These values are comparable to previous studies (Rodríguez-Antona et al., 2007;Naraharisetti et al., 2010), however, compared to our studies on other liver drug metabolizing enzymes [e.g., CYPs 2D6 (Zanger et al., 2001), 2B6 (Hofmann et al., 2008), and 3A4 (Wolbold et al., 2003)], CYP2C8 variation appeared to be somewhat less pronounced. All three CYP2C8 phenotypes were significantly, but moderately correlated to each other (Figure 1). We also confirmed a significant correlation between CYP2C8 and CYP3A4 (e.g., r s = 0.51, P < 0.0001 for protein level; Naraharisetti et al., 2010;Achour et al., 2014).
Non-genetic Factors Influencing CYP2C8 Expression
Available documentation to the liver donors included demographic, clinical, and self-reported data as previously reported . We used multivariate modeling to analyze whether any of these parameters had an impact on CYP2C8 phenotype. We identified cholestasis and increased bilirubin and GGT levels as factors being significantly associated with at least one microsomal CYP2C8 phenotype (
Hepatic Expression and Genotype of PPARA Correlates with CYP2C8 Phenotypes
Based on our previous studies describing impact of PPARα on the regulation of P450 enzymes, we assessed the correlation between PPARα expression and CYP2C8 phenotypes in our human liver cohort. As shown in Figure 2A, mRNA expression of PPARA was moderately but significantly correlated to mRNA (r s = 0.42; p < 0.0001), protein (r s = 0.24; p < 0.005) and activity (r s = 0.25; p < 0.005) of CYP2C8, while we did not observe any significant correlation between PPARα protein and any phenotype of CYP2C8. Additionally, we assessed the impact of two linked PPARA variants, rs4253728 and rs4823613, previously shown to correlate with CYP3A4 (Klein et al., 2012), on the expression and activity of CYP2C8 by univariate analysis, applying three different genetic models (dominant, recessive, or additive, see Materials and Methods). The additive model is based on the assumption of a gene-dose effect, such that heterozygotes are phenotypically intermediate between homozygous wild-types and mutants. Without correction for non-genetic factors, we found for both SNPs significantly decreased levels of CYP2C8 protein expression in homozygous carriers of the minor allele ( Figure 2B). However, after correction for non-genetic factors and adjustment for multiple testing these relationships did not remain significant.
Ligand-mediated PPARα Activation and PPARA Gene Knockdown Modulates CYP2C8 Expression in HepaRG Cells
To directly investigate the functional impact of PPARα on CYP2C8 we applied two available strategies, namely stimulation with the canonical PPARα ligand, WY14,643, and depletion of PPARα using siRNA-mediate gene knock down. As shown in Figure 3A, treatment of HepaRG cells with 100 μM WY14,643 significantly induced the expression of CYP2C8 at mRNA (more than threefold), protein ( Figure 3B, lane WY14,643) and activity (over twofold) levels, confirming earlier observations by Prueksaritanont et al. (2005). In the same experimental setup, transfection of HepaRG cells with PPARα-targeting siRNA resulted in >50% reduction in the expression of mRNA and >40% decrease in protein levels ( Figure 3B, lane siPPARa) of CYP2C8 as compared with cells treated with non-silencing siRNA. The measurement of corresponding CYP2C8 enzyme activity after PPARA gene silencing resulted in an average amodiaquine N-desethylation reduction over 45% as compared with non-targeting control. These findings demonstrated that PPARα mediates both basal and inducible regulation of CYP2C8 in human hepatocytes.
Chromatin Immunoprecipitation (ChIP) Identifies a CYP2C8 Promoter Region Occupied by PPARα in HepaRG Cells
In silico analysis of the CYP2C8 promoter and upstream region (10 kb) identified a number of putative direct repeat DR1 and DR2 motifs with different degrees of homology to the consensus peroxisome proliferator response element, PPRE, AGGTCA half site, although no 100% consensus motif was found. Thus, we systematically screened ∼10 kb of upstream region by ChIP of isolated chromatin from HepaRG cells using a total of 12 primer pairs. As evident from Figure 4, one region designated as PPARα-binding region (PBR-C, spanning between -2500 and -3300 bp) showed significant enrichment of promoter binding by PPARα compared with unoccupied intermediary gene regions and negative control (n.c.). Interestingly, pre-treatment of hepatocytes with 10 μM of CYP2C8 substrate, amodiaquine, resulted in ≈40% higher enrichment of PPARα occupation within this region (Figure 4, red bars).
PPARα Directly Binds to the Specific Motifs of CYP2C8 Promoter and Activates its Expression.
We next investigated whether PPARα/RXRα heterodimer can directly bind to their bioinformatically predicted potential sites using fluorescence-based EMSA. Figure 5A shows results for a selection of six motifs with the highest score for binding to the in silico predicted DR1/DR2 motifs out of a total of 15 tested motifs (Supplementary Table S1). We found that PPARα/RXRα specifically bound to two identified motifs, one of which is the DR-4 PXR/CAR-binding site, TCAACTTTGATGACCC positioned between (-8806/-8822), previously identified by Ferguson et al. (2005). However, the newly identified DR1 PPRE motif, AGTTCGAAGTTCA within the identified PBR-C positioned between -2762/-2775 bp was bound with much higher apparent affinity ( Figure 5A).
Furthermore, results of HuH7 cell cotransfection experiments performed in the absence and presence of WY14,643 are shown in Figure 5B. Luciferase reporter gene analysis of the CYP2C8 upstream promoter region incorporating PBRs-A and C showed over fourfold induction of PPARα activation by WY14,643. Furthermore, mutation of sites DR4-A and DR1-C completely abolished inducible PPARα-dependent transactivation of CYP2C8. These data further confirm functional activity of two motifs within CYP2C8 promoter, which can be bound by PPARα.
WNT/β-Catenin Functions as an Inhibitor of PPARα-mediated CYP2C8 Induction
We recently reported on the crosstalk between the PPARα and WNT/β-catenin pathways in the regulation of P450 enzymes which results in an inhibitory influence of β-catenin on FIGURE 2 | Influence of PPARα expression and genotype on the CYP2C8 phenotype. (A) Scatter plots of CYP2C8 phenotypes (amodiaquine N-desethylation, CYP2C8 protein and mRNA expression) vs. PPARα mRNA (top) and protein (bottom) expression. Spearman's rank correlation coefficient r s and corresponding p-values are given. (B) Box-and-whisker plots of CYP2C8 phenotypes (Amodiaquine N-desethylation, CYP2C8 protein and mRNA expression) for two previously described intronic PPARA variants. Genotypes are indicated as G/G, G/A, and A/A (for rs4253728) and A/A, A/G, and G/G (for rs4823613). P (rec.), unadjusted p-value of Wilcoxon-Mann-Whitney test for recessive genetic model; N, number of individuals per group; numbers for the three genotype groups do not add up to 150 due to missing values. Outliers were included in all calculations.
PPARα-mediated induction of CYP3A4 as well as CYP2C8 mRNA (Thomas et al., 2015b). Here we simultaneously analyzed mRNA and protein expression of CYP2C8 following PPARα activation by WY14,643, either in the presence of β-catenin targeting siRNA, siCatenin, or non-targeting siRNA, siCTR (Figure 6). In line with our previous observations, knockdown of β-catenin in the presence of WY14,643 led to increased CYP2C8 expression by activated PPARα (Figure 6A) on the mRNA (gray bars) and protein (black bars) level ( Figure 6B). Thus it can be concluded that in HepaRG cells β-catenin exerts a similar inhibitory modulation on PPARα-mediated CYP2C8 induction as previously shown for CYP3A4 regulation.
DISCUSSION
In the present study, we demonstrated direct binding of the nuclear receptor PPARα to the CYP2C8 promoter resulting in its transcriptional activation and regulation of CYP2C8 expression in hepatocytes and in human liver. We identified two specific regulatory elements that are essential for PPARα-mediated transactivation. One of them corresponds to the previously identified CAR/PXR-binding site at -8806 bp (DR-4) that had been shown to be essential for the activation of the CYP2C8 promoter by both the PXR ligand rifampicin and the human CAR ligand, CITCO (Maglich et al., 2003). In addition we identified FIGURE 4 | Binding of PPARα to the CYP2C8 promoter in vivo. Precipitated DNA from HepaRG cells without (w/o) treatment or after treatment with amodiaquine (10 μM for 6 h) was purified and was used, together with input DNA, as template for Sybr-Green PCR using a total of 12 primer pairs spanning approximately 10 kb of the CYP2C8 promoter region. Raw Ct (cycle threshold) values were normalized to input DNA to calculate the percentage of DNA immunoprecipitated. Primers encompassing the PPRE of the human HMGCR gene were used as positive control. Means relative to negative control primer pair (n.c.) are shown. (A-F) Schematic representation of CYP2C8 promoter regions, which were subjected for the ChIP analysis. Promoter scheme includes binding sites of previously described transcription factors. a novel PPRE DR-1motif at -2762/-2775 bp region, which is independently of the former site sufficient for PPARα-mediated regulation.
For the direct regulation of CYP2C8 expression by PPARα we provide three lines of evidence: first, ChIP in HepaRG cells revealed a PPARα binding region between -2500 and -3500 bp upstream of transcriptional start site in vivo; second, two motifs for PPARα/RXRα binding (DR4-A and DR1-C) were identified and further confirmed by EMSA; and third, reporter gene analysis demonstrated that both of these motifs are essential and sufficient for transcriptional activation by PPARα. The DR4-A motif was previously shown to be a binding site for PXR and CAR. Although EMSA revealed a relatively weak signal of PPARα/RXRα binding to this region, luciferase activity assay further supported the relevance of this site for PPARα-mediated regulation ( Figure 5B). We hypothesize that probably a multifactorial complex containing PXR/CAR and PPARα is responsible for the transcriptional regulation on this motif. The DR1-C site displayed the strongest binding affinity, in agreement with its strong functional role, as demonstrated by mutational analysis. The presence of several nearby and overlapping transcription factor binding sites may lead to protein-protein interactions that could be explained by the robustness of the regulatory system by several nuclear receptors. In general, our experiments suggest both constitutive and inducible transactivation of CYP2C8 by PPARα. (A) Electrophoretic mobility shift assays using in vitro translated proteins was used for assessment the binding of fluorescence-labeled double-stranded oligonucleotide probes corresponding to the indicated regions within CYP2C8 promoter (selection out of 15 tested probes is shown). As a positive control for binding, the known PPARα/RXRα binding site of the rat ACOX1 gene was used (Thomas et al., 2013). Complexes of PPARα/RXRα heterodimers with the oligonucleotides are marked by an arrow. (B) Luciferase reporter gene constructs containing the sequences of the CYP2C8 promoter, PBR-A (-9500/-8500 bp) and PBR-C (-2500 to -3500 bp) were cotransfected with a PPARα expression plasmid and renilla-luciferase expression vector into HuH7 cells. Cells were treated with either 100 μM WY14,643 or vehicle DMSO for 48 h before measurement of firefly/renilla luciferase activities. Firefly luciferase activities were normalized to renilla luciferase activities to consider changes in the transfection variability and to DMSO control, set as 1. Data are means of three independent experiments, each performed in triplicates in 96-well format; (mut), mutated sites; * , statistically significant (P < 0.05) compared with DMSO treatment set to 1.
Interestingly, pretreatment of hepatocytes with the CYP2C8 canonical substrate, amodiaquine, further increased enrichment of PPARα within PBR-C as assessed by ChIP assay. Although the exact molecular mechanisms of this increased occupation are currently unclear, it should be noted that WY14,643 did not show such an effect on the CYP3A4 promoter occupancy (Thomas et al., 2013).
For the characterization of genetic and non-genetic factors influencing interindividual variability of hepatic CYP2C8 expression we used our well characterized human liver cohort including 150 white individuals with comprehensive clinical documentation. We observed that environmental and other nongenetic factors have a moderate influence on hepatic CYP2C8 phenotype. Association of CYP2C8 with deregulated bile acid homeostasis and drug induced cholestasis have been reported before (Daly et al., 2007;Rieger et al., 2013). In particular for diclofenac-induced cholestasis, it was suggested that allelic FIGURE 6 | Inhibitory role of β-catenin in the ligand-mediated induction of CYP2C8 by PPARα. (A) mRNA (gray bars) and protein (black bars) expression levels of CYP2C8 were analyzed by quantitative real-time qRT-PCR and Western blotting, respectively, following treatment of HepaRG cells with 100 μM of WY14,643 in the presence of β-catenin-targeting siRNA (siCatenin) or non-targeting control (siCTR). Shown are mean values of three independent experiments ±SD. * , Statistically significant (P < 0.05) compared with DMSO treatment set to 1; #, statistically significant (P < 0.05) when siCatenin-treated cells compared to control siCTR-treated cells. (B) Representative western blot analysis of CYP2C8 protein expression following WY14,643-mediated activation of PPARα in the absence (lane siCatenin + WY14,643) or presence (lane siCTR + WY14,643) of β-catenin. variants in UGT2B7, CYP2C8, and MRP2 may cause an increase in the level of reactive metabolites leading to protein-diclofenac adducts that then produce toxicity. The observation that the two linked PPARA variants, rs4253728, and rs4823613, previously shown to influence CYP3A4 expression and function (Klein et al., 2012), also affect expression and activity of CYP2C8 is well explained by the direct transcriptional regulation of CYP2C8 by PPARα and further confirms the major finding of this study. However, whether the influence of these polymorphisms is sufficient to improve CYP2C8 pharmacogenetic prediction remains to be studied.
In addition to the significance for drug-drug interactions, our findings may have further clinical relevance for the treatment of lipid disorders and plasma dyslipidemia with PPARα ligands. As a variety of dietary and endogenous lipids, including saturated and unsaturated fatty acids, phospholipids, eicosanoids, and many derivatives and metabolites, have been implicated in PPARα activation, our findings suggest an intricate interplay between intermediary metabolism, nutritional status, and biotransformation. Of note, in our previous study we found induction of several P450s including CYP2C8 by the common nutritional phospholipid, POPC (Thomas et al., 2013). The current data on direct P450 induction through PPARα suggests a link between endogenous substances and the regulation of drug biotransformation in human liver and stresses a clear need to readdress the potential for drug-drug interactions, which may depend on nutritional status. This is of particular importance for newly developed PPARα ligands to target obesity, insulin resistance, and diabetes (Lalloyer and Staels, 2010).
CONCLUSION
We have elucidated the mechanistic basis for constitutive and inducible transcriptional regulation of CYP2C8 by PPARα. PPARα thus contributes to a transcriptional network so far including CAR, PXR, GR, and HNF4 as direct regulators of CYP2C8 expression, while inducibility is also modulated via WNT/β-catenin pathway. Through these studies, we have identified two specific elements that are essential for transcriptional activation by PPARα, and unraveling this cellular signaling pathway will help to better understand the physiological role of the CYP2Cs and the factors that control their inducibility and contribute to the variability observed in humans.
AUTHOR CONTRIBUTIONS
MT, SW, BK, MT (Oulu), and KK structured and conducted the experiments and analyzed the data. MT, MS, and UZ designed the study and wrote the manuscript. | 2016-05-04T20:20:58.661Z | 2015-11-04T00:00:00.000 | {
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216534095 | pes2o/s2orc | v3-fos-license | ISLAM, HUMAN RIGHTS AND GLOBALIZATION
The objective of Islam is to establish economic equality, social justice, and fairness in all spheres of the life of individuals and societies. Islam put a very clear prohibition on slavery and discriminated social grades and caste-systems, and asserts on the human dignity through the golden principles of universal brotherhood among all human and gave a special honor to the women. These principles are considered integral parts the UN charters and constitutions of civilized world for establishing exploitation free society. In the view of Islam, in the divine religions as well, all races of human, white and black or brown are born from Adam and Eve. Allah, the almighty creator, has made them vary in colour, language and has placed them in different lands, but before Him they are all one and cherished by Him alike, and he is most honoured who is most righteous. Islam described the rights and duties of all kinds of people as all are equal in gender as human being. This paper examines the rights of individual in the lights of Holy Quran specially Surah al-Nisa verse 36 and the Sunah specially the last sermon of the Holy Prophet Muhammad (PBUH).
and He sent down them with the true Book, so that it might judge between the people. In that in which they had differed. And none differed about it but the very people who were given it, after clear signs had come to them, revolting among themselves; whereupon Allah guided, by His will, those who believed to the truth about which they differed. And Allah guides whom He pleases to the straight path. 1 This verse describes that human being is required to follow a prescribed set of teachings and instructions and explains why religion was promulgated and human beings are obliged to follow it, and why differences occurred in thoughts and believes.
Human as living social beings, having been created with a natural urge to live together and remain cooperative with each other, were in the beginning one single group. Then with passage of time and increase in the population occurred differences because of the acquisition of the necessities of life. These differences could only be settled by creating social laws and regulations to give each one his right and to make him abide the respect and the rights of others. Allah has ordained the principles and sent it down as religion, accompanied by good tidings of reward for those who obey and a warning of punishment for the offenders. All this was accomplished by sending the prophets and the apostles.
After that, people differed again this time about the knowledge of religion, or about matters concerning the beginning and end of mankind.
Thus, religious unity was disrupted and various groups of thoughts THE SCHOLAR (July -December 1029) appeared on the scene, and their differences contaminated the other aspects of life.
These second differences only occurred because of the revolt of the very people who were given the book, after the fundamentals and characteristics of religion had been fully explained to them and the proof of the existence of one Creator of this universe had been completed for them.
It is clear that there were two differences: First, the difference about worldly gains, which was of physical instinct and worldly needs; second, the difference about matters of religion which was of metaphysical instinct and based on logic, not on nature but on the revolt of mischiefmakers. Then Allah guided the believers to the truth about which they differed; and this guidance was done by His Will; and Allah guides whomsoever He pleases to the righteous path.
The divine religion is the only means of happiness and felicity for the human and it keeps life in order. It creates a balance between human instincts and urges, and keeps them on the middle path, preventing them from going towards either extreme. Thus, there appears the best system and the highest discipline in the human life both of this world and of the Hereafter, the material as well as the spiritual.
CLASSES AND GRADES OF THE PEOPLE AND THEIR RIGHTS
Hazrat Ali describes the classes and grades of the people and their rights as follows: "You must know, Malik, that the people over whom you rule are divided into classes and grades and the prosperity and welfare of each class of the society individually and collectively are so interdependent upon the well-being or the other classes that the whole set-up represents a THE SCHOLAR (July -December 1029) closely women net and a reciprocal aspect. One class cannot exit peacefully, cannot live happily and cannot work without the support and good wishes of the other. Among them there are (i)soldiers of the Army of Allah who defend His cause, (ii)next class is that of the Secretaries of State to whom duties of writing and issuing special or general orders are assigned, (iii) third class of Judges and magistrates who administer justice, (iv) the fourth is of officers who maintain law and order and guard the peace and prosperity of the country, (v) Then there are common men, Muslims who pay the taxes levied by the government (vi) and non-Muslims who pay the tribute to the State in lieu of taxes, (vii) then the class of men who carry on various professions and trades, (vii) and the last but not the least are the poor and the have-nots who are considered at the lowest class of the society. The merciful Allah has fixed rights and duties of each one of them. They have been either mentioned in His Book or explained through the instructions of the Holy Prophet .A complete code of preserved with us. 2
KINDS OF RIGHTS
In the view of Islam there are two types of Rights: Rights to the Creator i.e Almighty Allah and rights to the creation i.e. human beings.
Quran says: "And worship Allah and do not associate anything with Him, and do good to the parents, and to the kindred, and the orphans and the needy, and the neighbor close to you, and the neighbor who is a stranger, and the companion in a journey, and the way farer, and the slaves whom 2 Imam Ali (a.s) Nahj-al-Balagha. (Peak of Eloquence)Letter No. 53,page No. 251) your right hands possess. Verily Allah does not love the one who is proud, boastful." 3 In this verse, two types of rights are spoken about. They are irrespective of the rights of Allah, and the rights of people, and also some civilities of social life. The prime nature and the substance of Islam is to worship the only the Allah and else to do good and to be good to mankind in particular, and to all our fellow creatures in general. The verse alludes to a series of rights and instructions to observe such rights. These rights, the observance of which is obligatory and incumbent upon us all, are the rights of the creator and those of His creatures, and some ten instructions can be deduced from the verse as under mentioned:
-Rights of Allah:
The first theme therein is that it invites people to worship and servitude to Allah while they should abandon idolatry and infidelity. This Allah manner is the root of all Islamic agenda. The act of following the notion of Unity and theism, purifies the soul, clarifies the intention, strengthens the will, and tightens the decision for performing any right and useful action in the cause of Allah. Since the verse is to state a series of Islamic rights, before referring to anything else, it points to the right of Allah upon people. "And worship Allah and do not associate anything with Him," Imam Ali ibn Hussain Zainul Aabiden (a.s) says in Risalatul Huqooq: "The greatest right of Allah is to worship Allah and do not associate anything with Him." 4 3.2 -Rights of parents: Then, it adds:" … and do good to the parents ..." The right of parents is one of the subjects which have been repeatedly emphasized in the holy Quran. There are fewer matters that have been recommended so much in it.
-Rights of Relatives:
Next to that, it continues saying:" ... and do good to the relatives," This subject is also one of the themes that have been emphasized abundantly in the Quran. It has sometimes been referred to as `blood ties', and sometimes has been enjoined under the commandment of doing good to the kindred '. In this regard our Holy Prophet said: a person can not enter in paradise who does not care of his relatives. 5
-Rights of Orphans:
Then it pays to the rights of orphans, and encourages the believing people to doing good unto the `orphans'. The reason of this emphasis is that, as a result of different incidents, there always exist some orphan children in every society that forgetting them, not only spoils their condition but also puts the situation of the society in danger. " ... and do good to the orphans, ..." 3.5 -Rights' of the Needy ones: Next to that, the Holy Quran reminds us the `rights' of the needy ones. " and do good to the needy, ..." The reason of this remembrance is that: in every society there are usually some handicapped persons, some feeble ones, and the like of them that leaving them out is against all the principles of humanity.
-Rights of near Neighbours:
After that, the verse recommends to doing good unto the neighbours who are near to us in house to house or in country to country. It says: ".. and do good to the neighbor close to you, .."
-Rights of Stranger Neighbours:
The neighbours who are strangers to us are then recommended. It says: " ... and do good to the neighbor who is a stranger," The 'right of neighborhood' is so important in Islam that Amir-ul-Mu'mineen Imam Ali (a.s) has stated about it thus: "The Prophet Muhammad (PBUH) put so much emphasis and stress on the abide of the rights of the neighbours that we thought he would finally order the neighbours to inherit one another." 6 Another tradition denotes that one day it happened that the holy Prophet three times said: "By Allah, he does not believe." A person asked him whom he meant, and the Prophet said: "The person whose neighbor is not in security from his molestation." What a dignified command of Islam it is? If the neighbor of House or Country is secured from one another subsequently the entire world would be in peace and safety.
-Rights of Companions:
After that, the Quran has recommended about those who are friends and companions. It says: ".. and do good to the companion in a journey,.." The Arabic phrase:/ as-sahib-il-janb/, of course, has a larger scope of meaning than `friend' and `companion'. Thus, the verse conveys a general and inclusive command regarding to having good manner due to those who somehow connect with us irrespective of real friends, fellow-workmen, fellow-travelers, those who ask us for something, students, counselors, and waiters etc.
-Rights of Travelers:
Another group, whom are recommended about here, are those who will be in need, because of some reasons, when they are in journey and are far from their own home, although they may be rich in their own city. So, it says: " ... and way farer, ..."
-Rights of Slaves:
The final recommendation is about doing good unto the slaves. It says: " ... and the slaves whom your right hands possess.
..." In fact, the above verse begins with the subject of the right of Allah and concludes with the rights of slaves '. Not only in this verse the slaves are recommended about, but also many other verses of the Quran are upon this matter. 7
CONCEPT OF HUMAN RIGHTS IN ISLAM
Human rights in Islam it means that these rights have been granted by Allah and also all rights are taken out from the rights of Allah, because Allah is the creator of all things so He has granted the rights to His creatures from His rights: Hazrat Ali (a.s) says: "A further sign of His Grace is that He gave to the rights of man over another man an importance equal to His Rights over Human beings. He granted these rights reciprocally to all human beings". 8 These rights have not been granted by any king or by any legislative assembly. The rights granted by the kings or the legislative assemblies, can also be withdrawn in the same manner in which they are conferred. The same is the case with the rights accepted and recognized by 7 Tafseer Noor ul Quran , Traslated by Syed Abbas Sadr Aamuli Isfahan, Iran, 9 th edition printed in 1383, vol 4 , page 41-42 8 Nahj-al-Balagha, commentary by Sheikh Muhammad Abduh, sermon 211,volume 2, page 224 the dictators. They can confer them when they please and withdraw them when they wish; and they can openly violate them when they like. But since in Islam human rights have been conferred by Allah, no legislative assembly in the world, or any government on earth has the right or authority to make any amendment or change in the rights conferred by Allah. No one has the right to abrogate them or withdraw them. Nor are they the basic human rights which are conferred on paper for the sake of show and exhibition and denied in actual life when the show is over. Nor are they like philosophical concepts which have no sanctions behind them.
Islam does not accept the rules made by other than Allah. Quran describes three commands for those persons who make their own decision against Allah(i) "Those who do not judge by what Allah has sent down are the disbelievers.". (ii) "They are the wrong-doers (zalimun)" and (iii)"They are the evil-livers (fasiqun)" 9
ALL HUMAN BEINGS ARE EQUAL IN RIGHTS
The first thing that we find in Islam in this connection is that it says some rights for man as a human being. In other words it means that every man whether he belongs to this country or that, whether he is a believer or unbeliever, whether he lives in some forest or is found in some desert, whatever be the case, he has some basic human rights simply because he is a human being, which should be recognized by every Muslim. In fact it will be his duty to fulfill these obligations.
Islam not only recognizes absolute equality between men irrespective of any distinction of colour, race or nationality, but makes it This is addressed to all human beings and not to the Muslims only, though it is understood that in a perfect world the two would be equal. All races of men, white, brown and black are His creation. Allah has made them to vary in colour, language and mode of life, and has placed them in different lands, but before Him they are all one and cherished by Him alike, and he is most honoured who is most righteous.
This means that the division of human beings into nations, races, groups and tribes is for the sake of distinction, so that people of one race or tribe may meet and be acquainted with the people belonging to another race or tribe and cooperate with one another. This division of the human race is neither meant for one nation to take pride in its superiority over others nor is it meant for one nation to treat another with contempt or disgrace, or regard them as a mean and degraded race and usurp their rights.
Our Holy Prophet said in his last sermon: O, mankind! Verily your Creator and Nurture is One, your Father is one, you must remember that there is no any preeminence to Arab on non-Arab, nor to non-Arab on Arab, not to beautiful on ugly, nor to ugly on beautiful but the pious one. 11 The Islamic policy is to create justice, fairplay and harmony in the human society. Abolition of slavery and caste system, clear assertion of human rights, brotherhood among all people of the world, rights of women, social welfare, establishment of a society free from exploitation of man by man and belief in one Allah are some of the Islamic teachings which are now part and parcel of all the constitutions of the civilized countries.
In this manner Islam established equality for the entire human race and struck at the very root of all distinctions based on colour, race, language or nationality. According to Islam, Allah has given man this right of equality as a birthright. Therefore, no man should be discriminated against on the ground of the colour of his skin, his place of birth, the race or the nation in which he was born.
PROTECTION OF THE LIFE OF CITIZENS
The first and the foremost basic right is the right to live and respect human life. The Holy Quran says: "Whosoever kills a human being without any reason like man slaughter, or corruption on earth, it is as though he had killed all human beings. . 13 The person who slays and kills an innocent human being, has such a preparation, in fact, to kill some other innocent persons, too. This person is, indeed, a homicide whose prey is innocent human beings. And, we know that there is no difference between the innocent persons from this 'The Right to Life' has been given to man only by Islam. You will observe that the people who talk about human rights if they have ever mentioned them in their Constitutions or Declarations, then it is clearly implied in them that these rights are applicable only to their citizens or they have been framed for the white race alone. This can clearly be gleaned by the fact that human beings were hunted down like animals in Australia and the land was cleared of the aborigines for the white man.
Similarly the aboriginal population of America was systematically destroyed and the Red Indians who somehow survived this genocide were confined to specified areas called Reservations. They also penetrated into Africa and hunted down human beings like wild animals. All these instances go to prove that they have no respect for human life as such and if they have, it is only on the basis of their nationality, colour or race.
Contrary to this, Islam recognizes this right for all human beings. If a man belongs to a primitive or savage tribe, even then Islam regards him as a human being. Almighty Allah has said in the Holy Quran: "Anyone who kills a believer deliberately will receive as his reward (a sentence) to live in Hell for ever. Allah will be angry with him and curse him, and prepare dreadful torment for him." 16
THE SECURITY OF LIFE AND PROPERTY
The Prophet has also said about the dhimmis (the non-Muslim citizens living in the land conquered by the Muslim): "One who kills a man under covenant (i.e. a dhimmi) will not even smell the fragrance of Paradise". 17 Islam prohibits homicide but allows only one exception that the killing is done in the due process of law which the Quran refers to as bi alhaqq (with the truth). Therefore, a man can be killed only when the law demands it, and it is obvious that only a court of law can decide whether the execution is being carried out with justice or without justification. In case of war or insurrection a just and righteous government alone, which follows the Shari'ah or the Islamic Law, can decide whether a war is just or unjust, whether taking of a life is justified or not; and whether a person is a rebel or not and who can be sentenced to death as a punishment. These
ISLAM AND SOCIAL JUSTICE:
Quran says: "0 you who believe! Be maintainers of justice bearers of witness for Allah's sake, though it may be against your own selves or) your (parents or near relatives; if he be rich or poor, Allah is nearer to them both in compassion; therefore, do not follow) your (low desires, lest you deviate; and if you swerve or turn aside, then surely Allah is aware of what you do." 20 Here a general instruction has been issued on account of social justice a commandment that all have to observe without any exception.
That is, in all our judgments we must have our Lord as a witness besides us. We must seek Allah's consent and pleasures in our judgment, and swerve not even though our decree has to be issued against our own interests or against the interests of our parents and our nearer and dearer.
And when we have to be a witness and testify to something, we must be strict, straight, and not drooping. We must be a witness for Allah, because we act in the presence of the Lord Allah Who is aware of whatever we do. The verse then points at the causes of swerve, that some people may be inclined to favour a rich man of whom they may expect some aid; and a judge may want to favour a poor for being helpless.
"Be you witness for Allah, even if it be against you or against your parents or kinsfolk; be the rich or be the poor, Allah is more deserving than them." Be they poor or rich, you have to observe justice and to avoid both extremes, because all of them are under the care and protection of Allah and Allah more deserves to be cared than the others.
This verse may also show the importance of justice in the sight of Allah and Islam. Shiites are of opinion that Justice is an attribute of Allah, and it is one of the tenets and five principles of religion; and that, to stand firm for justice is to be a witness of Allah, and being Allah.
In another verse, Quran says: "0 you who believe! Be upright for Allah, bearers of witness with justice and let not hatred of a people incite you not to act equitably" 21 This verse is similar to the verse 135 of " Al-Nisa", However, there is a subtle difference between the two verses. The verse of" Al-Nisa" forbids deviation from justice in bearing witness because of some low desires; the person loves the man for whom he bears witness because of some relationship or friendship, etc. and therefore, gives evidence in his favour in order that he might get some undue benefits. Conversely, this verse of "Al-Maida" prohibits deviation from justice while bearing witness because of hatred and enmity for the person against whom evidence is 21 21 Al-Quran 5: 8 This difference of themes has brought difference of stipulations in the clauses of the two verses. The verse in " Al-Nisa" says: Be maintainers of justice, bearers of witness for Allah's sake; while this verse in "Al-Maida" turns the restrictions around and says:" be upright for Allah, bearers of witness with justice." The main purpose of this verse is to restrain the believers from going against truth in bearing witness because of some enmity that the witness might be entertaining against the party concerned. Therefore, the evidence is attached to justice, meaning that one should observe justice while giving evidence; the evidence should not contain even an iota of injustice, even if the person concerned is one's enemy. On the other hand, evidence in favour of someone because of love or relationship) even if it goes against the right (is not counted such a deviation from justice although in reality it is not free from injustice and deviation. Therefore, the verse of" Al-Maida" enjoins bearing witness with justice and it has been based on the order of being upright for the sake of Allah; while the order of" Al-Nisa" enjoins giving evidence for the sake of Allah, that is, without following base desires and it is based on the order of being upright with justice.
The same is the reason why in the verse of" Al-Maida" the order to bear witness with justice leads to the order of acting equitably as Allah says: act equitably, that is nearer to piety; it calls to justice and counts it as a means of acquiring piety. And in the verse of " Al-Nisa", the order has been reversed and the command to bear witness for Allah is followed by the phrases: do not follow your) low (desires lest you deviate. Thus, Allah prohibits the believers from following one's low desire and discarding of
ISLAM AND UNITED NATIONS ORGANIZATIONS
The charter and the proclamations and the resolutions of the United Nations cannot be compared with the rights sanctioned by Islam; because the former is not applicable to anybody while the latter is applicable to every believer. They are a part and parcel of the Islamic Faith. Every Muslim or administrators who claim themselves to be Muslims will have to accept, recognize and enforce them. In his opinion the difference between the two set of rules is due to four following reason s: i. The charter of the United Nations was drafted by thousands of intellectuals belongs to almost all the countries of the world whereas the Alavi rules were enunciated by only one person viz Ali ibn Abi Talib.
ii. Ali arrived in this world fourteen years ago.
iii. Those that drafted the U.N Charter or in fact collected the requisite material for it indulged in too much extravagant talk and self-praise and boasted that world was indebted to them on this account. On the contrary Ali showed humility before God and was modest before the People he did not seek greatness or superiority. He always prayed to God and also wished the People that his acts of commission and omission might be over looked.
iv.
The difference which is more important than three above is that many Nations, out of those which participated in the U.N Declaration of human rights and endorsed it, violated this declaration and started armed conflicts to nullify and destroy it, but wherever Ali placed his foot, and whenever he said anything, or unsheathed his sword, he did so to destroy tyranny and oppression and leveled the grounds to march forward on the faith of truth and justice so much so he met his martyrdom in defense of human | 2020-04-02T09:34:25.036Z | 2019-12-16T00:00:00.000 | {
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271087222 | pes2o/s2orc | v3-fos-license | Fairness in Predicting Cancer Mortality Across Racial Subgroups
This cohort study evaluates whether racial bias exists in a machine learning model that identifies cancer mortality risk among patients with solid malignant tumors.
Introduction
Machine learning (ML) models can transform cancer care by providing oncologists with more accurate and accessible information to augment clinical decisions.There are a variety of use cases for incorporating ML in oncology. 1,2Implementation of a mortality predictive model integrated into a decision support system that encourages clinicians to discuss end-of-life care with patients improved rates of these conversations. 35][6][7] For example, patients from minoritized racial and ethnic groups have poor access to health care, 8 resulting in low sample sizes or missing or incomplete data for training datasets for ML.The use of models based on data lacking detail may further propagate inequities.Such models may demonstrate promising performance in the overall population but fail to meet standards for specific subgroups.While it is possible to create multiple models for each group, small sample sizes for specific groups may not allow for comprehensive implementation.Because race is socially constructed, its inclusion as a variable within prediction tools may lead to unwanted effects, including perpetuation of disparities. 9,10Conversely, excluding race may overlook important factors, such as social determinants of health, and continue to bias the algorithm.
When developing an ML model, it is important to evaluate for fairness using a variety of methods.President Biden's executive order on artificial intelligence (AI) highlighted the importance of developing AI tools that do not contribute to discrimination in health care. 11Approaches include examining the data used in development to identify issues in subgroup data quality that may limit model generalizability; measuring performance metrics to ensure there are no between-group discrepancies 8 ; and calculating fairness metrics, which provide additional measurements of the extent of bias, 12 with the most common metrics being equal opportunity, equalized odds, and disparate impact.Equal opportunity asserts that the true-positive rates (TPRs) between groups should be equal.Equalized odds specifies that the TPRs and the false-positive rates (FPRs) between groups should be equal.Disparate impact measures whether the positivity rate (ie, percentage of predictive positive [PPP]) is equal between groups. 12cologists may incorporate a predictive model in decision-making to identify patients for serious illness conversations (SICs), which occur between clinicians and patients and/or family members to elicit patients' values, preferences, and goals for medical care. 13Patients with cancer who engage in SICs often cite a better quality of life and goal-concordant care. 14However, most patients from minoritized racial and ethnic groups who have cancer die without a documented conversation. 15Oncology practitioners have identified many barriers to SICs, with patient and familial factors-such as difficulty accepting a poor prognosis and lack of agreement on decision-makingbeing among the most common. 16Uncertainty in estimating prognosis is another contributor; clinicians cannot identify patients at risk of short-term mortality using existing tools and often overestimate prognosis. 17Prognostic uncertainty, racial bias, and structural racism may lead clinicians to assume that patients from minoritized racial and ethnic groups do not want to have these
JAMA Network Open | Oncology
Fairness in Predicting Cancer Mortality Across Racial Subgroups conversations. 18Machine learning has the potential to help clinicians prognosticate, which can help prioritize patients for SICs.Caution is warranted in building models that do not exacerbate existing bias.
This study describes assessments to identify racial bias (measurable statistical variation in model performance and/or fairness metrics across racial groups) during the development of an ML model that predicts mortality among patients with solid tumors.The model was developed with intention for clinical use to identify patients for SICs, and the racial bias assessments were an important step to ensure safety prior to implementation.
Data Sources and Patient Selection
Patients were included in this cohort study if they had a date of cancer diagnosis recorded in the Mount Sinai Health System (MSHS) cancer registry between January 2016 and December 2021, were 21 years of age or older, and received care at an ambulatory cancer clinic within the MSHS.Data were obtained by matching records from the MSHS cancer registry, Social Security Death Index, and electronic health record and included all available retrospective data up to the date when databases were accessed for cohort extraction (February 2022).Our study followed the Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis or Diagnosis (TRIPOD) reporting guideline. 19The MSHS institutional review board approved this study and waived informed consent due to minimal risk based on the protected health information that was accessed.
The MSHS cancer registry is part of the national cancer registry; all data were abstracted by certified tumor registrars to adhere to the North American Association of Central Cancer Registries' standards. 20The Social Security Death Index was obtained from the Social Security Administration's death information master file and was used to calculate the date of death.Variables included were admission-discharge-transfer events, laboratory results, assessments from nursing flow sheets, cancer stage, and cancer treatment plans (the eTable in Supplement 1 shows the full list).Cancer stage and status and patients' race and ethnicity were obtained from the cancer registry.Race categories were Asian, Black, Native American, White, and other (not broken down further in the database accessed) or unknown, and ethnicity categories were Hispanic, non-Hispanic, unknown, and missing.Cancer stage was specified as the stage at the time of incident cancer diagnosis.Cancer status was defined as present or not currently detectable (cancer was cured or in complete response from treatment).Race and ethnicity were recorded by certified tumor registrars as part of routine operations.Based on data availability and interval of measurements, different sampling logics were used to extract measurements for each variable (the eTable in Supplement 1 includes the number of measurements sampled).Missing values were imputed by using the median value of the variable over the entire cohort at the time of sampling.
Model Creation
The target variable was 180-day mortality from the date of prediction.The period for prediction was set as 6 months leading up to the most recent recording of the patient's vital status (eg, if the patient's last recording was in December 2021, the prediction period was defined as from June to December 2021).For the retrospective validation cohort, each patient received 1 prediction.To accentuate the differences in data between deceased and living patients, we structured the death case profiles using data from the 30 days preceding death.This strategy was implemented to reduce the cosine similarity in the data representation, thereby enhancing the ML process during training.
Structuring the profiles differently for records of alive vs dead individuals was chosen because the objective of the model was to predict the patient's chance of dying at any time within the next 180 days rather than to predict patient status specifically at day 180.
The dataset was randomly split into the training dataset (70%) and the test dataset (30%).
Within the preliminary dataset, it was noted that there was a higher cumulative prevalence of records with a status of alive compared with dead.There were concerns that a training dataset that overrepresented a class in the target variable may lead to model underperformance.To manage this, the training dataset was adjusted using random undersampling; we randomly removed excess records of the alive patients until both classes in the target variable were equally balanced.10-fold cross-validation was used to train the model by using the random forest algorithm from the opensource Apache Spark project ML library, and recursive feature elimination was used for feature selection. 21,22The importance of each feature was calculated using the Gini coefficient.Variable importance is the sum of the Gini coefficients for each measurement of a variable.
Model Assessment
The primary outcomes for the study were discriminatory performance and fairness metrics among each race category (Asian, Black, Native American, White, and other or unknown) in the test dataset.
Overall model discriminatory performance was measured using the F1 score and the area under the receiver operating characteristic curve (AUROC). 23,24Fairness metrics were equal opportunity, equalized odds, and disparate impact.Based on consensus among the clinical operational leadership (including C.B.S.) who stewarded the planned implementation of the tool, it was prespecified that a threshold of 80% (ie, a fairness metric ratio between 0.80 and 1.25 [1/0.8]) was evidence of no racial bias.We also considered stricter thresholds of 90% (ratios between 0.90 and 1.11) as a sensitivity analysis.
Statistical Analysis
Descriptive statistics were used to evaluate patient characteristics in the test cohort.The predicted mortality was compared with the actual mortality.Equal opportunity ratio was calculated as the ratio of TPRs.Equalized odds ratio was both the ratio of TPRs and the ratio of FPRs.Disparate impact ratio was the ratio of the positivity rate.Ratios of TPRs, FPRs, and PPPs and their 95% CIs were computed.
The formula used for 95% CIs was exp(ln[ratio of proportions] ± [Z × SE]).All statistical calculations were performed using Python, version 3.9.13(Python Software Foundation).
Results
The 19.2%, other or unknown race; and 0.1% had missing race data.A total of 9.6% were Hispanic and 83.6%, non-Hispanic; 6.7% had unknown ethnicity, and 0.1% had missing ethnicity data (Table 1).
The Native American cohort was small (n = 45) and was not included in subsequent subgroup analysis.The most common cancers were breast (20.3%), genitourinary (19.9%), or gastrointestinal (19.1%).Among patients who were dead compared with those who were alive, there was a higher proportion of patients who were older than 65 years (63.8% vs 47.8%), were Black (25
Discussion
This study comprehensively assessed potential racial bias in a model predicting mortality for patients with solid tumors.In anticipation of clinical implementation, the AUROC and F1 score metrics demonstrated good agreement across the 4 analyzed racial subgroups, indicating similar performance for all groups (ie, no evidence of racial bias).Furthermore, all 6 comparisons of racial categories met all 3 fairness metrics within our safety threshold established prior to expected deployment.It is crucial to recognize that fairness metrics are descriptive tools for operational leaders, not prescriptive mandates.The decision to implement this model ultimately hinges on the interpretation of these metrics within the specific clinical context.While some numerical variation was observed in the fairness metric scores, all ratios remained within the prespecified range of 0.80 to 1.25 (1/0.8),suggesting that the prediction model is fair.
Our workflow prioritized achieving equal opportunity over the stricter equalized odds criterion if the latter proved to be impractical.A positive prediction triggers an SIC, which focuses on understanding the patient's goals of care.False positives leading to SICs for low-risk patients are unlikely to cause harm, as the intervention is merely a conversation.Furthermore, the prediction is meant to supplement clinical judgment, not to replace it.Clinicians can initiate conversations with any patient for whom they deem the conversation to be necessary regardless of the model output.
Therefore, a slight discrepancy in FPRs across race categories might not be significantly detrimental in this context.
Table 3 .
Fairness Metrics Comparisons Across Races a Other race was not broken down further in the database accessed. | 2024-07-11T06:15:49.999Z | 2024-07-01T00:00:00.000 | {
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67860553 | pes2o/s2orc | v3-fos-license | Simplifying the Milestones
Supplemental Digital Content is available in the text.
METHODS
Ten procedure-specific rating instruments were developed for sentinel cases, each consisting of critical procedure-specific steps based on literature review and faculty focus group consensus. Sentinel cases were chosen based on review of the American Council of Graduate Medical Education Milestones and resident logs of the most commonly performed plastic surgery procedures, both at our institution and nationally. The degree of guidance required from the attending surgeon is recorded for each step. General operative performance competency is evaluated from validated items developed by the University of Toronto. 1 All items use a 5-point Likert scale with behavioral anchors.
The OPRS assessments will be incorporated into the internet-based resident management platform New Innovations. Sentinel procedures for evaluation will be identified on a weekly basis by the residency coordinator, based on resident operative assignments (postgraduate year 2-6) organized by the chief resident. OPRS assessment forms will be available electronically immediately following the procedures, with an e-mail reminder notification 24 hours later to help encourage compliance.
In addition, resident self-assessment using the same OPRS will be conducted and correlated with faculty OPRS evaluations.
Each OPRS assessment will be evaluated for internal consistency reliability and inter-item correlation. Inter-rater reliability will be measured by faculty assessment of videotaped sentinel procedures using the appropriate OPRS instrument. Performance variation based on resident PGY level will be analyzed using 1-way analysis of variance. Feasibility will also be determined based on attending and resident Development of an Operative Performance Rating System for Plastic Surgery Residents ACAPS Abstracts Supplement response rates and time to completion for the OPRS evaluations, as well as a short written survey to assess resident and attending satisfaction and obtain feedback following OPRS implementation.
CONCLUSIONS
A web-based OPRS provides timely and objective feedback to improve residents' technical and decision-making skills, as demonstrated by the experiences of other surgical specialties. 2 This instrument will provide both formative and summative resident feedback, encouraging faculty and residents to focus on demonstrated competencies and areas for improvement. 3 Furthermore, resident operative performance can be monitored across time and residents, allowing program directors to have a long-term objective method of evaluating resident technical performance. 3 A reliable and valid OPRS may provide a feasible method of intraoperative assessment that could be implemented across all plastic surgery training programs.
INTRODUCTION
The partner hospital model identifies hospitals in the developing world to educate and enable local surgeons to deliver effective cleft care. This study aimed to determine the outcomes of this model on safety, education, and quality of surgical care.
MaTERIaLS aND METHODS
Twelve partner hospitals, sponsored by Smile Train for 5 or more years and distributed over 4 continents, were selected. Activities at each institution were evaluated using cleft surgical data, and electronic surveys were completed by hospital leadership.
RESULTS
A mean of 82% of patients with cleft at partner hospitals underwent sponsored surgeries. After partnership, all 12 hospitals implemented preoperative checklists for cleft surgery, and 5 hospitals implemented checklists for other sur-Improving Education and Standards for Cleft Care in the Developing World: The Partner Hospital Model | 2019-03-11T17:21:58.536Z | 2015-03-01T00:00:00.000 | {
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209341423 | pes2o/s2orc | v3-fos-license | Suppression of NB‐LRR genes by miRNAs promotes nitrogen‐fixing nodule development in Medicago truncatula
Abstract Plant genomes contain two major classes of innate immune receptors to recognize different pathogens. The pattern recognition receptors perceive conserved pathogen‐associated molecular patterns and the resistance genes with nucleotide‐binding (NB) and leucine‐rich repeat (LRR) domains recognize specific pathogen effectors. The precise regulation of resistance genes is important since the unregulated expression of NB‐LRR genes can inhibit growth and may result in autoimmunity in the absence of pathogen infection. It was shown that a subset of miRNAs could target NB‐LRR genes and act as an important regulator of plant immunity in the absence of pathogens. Plants not only interact with pathogens, but they can also establish symbiotic interactions with microbes. Nitrogen‐fixing symbiotic interaction and nodule formation of legumes may also require the suppression of host defence to prevent immune responses. We found that upon symbiotic interactions, miRNAs repressing NB‐LRR expression are upregulated in the developing nodules of Medicago truncatula. Furthermore, we show that the suppression of the activity of the NB‐LRR genes targeted by these miRNAs is important during nodule development. Our results suggest that the downregulation of NB‐LRR resistance genes in the developing nodule produces a suitable niche that facilitates bacterial colonization and the development of an N‐fixing nodule.
nucleotide-binding (NB) and leucine-rich repeat (LRR) domains (Meyers, Kaushik, & Nandety, 2005), which function as a second line of defence, that recognize specific pathogen effectors and trigger resistance responses. As a consequence, R genes are protecting plants from diseases caused by devastating pathogens. However, the unregulated expression of NB-LRR-type resistance genes can inhibit growth and may result in autoimmunity in the absence of pathogen infection (Michael Weaver, Swiderski, Li, & Jones, 2006).
In addition to innate immune receptors, in plants and other eukaryotes, small RNA (sRNA) systems mediate gene silencing which is thought to have evolved as a defence system against viruses and other molecular intruders (Ding, 2010;Ding & Voinnet, 2007). sRNAs are also important gene expression regulators, involved in many developmental processes and stress responses. These molecules are generally 21-to 24-nt-long and based on their biogenesis we distinguish two main classes: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs function in a post-transcriptional manner by downregulating target mRNAs in a variety of cellular processes (Rogers & Chen, 2013). The large majority of the plant sRNAs are siRNAs and one class of siRNAs termed phased siRNAs (phasiRNAs) (Zhai et al., 2011) regulate proteincoding genes in a similar fashion as miRNAs.
Plant genomes contain large numbers of NB-LRR-type resistance genes that recognize specific pathogen effectors and trigger resistance responses (Meyers, Kozik, Griego, Kuang, & Michelmore, 2003), however, the unregulated expression of NB-LRR genes may be harmful to plant growth and to immune responses (Michael Weaver et al., 2006). It was shown that a subset of plant miRNAs (miR482/2118 superfamily, miR1507, miR2109) can target NB-LRR genes and regulate plant immunity (Deng et al., 2018;Li et al., 2012;Shivaprasad et al., 2012;Su et al., 2018;Yang et al., 2015;Zhai et al., 2011). The miR482/2118 superfamily is the most ancient among the NB-LRRregulating miRNAs and it emerged first in Gymnosperms, followed by extensive radiation in seed plants. This gene family is present in the sequenced legume genomes except in Lotus japonicus (Chávez Montes et al., 2014;de Vries, Kloesges, & Rose, 2015). The miR1507 and miR2109 (also known as miR5213 in Medicago truncatula) also target NB-LRR genes and they are prevalent in Fabaceae (Taylor, Tarver, Hiscock, & Donoghue, 2014;Zhang, Xia, Kuang, & Meyers, 2016). Some of the NB-LRR-targeting miRNAs are capable of triggering the production of phasiRNAs from their cleaved target mRNAs and target a large number of NB-LRR mRNAs by phasiRNAs (Zhai et al., 2011). Moreover, this regulation by miRNAs is proposed as a mechanism for reducing fitness costs associated with NB-LRR genes by targeting them in the absence of pathogens (Shivaprasad et al., 2012). It was proposed that in the absence of pathogens, the NB-LRR resistance genes are transcribed but cleaved by miRNAs/phasiRNAs keeping their translation at a very low level. During pathogen attack, the levels of miRNAs and phasiRNAs are reduced resulting in an increased level of resistance gene transcript (Shivaprasad et al., 2012). The NB-LRR proteins are normally associated with effector-triggered immunity (Jones & Dangl, 2006), however, if the NB-LRR proteins are overexpressed (Bendahmane, Kanyuka, & Baulcombe, 1999), defence can also be induced independently of protein-based recognition mechanisms. Therefore, the elevated levels of NB-LRR proteins accelerate the activation of the defence pathways in non-race-specific defence against viral and bacterial pathogens (Shivaprasad et al., 2012).
Legumes establish symbiotic interactions with microbes, however, it is not fully understood how they differentiate between symbiotic or pathogenic microbial partners. Based on recent research achievements, symbiotic interaction and nodulation require the suppression of host defences to prevent immune responses (Yang, Tang, Gao, Krishnan, & Zhu, 2010). The M. truncatula genome encodes around 540 NB-LRR genes, and more than 60% of them could be targeted by the NB-LRR-targeting miRNAs such as miR1507, miR2109, and miR2118 (member of the miR482 superfamily) or by the phasiRNAs that are produced from at least 114 phasiRNA producing loci by the action of these miRNAs (Zhai et al., 2011).
Legumes invite symbiotic soil bacteria, termed rhizobia to enter into their roots and induce the development of nitrogen-fixing symbiotic nodules. It is a long-lasting vital question of how the host plants differentiate between interacting beneficial and detrimental microbes.
Here we report, that the expression level of the NB-LRRregulating miRNAs (miR1507, miR2109, and miR2118) is induced at the early phase of symbiotic interaction in M. truncatula and this induction is maintained in the symbiotic nodule, co-localizing with symbiotic bacteria in colonized nodule cells. Furthermore, the target NB-LRR mRNAs of these miRNAs are downregulated in the symbiotic nodules. We show that modification of the expression level of the NB-LRR-regulating miRNAs (either upregulation or downregulation) significantly changed the number of the symbiotic nodules on M. truncatula plants. These results indicate that nodulation requires the fine-tuned differential regulation of NB-LRR genes by miRNAs during symbiotic interactions to prevent the host's immune responses.
| Plant materials and growth conditions
Medicago truncatula Jemalong genotype was used in all experiments.
| Treatment of M. truncatula seedlings with flg22
Roots of 7-day-old M. truncatula plants were immersed in 1 μM flg22 (Ezbiolab) solution diluted in Fahräeus medium or in Fahräeus medium as a mock control for 6 hr. Then roots were washed with distilled water and incubated in Fahräeus medium for an additional 24 hr. RNA was isolated from roots and cDNA was synthesized for qPCR. As markers of the activation of plant defence responses, expression of PATHOGENESIS-RELATED1 (PR1, Medtr2g435490) and PATHOGENESIS-RELATED10 (PR10, Med-tr2g035150) genes were tested by qPCR (Domonkos et al., 2017).
| Bacterial strains and inoculation of M. truncatula roots
Five-day-old plants were infected with wild-type Sinorhizobium (Ensifer) meliloti 1021 or its exoY mutant derivative defective in succinoglycan production (Reuber & Walker, 1993). Rhizobial strains were grown for 24-48 hr at 30 C in tryptone yeast (TY) medium supplemented with 6 mM CaCl 2 , and inoculations were carried out as described previously . In hairy root transformation experiments, Agrobacterium rhizogenes ARqua1 strain was used.
| Microscopy
In situ hybridized nodule sections were observed with ZEISS axiostar plus M light microscope and images captured using a euromex HD ultra 1080p digital camera. To analyse the presence of rhizobia in nodules, in situ sections were stained with 5 μM SYTO™ 13 in PBS (pH 7.4) for 20-60 min and were analysed with confocal laser scanning microscopy.
Confocal laser scanning microscopy was performed using a Leica SP5AOBS confocal laser scanning microscope (Leica, Germany) on DMI6000 microscope base using HCX PL FLUOTAR ×5 dry objective with a numerical aperture of 0.15. SYTO™ 13 fluorescence was detected between 502 and 587 nm using 488 nm laser excitation. In situ hybridization signal of the same sample was detected using a Zeiss Axiocam MRc-5 colour camera (Carl Zeiss MicroImaging GmbH, Germany) connected to the side port of the confocal microscope.
| RT-qPCR assays
Total RNA samples were treated with DNase I (Thermo Scientific).
The cDNA synthesis was performed with 500 ng total RNA by High- (Dai & Zhao, 2011). Primers for the miRNA target genes were designed around the miRNA target sites using eprimer3, part of the EMBOSS software package (Rychlik, 1993). promoter. The base of the destination vector was the pKGW-R vector (obtained from Plant Systems Biology, VIB-Ghent University) in which the EF1α promoter was inserted between the SpeI and HindIII sites, the box containing the bacterial selection marker, chloramphenicol resistance, and the two homologous recombination sites (AttR1-CmR-ccDB-AttR2) were inverted (pKGW-R-pEF1). We used short tandem target mimic (STTM) construct to decrease miR2118abc miRNA levels (Fei et al., 2015) and IPSbased target mimicry construct (Franco-Zorrilla et al., 2007) to decrease the miR2118a level (Table S1). in vitro synthesized STTM sequences (Integrated DNA Technologies) and the amplified nos terminator was cloned into SpeI and AatII site of pKGW-R-pEF1 plasmid, using infusion technology (In-Fusion ® HD cloning kit Clontech). For analysis of the expression of miRNA regulated NB-LRR target genes, plants were treated with A. rhizogenes as described earlier (Boisson-Dernier et al., 2001). After 6 days on Fahräeus agar medium, the transformed seedlings were transferred to Fahräeus agar complemented with kanamycin (25 μg ml −1 ) and cefotaxime (250 μg ml −1 ) for 2 weeks. Kanamycin was used as a selection marker for the vector and the constructs, while cefotaxime was used for eliminating Agrobacterium from the transformed hairy roots. The plants were kept sterile until the red fluorescent transformed roots were collected for RNA preparation and qPCRs were performed on NB-LRR target genes.
| Construct design and plant transformation
For the quantification of infection events and nodule numbers, all constructs were introduced in M. truncatula Jemalong plants using A. rhizogenes hairy root transformation (Boisson-Dernier et al., 2001).
DsRed was the fluorescent selection marker of transformed hairy roots.
Transformed plants were planted in zeolite substrate and inoculated with S. meliloti 1021 strain carrying the pXLGD4 plasmid constitutively expressing the lacZ reporter gene for counting infection events or F I G U R E 1 Expression of the NB-LRR-regulating miRNAs in Medicago truncatula roots and nodules during pathogen attack (a) and symbiosis (b, c). (a) Small RNA northern blot analysis showed the suppression of miR2118a, miR1507, and miR2109 expression in M. truncatula cotyledons 4 days post-inoculation (dpi) with Alfalfa mosaic virus (AMV) compared to mock samples. The expression of the same miRNAs was analysed in M. truncatula seedlings 24 hr after treated with the synthetic flg22-peptide for 6 hr. (b) Small RNA northern blot analysis of miR2118a, miR1507, and miR2109 in mocktreated M. truncatula roots and roots inoculated with exoY mutant and wild-type (wt) strains of Sinorhizobium meliloti at 1 dpi. The RNA was transferred to a membrane and probed with radiolabelled DNA oligonucleotides for miR2118a, miR1507, miR2109, and U6 snRNA as a loading control. (c) Spatial distribution of miR2118 in M. truncatula nodules. In situ hybridization of longitudinal sections of nodules either with 5 0 digoxigenin (DIG)-labelled LNA-modified oligonucleotide probe for miR2118 or chicken miR449 as a negative control. Nodules were harvested at 4, 7, and 14 dpi with wt S. meliloti, and 14 dpi with exoY mutant of S. meliloti. (d) The images show the symbiotic phenotype of nodules induced by the wild-type and the exoY mutant S. meliloti at 14 dpi. Scale bars: in panel c-100 μm; in panel d-1 mm S. meliloti 1021 (wt) for nodule number analysis. To quantify microcolonies and infection threads, at least 15 red fluorescent transformed roots per construct were fixed and stained with 0.1% (wt/vol) X-Gal solution as described earlier (Domonkos et al., 2017). Blue-stained microcolonies and infection threads were counted at 5 dpi. Numbers of infection events were normalized to root length (counts per mm of root).
Nodule number was counted at 4 weeks post-inoculation (wpi) with S. meliloti 1021. Following quantification, the roots were individually frozen in liquid nitrogen and total RNA was isolated for northern analysis of sRNAs.
| Statistical analysis
In the case of RT-qPCR analysis and nodule number quantification, unpaired, one-tailed t tests were performed to estimate statistical significance of the difference between the samples; infected plants were compared to mock-treated ones or transgenic roots were compared to empty vector-transformed roots. In the RT-qPCR experiments, four independent biological replicates were measured with two technical repeats the mean of which was used in the statistical analysis. In the nodule number quantification, at least 45 (miRNA target mimicry lines) or 15 (miRNA overexpression lines) transgenic roots were counted.
The statistical tests were performed in Microsoft Excel. Statistical significance of the observed difference was marked in the figures with asterisks according to the following categories: ***p ≤ .001; **p ≤ .01;*p ≤ .05, ns = not significant.
| Promoter analysis
The pri-miRNA sequences were identified by PatMan (Prüfer et al., 2008) using the mature miRNA sequences as the query patterns and the r5.0 version of the M. truncatula A17 annotated transcripts (Pecrix et al., 2018) as the database. Promoters of the five MIRNA genes were obtained by extracting the sequences 2 kb upstream of the transcription start site (TSS) of the identified transcripts using BEDTools v2.26.0 (Quinlan & Hall, 2010). Promoter analysis was conducted using the PLACE tool (Higo, Ugawa, Iwamoto, & Korenaga, 1999). The lists of motifs of the five MIRNA promoters were compared and drawn with an online Venn-diagram drawing tool (http://bioinformatics.psb. ugent.be/webtools/Venn/). The selected common elements were plotted with a custom R script and further edited with Inkscape.
| Both pathogen infection and symbiotic interaction modulates the expression level of NB-LRRregulating miRNAs
To test the effect of pathogen infection on the expression level of NB-LRR-regulating miRNAs (miR1507, miR2109, and miR2118) in legumes able to form symbiotic N-fixing interaction, we infected M. truncatula plants with AMV (Salamon et al., 2018). The level of the NB-LRR-targeting miRNAs was monitored in the inoculated cotyledons of mock and virus-infected plants with northern blots 4 dpi ( Figure 1a). We found that similarly to previous reports in Solanaceae species (Deng et al., 2018;Li et al., 2012;Shivaprasad et al., 2012;Yang et al., 2015), the expression level of these three NB-LRR- (Zipfel et al., 2004). We tested the effect of the synthetic flg22 on the level of NB-LRR-regulating miRNAs (miR1507, miR2109, and miR2118). We immersed M. truncatula seedlings into media containing 1 μM flg22, and after incubation for 6 hr, the seedlings were transferred into a new media with no flg22. Following incubation for a further 24 hr, RNA was extracted from the seedlings and subjected to northern blot to analyse the expression level of the NB-LRR-regulating miRNAs. We found that the expression level of all three miRNAs decreased as a consequence of flg22 treatment (Figure 1a). This result shows that the activation of PTI with flg22 can trigger the plant defence signalling to suppress the level of NB-LRR-regulating miRNAs in M. truncatula.
The quick and precise regulation of plant immunity is not only paramount during pathogen attack, but it could also be very important during the recognition of beneficial microbes to establish symbiotic interaction with nitrogen-fixing rhizobia. To test this hypothesis, we used the M. truncatula-Sinorhizobium meliloti symbiotic interaction as a model system to investigate the role of miRNAs in regulating NB-LRR genes during symbiosis. In our experiments, we monitored the expression level of the three NB-LRRregulating miRNAs at the early stage of the symbiotic interaction.
Five-day-old M. truncatula seedlings were inoculated with wildtype S. meliloti strain 1021 and RNA samples were collected from non-inoculated roots (mock) and inoculated roots at 1 dpi. The collected RNA samples were subjected to sRNA northern blot, and the expression level of all three NB-LRR-regulating miRNAs was monitored ( Figure 1b). We found that the basal expression of all three miRNAs increased at 1 dpi. During this experiment, we also inoculated M. truncatula seedlings with the exoY mutant derivative of S. meliloti deficient in succinoglycan (exopolysaccharide I) production (Reuber & Walker, 1993). The exoY mutant of S. meliloti is defective to induce infection thread formation and hence nodule primordia lacking rhizobia develop on the roots of M. truncatula (Jones et al., 2008). We collected RNA samples from noninoculated roots (mock) and inoculated roots at 1 dpi with the mutant rhizobia. The expression level of all three NB-LRR-regulating miRNAs was tested with sRNA northern blot. We found that the expression level of miR2118a, miR1507, and miR2109 was also increased in roots inoculated with S. meliloti exoY mutant, similarly what was found with wild-type rhizobia (Figure 1b). These results indicate that the miRNAs regulating NB-LRR genes could play a role during the establishment of symbiotic interaction with rhizobia by the post-transcriptional downregulation of the NB-LRR genes.
| Promoters of Mtr-MIR2118a,b,c, Mtr-MIR2109, and Mtr-MIR1507 genes contain both pathogen-responsive and nodulation-regulated motifs
The observed dual regulation of the miRNA levels raised the possibility that the promoters of these NB-LRR-regulating miRNAs respond both to pathogens and symbionts. We searched for known regulatory motifs in the promoters of the five miRNAs with PLACE (Higo et al., 1999) and found several sequence motifs that were associated either with pathogen response or nodulation, and other functions as well (Table S2). We wondered if there were motifs that were present in all the five promoters, therefore we analysed the sets of motifs and visualized the result in a Venn-diagram ( Figure 2a). There were 44 common sequence motifs present in the promoters of the five miRNAs, eight of which were associated either with pathogen-response (i.e., W-boxes) or nodulation (Table S2). For simplicity, we grouped these sequence motifs into two categories and visualized their location on the 2 kb sequence upstream to the TSS of the corresponding MIRNA genes (Figure 2b). (Lohar et al., 2006). In addition, the involvement of an NB-LRR disease protein controlling nodulation in a strain-specific manner in soybean has been reported indicating the role of effector-triggered immunity in the rhizobial symbiotic interaction (Yang et al., 2010). Also, during the later events, when the intracellular rhizobial invasion of nodule cells occurs, the regulation of plant immunity is also required. To monitor the presence of NB-LRR-targeting miRNAs during nodule development, we carried out in situ hybridizations of these miRNAs in developing nodules at different time points post-inoculation with rhizobia.
We inoculated M. truncatula roots with wild-type and exoY mutant S. meliloti and investigated the distribution of miR2118 in 4-, 7-, and 14-day-old nodules with in situ hybridization (Figures 1c and S1). At 4 dpi, strong expression of miR2118 was detected only in the symbiotic cells of nodule primordia. In the 7-and 14-day-old nodules, the miR2118 expression localized to the nodule meristem and the infected nodule cells. The presence of bacteria detected by the DNAbinding SYTO™ 13 staining and the signal of miR2118 expression predominantly overlap in 7-and 14-day-old nodule sections indicating the elevated expression level of miR2118 in colonized nodule cells F I G U R E 2 Promoter analysis of the NB-LRR-targeting MIRNA genes. Promoter analysis was conducted using the PLACE tool (Higo et al., 1999). The lists of motifs of the five MIRNA promoters were compared with each other using an online Venn-diagram drawing tool (http://bioinformatics.psb.ugent.be/webtools/Venn/). There were 44 common elements in all the five promoters (a). From them, we selected the potential nodulation-regulated motifs (NODCON2GM, OSE2ROOTNODULE) and the pathogen-responsive elements (BIHD1OS, WBOXATNPR1, WRKY71OS, WBOXHVISO1, WBOXNTERF3, GT1GMSCAM4), and plotted them by position within the promoter (b). The downward-and upward-pointing triangles mark the sites found on the forward and the reverse strands, respectively ( Figure S1). This result also revealed that no signal of miR2118 expression was detected by in situ hybridizations in root tissue and nonsymbiotic cells of root nodules. Furthermore, we tested the expression pattern of miR2118 by in situ hybridization in M. truncatula nodules induced by the exoY mutant of S. meliloti. No expression of miR2118 was found in nodules devoid of any bacteria further suggesting the incidence of bacterial presence and miR2118 expression (Figure 1c). These results indicate that (i) cells colonized by rhizobia could have a lower NB-LRR level than non-infected cells and (ii) higher NB-LRR gene expression level in non-symbiotic nodule cells might block the colonization of these cells by rhizobia.
Next, we investigated the spatial distribution of all of the other NB-LRR-targeting miRNAs during different stages of nodule development by in situ hybridization. In 4-day-old nodules, the expression of all NB-LRR-targeting miRNAs (miR2118, miR2109, and miR1507) was detected and distributed uniformly in the developing nodules, and no expression of the miRNAs was detected in the non-symbiotic tissues of the nodule ( Figure S2a). In 7-day-old nodules, we were able to detect the expression of all three NB-LRR-targeting miRNAs ( Figure S2b). Similarly, all three NB-LRR-targeting miRNAs were expressed in 14-day-old nitrogen-fixing nodules, but they showed different spatial distribution. The miR2118 was evenly expressed in the nodule meristem and the different symbiotic zones of the 14-day-old nodules. The miR1507 expression showed a gradient expression predominantly localized to the meristem, the infection, and intermediate zones of the nodules, while a lower expression was found in the nitrogen fixation zone. The spatial expression pattern of miR2109 was mainly restricted to the apical part of the nodule ( Figure S2c). We also investigated the spatial distribution of all of the other NB-LRRtargeting miRNAs in 14-day-old nodules elicited by the exoY mutant of S. meliloti and similarly to miR2118a, we did not detect the expression of any of these NB-LRR-targeting miRNAs in nodules devoid of rhizobia ( Figure S2d). The nodules produced upon these infection processes are not able to fix nitrogen, because the exoY mutant S. meliloti bacteria are entrapped in the infection thread. Since the expression level of NB-LRR-targeting miRNAs is very low in these nodules lacking bacteria, the level of NB-LRR proteins could be increased to a level that would contribute to the block of colonization of S. meliloti exoY mutant in these nodules.
| miR2118, miR2109, and miR1507 silence NB-LRR mRNAs
The M. truncatula genome encodes approx. 540 NB-LRR genes and more than 60% of these genes can be targeted by the NB-LRRtargeting miRNAs or by the phasiRNAs that are produced by the action of the NB-LRR-targeting miRNAs (Zhai et al., 2011). The upregulation of all three NB-LRR-regulating miRNAs during symbiotic interactions in the nodules predicts that they decrease the level of the mRNAs of their target NB-LRR genes. To confirm that the targeted NB-LRR gene expressions are indeed downregulated, we selected two NB-LRR genes targeted by each miRNA (miR2118, miR2109, and miR1507), respectively. We measured their expression level in symbiotic nodule by qRT-PCR at 4, 7, and 14 dpi with S. meliloti. As it was expected, the expression level of all the six investigated miRNAtargeted NB-LRR gene was suppressed in the symbiotic nodules compared either to the symbiotically insensitive region of roots (Figure 3a) or to empty nodules elicited by S. meliloti exoY mutant (Figure 3b).
However, the activity of NB-LRR genes showed differential suppression and temporal variation (Figure 3). These results show that the three NB-LRR-regulating miRNAs control the expression level of the NB-LRR genes and this regulation could contribute to the successful induction of symbiotic nodule development by facilitating the infection and the bacterial colonization of the root and the developing nodule tissue.
| Symbiotic nodule number is regulated by the expression level of NB-LRR-regulating miRNAs
Our model predicts that if the reduced level of NB-LRR gene expression is necessary to the formation and proper development of symbiotic nodules, the higher level of NB-LRR gene expression will interfere with the process resulting in the reduction of nodule number. To test this hypothesis, we generated composite M. truncatula plants, using A. rhizogenes-mediated hairy root transformation, expressing short sequences mimicking miRNA target sites (Franco-Zorrilla et al., 2007;Yan et al., 2012) that lead to the degradation of targeted miR2118 and hence lowering the level of the NB-LRR-targeting miR2118. This observation was in line with a previous report (Fei et al., 2015) where the same short sequences mimicking miR2118 target sites (MIM2118) were reported to reduce the level of NB-LRR-targeting miRNAs. In MIM2118 transgenic roots, we checked the expression level of an NB-LRR gene targeted by miR2118 and its expression level was elevated as expected ( Figure S3a). Next, we examined the hairy roots of the composite plants for the characteristics of root growth and nodulation by S. meliloti. We also confirmed the downregulation of miR2118 in all of the transgenic roots by northern blot analysis (Figure 4a). We found that under our growing conditions, the lower level of miR2118 had no significant effects on root growth characteristics. To test the early infection events during the downregulation of miR2118, we counted the microcolonies ( Figure S4a) and infection threads ( Figure S4b) on MIM2118 transgenic roots at 5 dpi with S. meliloti. We found no significant differences in any early infection events compared to control roots. Nevertheless, when we scrutinized the nodule numbers in the transgenic hairy roots showing reduced miR2118 expression level 3 wpi with S. meliloti, we found a significant reduction in the number of mature nodules compared to the control roots transformed with empty vector (Figure 4b,c), irrespectively of which of the two mimicking constructs was expressed. The mature nodules were similar in size, infected with bacteria, and displayed normal morphology on both the MIM2118 transgenic and control roots ( Figure S5). Based on these results, we concluded that the expression level of NB-LRR-regulating miR2118 correlated with the nodule number but had no effect on nodule morphology and rhizobial colonization.
To further confirm this conclusion, we tested the effect of symptomless virus infection on nodulation. We have shown that AMV infection of M. truncatula plants reduces the expression level of the NB-LRR-regulating miRNAs (Figure 1a). Furthermore, it has been previously demonstrated that the inhibition of the function of these miRNAs (miR2118, miR2109, and miR1507) leads to the upregulation of their target mRNAs (Fei et al., 2015). Based on these results, we speculated that AMV infection leads to the downregulation of the NB-LRR-regulating miRNAs that might cause the upregulation of its target NB-LRR genes similarly to the target mimicry plants reported earlier (Fei et al., 2015). It is also important to mention that AMV infection, beside downregulating the NB-LRR-regulating miRNAs, does not induce any visible phenotypes of M. truncatula plants ( Figure S6a). Therefore, the mock and the AMV infected plants were indistinguishable from each other by visual inspection. We infected five-day-old To confirm this hypothesis, we generated the overexpressing constructs of the same miRNAs (miR2118OX, miR2109OX, and miR1507OX) that were shown previously (Fei et al., 2015) to increase the abundance of miR2118, miR2109, and miR1507 in M. truncatula roots, to test their effect on nodulation. We generated hairy root composite M. truncatula plants using A. rhizogenes-mediated transformation system. Next, these plants were examined for root growth characteristics and nodulation by S. meliloti. We verified the overexpression of miRNAs in DsRed-positive transgenic roots by northern blot analysis 4 wpi with rhizobia. The results confirmed that transgenic roots of the plants transformed by the three miRNA overexpressing constructs had significantly higher miRNA expression levels ( Figure 5a). Under our growing conditions, overexpression of these miRNAs (miR2118, miR2109, and miR1507) did not cause any significant changes in the characteristics of root growth. Compared with empty vector transformed controls, the miRNA overexpressing F I G U R E 4 The decreased level of miR2118 induced by target mimicry constructs led to the reduction of nodule number on Medicago truncatula roots inoculated with Sinorhizobium meliloti. Target mimicry constructs reducing the expression level of miR2118a,b,c (MIM2118abc) and miR2118a (MIM2118a) respectively, controlled by the constitutively active Arabidopsis thaliana EF1α gene promoter were introduced into in M. truncatula roots using Agrobacterium rhizogenes-mediated transformation. (a) Small RNA northern blot analysis of the same transgenic roots displayed in panel b showed a decreased abundance of mature miRNAs in transgenic lines at 3 weeks post-inoculation (wpi). RNAs isolated from M. truncatula roots were separated on a 12% (wt/vol) polyacrylamide gel and subsequently transferred to a membrane and probed with radiolabelled DNA oligonucleotides for miR2118, and U6 snRNA as a loading control. (b) Representative images of nodulated transgenic roots 3 wpi with S. meliloti, transformed with MIM2118abc (abc) and MIM2118a (a) target mimicry constructs or with empty vectors (EV) identified by expressing the DsRed fluorescent protein.
Scale bars: 1 mm. (c) Average nodule numbers of at least 45 transgenic roots were counted for each construct at 3 wpi with S. meliloti F I G U R E 5 Constitutive overexpression of miRNAs in Medicago truncatula hairy roots generated with Agrobacterium rhizogenesmediated transformation resulted in an increased nodule number. NB-LRR-regulating miRNAs (miR2118, miR2109, and miR1507) were driven by the constitutive Arabidopsis thaliana EF1α gene promoter in M. truncatula transgenic roots. (a) Small RNA northern blot analysis of the same transgenic roots showed an increased abundance of mature miRNAs. RNAs isolated from M. truncatula transgenic roots were separated on a 12% (wt/vol) polyacrylamide gel. The RNA was transferred to a membrane and probed with radiolabelled DNA oligonucleotides for miR2118, miR2109, miR1507, and U6 snRNA as a loading control. (B) Representative images of nodulated transgenic roots, overexpressing miR2118 (2118OX), miR2109 (2109OX), and miR1507 (1507OX) or transformed with empty vector (EV), identified by expressing the DsRed fluorescent protein 4 weeks post-inoculation (wpi) with Sinorhizobium meliloti. Scale bars: 1 mm. (c) Average nodule numbers of at least 15 transgenic roots for each construct were counted at 4 wpi with S. meliloti transgenic lines showed no obvious differences in root length or lateral root density. In miR1507OX, miR2109OX, and miR2118OX transgenic roots we checked the expression level of NB-LRR genes targeted by each of these miRNAs and their expression level was decreased, as it was expected ( Figure S3). To test the early infection events during the overexpression of NB-LRR-regulating miRNAs, we quantified microcolonies and infection threads on the miRNA overexpressing (miR2118OX, miR2109OX, and miR1507OX) transgenic roots at 5 dpi with S. meliloti. We found significantly more microcolonies with all three miRNA overexpression constructs ( Figure S4a) and a significantly increased number of infection threads on transgenic roots overexpressing miR2118 or miR2109 ( Figure S4b) compared to empty vector transformed control roots. Next, we examined the nodule numbers in the transgenic hairy roots 4 weeks after S. meliloti inoculation. We found increased nodule numbers on transgenic hairy roots overexpressing miR2118, miR2109, and miR1507 compared to control empty vector transformed roots at 4 wpi with S. meliloti (Figure 5b,c). The mature nodules were similar in size, infected with bacteria, and morphologically looked normal in all three miRNA overexpressing transgenic and control roots ( Figure S5). Our result is in line with a report showing that the overexpression of miR482, another member of the miR482/2118 superfamily increases soybean nodulation (Li et al., 2010). These results suggested that the NB-LRR-regulating miRNAs are important components of nodulation by regulating the expression level of NB-LRR genes and, hence, promoting bacterial infection and development of nitrogen-fixing symbiotic nodules.
| CONCLUSIONS
The expression level of the three miRNA families (miR2118, miR2109, and miR1507) that regulate NB-LRR mRNAs posttranscriptionally is affected by the nature of plant-microbe interactions in legumes (see proposed model in Figure 6). In pathogeninfected plants, the expression level of these NB-LRR-regulating miRNAs decreases and in line with previous findings (Li et al., 2012;Shivaprasad et al., 2012) The expression level of the three miRNA families (miR2118, miR2109, and miR1507) regulating NB-LRR mRNAs post-transcriptionally is modulated by the nature of plantmicrobe interactions in legumes. In pathogen-infected plants, the expression level of these miRNAs decreases advancing pathogen inducible expression of NB-LRR proteins and thus activating defence mechanisms against pathogen attack. In contrast, the interaction with the nitrogen-fixing symbiotic bacterial partner upregulates the expression level of the same set of miRNAs and resulting in the downregulation of the NB-LRR defence genes. As a consequence, the plant's innate immunity is suppressed in the nodule during symbiosis. Red tildes represent miRNAs, green wavy lines represent NB-LRR mRNAs, and light blue bubbles show NB-LRR proteins decrease the basal level of the NB-LRR-regulating miRNAs resulting in efficient defence response. However, during symbiotic interaction, the increased expression level of the NB-LRR-regulating miRNAs lowers the transcript level of defence genes and therefore can provide a suitable niche to the symbiotic bacteria to infect the roots and colonize the developing nodules. Because the NB-LRR-targeting miRNAs can respond to both pathogens and symbiotic partners in M. truncatula, they could function as an elegant molecular switch to provide a fast and adequate response by increasing or decreasing the level NB-LRR mRNAs according to the nature of plant-microbe interaction.
In M. truncatula, these miRNAs target at least 114 NB-LRR genes that produce phasiRNAs that can target additional NB-LRR genes and in this way, it is estimated that this regulating cascade can efficiently target at least 60% of the plant's NB-LRR mRNA repertoire post-transcriptionally (Fei et al., 2015;Zhai et al., 2011). The generation of phasiRNAs by these miRNAs, therefore, constitutes an efficient amplification system to modulate the expression of a large set of target mRNAs. By modulating the expression level of NB-LRRregulating miRNAs, legume plants are able to fine-tune their response to the microbial partner in a cost-effective way. This mechanism along with other regulatory systems (Djordjevic, Mohd-Radzman, & Imin, 2015;Imin, Patel, Corcilius, Payne, & Djordjevic, 2018;Mortier, Holsters, & Goormachtig, 2012) could contribute to the control of nodule numbers developed on legume roots. The spatial distributions of these miRNAs within the nodule furthermore indicate that they control the invasion of non-symbiotic nodule cells by the symbiotic bacteria. However, the regulation of the NB-LRR genes by a miRNA switch could have a fitness cost during special circumstances. Virus infection can trigger the accumulation of NB-LRR mRNAs by downregulating the miRNAs, and as a consequence, the interaction with the symbiotic partner could be blocked or attenuated. In line with this hypothesis, a reduced number of developing nodules was observed on the roots of virus-infected alfalfa (Medicago sativa), and these virus-infected plants showed growth reduction when the nitrogen source was limiting (Ohki, Leps, & Hiruki, 1986).
Since miR482/2118 superfamily is present in many plant families (González, Müller, Baulcombe, & Puigdomènech, 2015), it is tempting to speculate that they might contribute to the regulation of both pathogenic and symbiotic interactions besides the legume family as well. | 2019-12-14T14:02:22.347Z | 2019-12-13T00:00:00.000 | {
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248654000 | pes2o/s2orc | v3-fos-license | The Role of Conversational Implicature in Daily Conversations – What Matters, Content or Context?
— In daily conversation, sometimes dialogue includes terms that vary entirely from the common phrases. From a linguistic perspective, the conversational implicatures are the speaker's intended meaning of the utterance. Conversation implications are the specific conversations between the speaker and the receiver by following the communications principles. It is the most significant component that has undergone argumentation in conversation theory. Grice's theory of dialogue inference is the possibility of providing meaning to the literal. In other words, people apply certain cooperative principles to communicate cooperatively. Thus, conversational implicatures have become one of the top research areas in pragmatics. This paper intends to explore the importance of conversational implicatures in day-to-day conversations in various contexts. It focuses on certain dialogues collected and integrated from the routine conversation. The outcome revealed that context plays a vital role in interpreting utterances. Therefore, there is no possibility of a complete correspondence of one utterance to one context, which shows the conversational implicature cannot be context-independent. In addition, dialogues were classified into Generalized, Scalar, and Particular conversational implicatures.
I. INTRODUCTION
Conversational implicatures (CI) effects have been significant pragmatic issues (Khairunas et al., 2020). Separating senses and entailments from generic conversational consequences is a significant conceptual and methodological problem in semantics (Khairunas et al., 2020). The degree to which the context of the sentence decides what is said is a related topic. Grice developed an influential theory in order to anticipate and understand conversational consequences (Diliana, 2019). A core function of this theory is the Cooperative Principle (CP) and related maxims (Akmal & Yana, 2020). To some degree, Neo-Gricean theories replace the concepts of Grice and relevance theories with a communicative efficiency theory. Effervescent, lack of determinism, collisions, and speakers are the problems of principle-based theories. Usually, CI has fascinating properties, including calculability, cancelability, indeterminacy, and non-detachability (Suryadi & Muslim, 2019). These characteristics can be used to evaluate the definition of recognized implication in a conversation.
Language awareness encompasses all facets of human life (Yolanda, 2020). The taxonomy of CI is used to convey a special meaning to a conversation (Ali Milad, 2019; Khairunas et al., 2020). It involves exploring the benefits of creating good language skills a conscious understanding of the functionality of languages and how people learn and use them (Ismail, 2019;Pagin, 2019). Multiple research studies have been carried out in the linguistic field rather than in one interdisciplinary, concisely and complimentarily. Levinson (2000) described the CI as: (1) "Implicature stands as a paradigmatic example of the nature and power pragmatic explanations of the linguistic phenomenon." This shows an implicature is an additional meaning that indicates the influential aspect of pragmatics as a linguistic feature. (2) "It provides some explicit possibilities to mean (in some general sense) more than what is said". Here, implicatures provide an opportunity of interpreting the meaning of conversation in multiple dimensions. (3) "The notion of implicature seems likely to affect substantial simplifications in both the structure and the content of semantic descriptions". It means the concept of implicature deals with the simplicity of semantic components such as structure and content. (4) "Implicature … seems to be simply essential if various basic facts about language are to be accounted for properly". This reveals that the importance of implicature depends mainly on the consideration of different basic facts about language. Levinson (2000) asserts that implicature is a type of pragmatic inference related to some general principles of cooperative conversation. It goes above and beyond what is expressed explicitly in the process of interpretation. Moreover, Yule (1996) stated that "Implicature is an additional conveyed meaning." He means that an utterance can convey more than what is said. In other words, there are some implied meanings intended by the speakers which are not spoken explicitly in their utterances. Also, Allan k (2001) stated that CI is the principle device that allows speakers to minimize the quantity of language expressed. Chen (2020) defines CI as "Based upon non-truth-functional elements of the utterance, the addressee is permitted to make inferences about the views and intentions of the speaker. i.e., the audience must infer the expected and communicated meanings the speaker conveys. Morteza (2020) supports the definition by arguing that speaker communication is far richer than what they directly express. The linguistic meaning of a conversation radically underdetermines the message conveyed and understood.
The objective of the proposed study is to identify the Conversation Implicatures in daily conversations. It attempts to classify everyday conversations such as Generalized Conversation Implicatures, Particularized Conversation Implicatures, and Scalar Implicatures. It can support research scholars to expand their research in linguistics. In addition, the proposed study integrates conversations from academic and internet sources.
The organization of this study is as follows: Section I addresses the role of CI in expressing emotions and the objective of the research. Section II provides information about the existing literature and theoretical background of Pragmatics and CI. Section III covers the methodology and materials used in the study. Section IV and V discusses the outcome of the study. Finally, a summary of the work and future direction is presented in section VI.
A. Pragmatics
Pragmatics has been a significant area of linguistics since the early 1970s. However, it has still been debated and disputed whether it should be regarded as a field of linguistics (Na'mah & Sugirin, 2019). Despite this, pragmatics began appearing in "linguistic textbooks" only in the 1980s (Na'mah & Sugirin, 2019).
Grice, Searle, and Austin are considered to be supporters of pragmatics (Na'mah & Sugirin, 2019). They have either published a book or essay that significantly impacts the Pragmatics research, contributing to the growth of the field of study in linguistics. However, many linguists and critics stated linguistics as a branch of philosophy. The originators of linguists are philosophers, and they debated and implemented pragmatics from a metaphysical and logical view rather than a linguistic point of view. Grice's theory of conversational implicatures belongs to the prominent theories of conversations. Systematically, it explains the particular meaning of an utterance (Na'mah & Sugirin, 2019; Yolanda, 2020). Figure 1 illustrates the form of implicatures and their various forms. Pragmatics describes utterances that indicate particular events, deliberate actions of speakers at times, and locations with expression. Logic and semantics typically deal with the characteristics of terms of the token or their usage with the specific characteristic of their utterance to utterance.
The concept of Generalized Conversational Implicature (GCI) can be described as the inferences for the non-explicit interpretation that exists in any form of context (Grice, 1975). As long as no particular evidence rejects or contradicts it, knowledge is assumed in a prototypical manner. Particularized conversational implications (PCI), on the other hand, are THEORY AND PRACTICE IN LANGUAGE STUDIES 887 closely associated with particular or specific contexts, often called ad-hoc implicature. The effectiveness of these inferences is related to awareness about specific contextual details (Sadock, 1978;Grice, 1989;Frawley, 2003;Paltridge, 2006; Abdel-Karim A, 2020; Akmal & Yana, 2020). For context, if someone asked Shadan, "are you visiting Jaffer tonight," and Shadan responded, "I've to teach my kids," her response meant she isn't visiting Jaffer's house, even though she didn't say that. In contrast to non-explicit definitions, such as entailments or traditional consequences, PCI and GCI have one distinguishing characteristic. The scalar implicature (SI is centred on linguistic terms such as some or must, etc. Such phrases are indicative of an information-organized scale (Abdul-Kareem, 2019; Ali, 2020). Scales include:<Must/may>, <Many/some/all>, <Often/always/sometimes>, etc. are the instances of the information-organized scale (Chen, 2020). It lies between the utterance and its implication (Grice, 1975). For example, "Some of the employees did not get their salary", in this utterance, the term "some" implicates that only a few employees did not receive their salary, not all of them, which generates the SI.
Properties of Conversational Implicature
Several unique properties characterize conversational implicatures. Sadock (1978) briefly states these properties as: (1) Conversational implicatures can be "worked out" based on the Cooperative Principle. For instance, a. Nawaf has a flat. b. Nawaf has only one flat. c. Nawaf has one or more flats.
Here, (a) entails (c) but conversationally implicates (b). The hearer will assume that the speaker of (a) is following the conversational maxims. In particular, the Maxim of a quantity indicates that the speaker should be informative enough. Therefore, the hearer will assume that if the speaker knew that Nawaf had more than one flat, he would have said so. In addition, the hearer will understand the speakers' intention. Therefore, the hearer will deduce that the speaker has the correct information about how many flats Nawaf has. Consequently, the hearer will infer (b).
(2) Conversational implicatures are cancellable. For instance, I got some of these gifts from my friend -actually, I think I got most of them from her.
In the above utterance, by saying 'some' the speaker implicates that she did not get all of these gifts from her friend.
(3) Conversational implicatures are nondetachable. Expressions with the same linguistic meaning should generate the same implicatures relative to a fixed context. For instance,
a. Can you lend me $90 for a few days? b. Are you able to lend me $90 for a few days? c. Please, lend me $90 for a few days.
The above example shows that the three different linguistic expressions convey the exact intended meaning. Analysis of conversation includes the conversation among two or more speakers. It considers interaction and other types of human activity in exchange, for instance, look, gesture, body orientation and combinations. One of Grice core ideas is to ensure that the speaker complies with several principles when reading a sentence, which guarantees that communication is a cooperative endeavour. Grice names such values as the CP. As per the Maxim of quality, one cannot say anything without proper proof or evidence. According to the Maxim of quantity, a speaker should provide the most significant knowledge and not more than the requirement.
On the other hand, the Maxim of relevance signifies that a speaker should convey only relevant information. Lastly, the Maxim of manner teaches speakers to express orderly. To decipher an intention to communicate a speaker ("what's intended"), one draws knowledge on the speakers' state of mind and investigate whether they followed Grice's Maxim (Frawley, 2003;Paltridge, 2006; Abdel-Karim, 2020; Akmal & Yana, 2020).
B. Related Works
Ali (2019) presented a study applying CI and Grice theory to Arabic conversation. The author translated the conversation from Arabic to English and analyzed it using Grice cooperative principle. On the one hand, the concept of semantics deals with the meaning of an utterance. On the other hand, pragmatic concepts deal with using a term/word in a sentence. The author discussed the similarities and differences of both semantics and pragmatics. The outcome of the study shows that the Arabic speakers flouted the cooperative principle and generated CI in their conversation.
Diliana (2019) proposed a study on CI for investigating the conversation between Brebesnese friends. The author discussed the type of CI and its percentage of usage in conversation. Both GCI and PCI are frequently used in the conversation. However, the percentage of PCI is higher than the GCI. The results of the study have presented that the percentage of PCI was 72.2%, whereas the GCI was 27.7%. Furthermore, the study was conducted with limited number of participants. Thus, there is a possibility of variation in the percentage of GCI and PCI with a larger number of participants.
Ali (2020) proposed a study for investigating CI in English communications. In this study, the author focussed on the functional words, which are part of a dialogue in communication. A descriptive -pragmatic approach was followed for analyzing the communication. The author discussed the cooperative principle and its type. Internet and YOUTUBE were the sources for collecting the conversations. The outcome of this study shows that functional words are the key to generating CI.
Elizabeth and Radhika (2020) presented a study about an annotated dataset on CI. The authors have collected conversations related to CI from the resources of TOEFL (Test of English as a Foreign Language) and Internet Movie Script Database. They argued that a speaker could avoid an implicature by expressing a dialogue with explicit meaning. In addition, they classified the CI into SI, GCI, and PCI. The results of the study support researchers to utilize the dataset for further research in linguistics.
THEORY AND PRACTICE IN LANGUAGE STUDIES
According to the existing literature based on CI and CP related to linguistics, the following research questions are framed to analyze the role of CI in daily conversation.
Research Question 1 (RQ1) -Why are speakers using implicature in their conversations? Research Question 2 (RQ2) -What are the frequently used implicatures in daily conversations?
III. MATERIALS AND METHODS
In everyday conversation, communicators are utilizing CI frequently to express their opinion more concisely. Some conversations from daily situations were collected and analyzed. Also, the existing dataset for CI was used to extract some of the conversations. Moreover, some conversations were extracted from the implicature dataset (Bublitz & Lichao, 2010; George & Mamidi, 2020; Fitri et al., 2019; Ismail, 2019). The dataset is available online and free to access for researchers. A descriptive qualitative approach is adopted to classify the conversation into multiple types of CI and identify any violation of Grice's Maxim in daily conversation. The descriptive qualitative approach investigates each utterance in the conversation and classifies it into GCI, PCI, and SI. It is more comprehensive and includes gathering evidence from multiple sources so that the role of CI can be investigated in different viewpoints. It collects qualitative data, and the outcome is primarily qualitative. Induced data exploration is also required to define repeated subjects, designs or principles and explain the CI classifications. Thus, we investigated the violation of Grice' Maxim in the conversation in order to find why the speakers employ implicature. The crucial role of context or content in generating implicatures is also analyzed.
IV. FINDINGS
The non-literal definition refers to non-explicit significance. It is a term that includes many others, such as entailments, expectations, and consequences. Among them, special attention among pragmatists has been paid to the implications. Pragmatics describes utterances that indicate particular events, deliberate actions of speakers at times, and locations, generally with expression. The social rules play a crucial role in defining the features of the ideal communicative exchange (Grice, 1975). It decides the expectations of reasonable speakers about the other speakers' linguistic actions. In this section, we present the findings of this study by analyzing some frequent conversations.
A. Classification of Conversational Implicature
This study classified the CI into broader classifications, including PCI, GCI, and SI. Moreover, the cooperative principles are applied and find how speakers have violated Grice's Maxim in their conversation.
Particularized Conversational Implicatures
Particularized conversational implicatures are analyzed concerning special background knowledge. Yule (1996) states that most of the inferences are assumed in a particular context in which a conversation occurs. The researchers opine that the analysis of conveyed meaning needs inferences to particularized conversational implicatures. Some responses may deviate from relevance in most cases, as illustrated in the following Table 1.
TABLE 1 OUTCOME OF ANALYSIS OF CONVERSATION -PCI Conversation Implications
Amal: Are you coming to the party tonight? Rawan: I have exams.
As per Grice's theory, the terms yes or no would have been a relevant response to the question asked by Amal. However, Rawan's reply cannot be taken as completely irrelevant. Amal, being a student, is supposed to consider some background knowledge that would be mutually assumed. Rawan's reply could be interpreted as having to study for her exams. Therefore, she won't attend/ would not participate in the party tonight. Nasser: Did you enjoy your time? Did you like the movie? Naif: "There's nothing on at the movies." In this situation, Naif's response doesn't mean that there is nothing at all but nothing that he is interested in seeing. Doctor: the heart attack must be moved to the IC.U Nurse: I will do my best.
In this local context, to make the nurse's response relevant, the doctor has to draw on some tacit knowledge shared by all the participants involved in the medical field, excluding the co-patients and visitors. Suha: What on earth has happened to the grilled fish? Sara: Saly is looking very happy.
Suha derives the implicature " Saly ate the grilled fish " from Sara's statement in the above conversation. This is because Suha believes that Sara is observing the conversational Maxim of relevance in the specific context of Suha's question.
Therefore, the researchers derived the interpretations based on the study (Levinson, 2000). He stated that the Maxim of relevance is used in a particular situation, and the speaker utters a word relevant to a specific topic or issue. However, Abdul-Kareem (2019) claims that particularized implicatures are derived from the utterance and context. Thus, it is evident that PCI is context-dependent.
THEORY AND PRACTICE IN LANGUAGE STUDIES 889
Yule (1996) states that "when no special knowledge is required in the context to calculate the additional conveyed meaning, it is called a generalized conversational implicature", as illustrated in Table 2.
TABLE 2 OUTCOME OF ANALYSIS OF CONVERSATION -PCI Conversation Implications
Ilyas: Where were you? Mohamad: I entered a club, and a kid came running towards me.
In the above utterance, the indefinite article provides a clue that he is a guest or a visitor; therefore, the club and the kid do not belong to him. The researchers' view is that the speaker would have been more specific saying 'my club' and 'my kid'. Fawaz: How are we getting to the kingdom Tower tomorrow? Nawaf: Well, I'm going with Turki.
The use of "well" can conventionally implicate that what the speaker is about to say is not what the hearer is hoping to hear. In other words, Nawaf doesn't want to join with Fawaz as he is accompanying Turki.
Sultan: Can I get some sandwiches somewhere around here?
Hussam: There's a restaurant around the corner.
Hussam's response could be interpreted in different ways, as shown below: 1-Hussam is not interested in guiding Sultan.
2-
Hussam does not know what is served there. 3-Sultan can get sandwiches by himself from the restaurant around the corner. (Situation) The two friends were invited to a wedding party.
Suha: How much longer will you be?
Saja: Prepare yourself a cup of coffee.
Based on the conventional principle, Saja's indirect response indicates that Suha has to infer that Saja is not going to give a particular time or she intends to say ''Relax, it will take plenty of time to get ready".
To wrap up, in such type of implicature, inferences are made without taking into account the special contextual background knowledge of the utterance. It is evident that CI is context-independent (Fitri et al., 2019; Ismail, 2019). Mamidi (2020) confirms that generalized conversational implicature has little or nothing to do with the contextually relevant understanding of an utterance.
Scalar Implicatures
Scalar implicatures arise from specific words to express a scale of values to communicate information. Levinson (1983) listed the linguistic items from the highest to the lowest value: Speakers select values contextually suitable from the scales: <all, most, many, some, few >, < excellent, good >, < hot, warm >, < always, often, sometimes >, < certain that P, probable that P, possible that P >, < must, should, may >, < cold, cool >, < love, like > , < none, not all > and make decision on the basis of informativity and truthfulness. Table 3 presents the conversation and its implicature using SI. In this utterance, by using the word 'often', the speaker implicates that Omar does not always visit his grandmother on Friday. Otherwise, he prefers to see her on Friday rather than any other weekday.
Hayat: Did you complete your Ph.D.? Mona: I have written many research papers.
Using the word 'many', the speaker creates the implicatures (+> not all, +> not most). The implicature is that the speaker did not write all or most of the research papers that they must write. Yule (1996) In the above instances, how old are you? is interpreted differently following the context. In (A), it refers to Maha's age. Whereas in (B), it shows the father's anger towards his daughter's childish behaviour; and in (C), it shows that the doctor is surprised that the decision is being made by the husband instead of the wife.
V. DISCUSSION RQ1 aims to investigate the reason for employing CI in communication. The outcome of the current study presents that the implicature plays a pivotal role in everyday conversation. It reveals that speakers flouted the cooperative principle rather than unknown speakers. The known speakers know their environment, leading to use implicature to communicate successfully. For instance, Speaker A and B are long time friends. Thus, A communicates B with some previous inputs. Therefore, B replies A in a shorter form or utterance that implicates some meaning. The unknown speakers use GCI compared to PCI and SI. GCI is generated through standard terms, as discussed in the previous section.
To provide solutions for RQ2, the cooperative principle of Grice is applied to decide that context is playing a pivotal role in generating CI. Semantic interpretation can be a word (i.e. verbal or traditional meaning) and implicit meaning of a speaker (i.e. the importance of content to be conveyed by the speaker). Grice concluded that the hidden meaning of an utterance of speakers could be conveniently recovered due to the implied maxims set forth by the Cooperative principles. According to Grice, a hearer would consider both what the speaker said and what he may have said to measure the dialogue implication. The justification is based on the natural expression as well as its potential equivalents. Thus, the study's findings show that the conversations were made based on the context. According to the outcome, PCI is highly influenced by context, whereas GCI and SI partially depend on context.
VI. CONCLUSION
This study analyzed the role of conversational implicatures in daily conversation and how speakers violate the Grice maxims in their communication. Therefore, some conversational implicatures of everyday conversations in various contexts were analyzed. The study's outcome shows that context plays a critical role in generating implicatures rather than content. The outcome reveals that the particularized conversational implicature is mostly context-dependent. Moreover, generalized and scalar implicatures are partially context-dependent. The investigation of the violation of the Grice maxim supported that the conversational implicatures are context-dependent in most situations. Finally, the researchers stress that implicatures play a significant role in successful communication. Furthermore, the findings of this study are based on the conversations that were extracted from the sources. The future direction of this research is to 892 THEORY AND PRACTICE IN LANGUAGE STUDIES extend the analysis to the higher number of conversations and how a term is utilized in the conversation to imply multiple meanings. | 2022-05-10T16:03:52.215Z | 2022-05-04T00:00:00.000 | {
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225249553 | pes2o/s2orc | v3-fos-license | Wear of a high pressure layered pipe by a rough rigid bush
The article describes the solution to the problem of linear wear of a high pressure elastic pipe with an elastic coating by rigid bush worn on the pipe so that there is no gap between them. It is assumed that both the thickness of the coating in undeformed state and the shape of the inner surface of the bush are variable and depend on the coordinate along the axis of pipe. In the case when indicated characteristics are described by complex rapidly changing functions, standard approaches to solving such a problem turn out to be ineffective. The article describes an approach based on the use of special representations and basic functions that take into account the presence of complex characteristics. In the expression for contact pressures, which is the result of solving the problem and allows us to further evaluate the resource and durability of such a connection, the functions that describe the surfaces shapes are highlighted in explicit form. This allows us to perform exact calculations in cases where the forms are described by complex functions.
Introduction
In real constructions, pipes loaded with internal pressure are often used. External conditions determine the use of multilayer pipes, in which different layers serve for different purposes. Such pipes can be fastened with bushings, however, during operation due to vibration or torsion of the bush around the pipe, wear occurs, leading to a thinning of the outer layer. To assess the durability of such a connection, it is necessary to monitor the evolution of the stress-strain state of the pipe and evaluate its wear.
Mathematical model of contact-wear problem
Consider elastic pipe with inner radius r in and the thickness h in covered by thin elastic layer of variable thickness h out0 (z) and inner radius r out = r in + h in . We put rigid bush on such a tube. Inner radius of the bush is variable, depend on the coordinate z and less or equal to outer radius of pipe with coating in undeformed state, i.e. g(z) r out + h out0 (z). We assume that thickness h out0 (z) is much smaller than bush length 2a, inner radius of main pipe r in , and thickness of main pipe h in . The coating is considered soft compared to the lower layer (its rigidity not exceed the rigidity of lower layer [1]). It is also assumed that there is a smooth contact between layers and between bush outer layer along z axis. At the time τ 0 bush start to vibrate or rotate with angular velocity ω. Due to friction between the coating and the bush coating begins to wear out. At the same time we begin to load the pipe with internal pressure P in (t). The scheme for such a problem for the case of rotation is presented in the figure 1. It is obvious that the stress-strain state of the layered pipe and, in particular, the contact pressures under the bush will change over time. Further we will assume that the contact-wear region is constant and bounded by the boundaries −a and a (coordinates of left and right boundaries of the bush). We will construct solution only for the case when wear is less then coating thickness.
The movement of the outer boundary of the coating under the bush consists of displacement due to load and displacement due to wear.
A number of experiments show that the wear velocity linearly depends on the relative velocity of the contacting surfaces and on the normal load and is inversely proportional to the hardness of the material [2][3][4][5][6]: where H(z) is coating hardness, q(z,t) is contact pressure, k w is experimentally determined constant. The velocity V can be estimated with fretting-wear of surfaces, when the angular velocity is absent, however, the coating wears out due to vibrations of one body relative to another. In the case of rotation out out out ). Then the vertical displacement of outer surface due to wear can be determined by integrating of wear velocity over time from start moment until current time Based on the results of [7][8][9][10][11][12][13], it can be obtained that the displacement of the outer face of the coating, due to the load on the coating outer boundary and pressure on the pipe inner boundary, is represented by the expression ). , In this expression q(z,t) is contact pressure on outer surface of the coating (or normal load on outer surface on the region [−a,a]), E in and E out are Young's moduli of inner pipe and coating, ν in and ν out are , I 1 (u), K 0 (u), and K 1 (u) are Bessel functions. Note that a 2 << a 3 and E out < E in . Hence Θ a 1 /a 3 2(1 − ν 2 in )r 2 in r out /(r 2 out − r 2 in ). Since we assume that wear is much less than the thickness of the coating, then u w (z,t) << h out0 (z). Hence expression for displacement due to load (2) can be simplified Note that it is linear equation over q(z,t).
Summing displacements due to wear (1) and forces (3), we get following expression for displacement of outer surface of coating On the other hand, the displacement of the coating outer surface can be represented as follows where h out (z,t) determined by (2). Comparing expressions (4) and (5), we obtain the linear mixed integral equation of the problem We will make assumption that due to technological process of coating production its hardness H(z) is greater where coating thickness h out0 (z) is less (in areas with less thickness, the surface is hardened
Dimensionless form and solution of the problem
We introduce into (7) new variables and functions by the formulas Then mathematical model for contact-wear problem in dimensionless form Here I is identity operator. It is necessary to determine the function q*(z*,t*) from the obtained equation (8). Note that functions m*(z*) and g*(z*) in (8) relate with properties and forms of contacting surfaces; these parameters can be described by a rapidly changing function. Next, we will construct a solution for the dimensionless equation (8), and for brevity and convenience, we will omit the asterisks in notations of constants, variables, functions, and operators.
In integral equation (8), we make new kernel k(z,ζ) and unknown function Q(z,t) where R is Volterra operator with kernel −Ve −V(t − τ) (note that −Ve −V(t − τ) is kernel of resolvent for Volterra integral equation with kernel −V, see [14]). Then equation (8) take a form 5 We will see the solution of our problem in the class of time-continuous functions in a Hilbert space L 2 [−1,1] (e.g., see [15]). To this end we will construct orthonormal basis in L 2 [−1,1] from the linearly independent system of functions } , ) (12) Note that p j (z) (j = 0, 1, 2, ...) are polynomials. So we will seek the solution in the form where f k (t) is the desired function and the eigenfunctions Φ k (z) of the operator F are determined by solving the spectral problem for the operator F, The eigenfunction system Therefore, solving the spectral problem is reduced to solving the system of algebraic equations We substitute the representation (13) into the integral equation (10) and use (11), (12), (14), and (15) to obtain following expression for the functions f k (t), where W k are the Volterra operators whose kernels ] exp[ ) , are resolvents of the kernels −V/(1 + γ k ). Then the functions f k (t) can be represented as As a result, by using (9), (11)-(13), (15), we finally obtain the expression for the contact stresses | 2020-08-20T10:12:28.536Z | 2020-08-20T00:00:00.000 | {
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252210535 | pes2o/s2orc | v3-fos-license | Mathematical analysis of the effect of process conditions on the porous structure development of activated carbons derived from Pine cones
This paper presents the results of a study on the influence of the degree of impregnation and activation temperature on the formation of the porous structure of activated carbons (ACs) obtained from Pine cones by the chemical activation process using potassium hydroxide as an activator. The advanced new numerical clustering based adsorption analysis (LBET) method, together with the implemented unique numerical procedure for the fast multivariant identification were applied to nitrogen and carbon dioxide adsorption isotherms determined for porous structure characterization of the ACs. Moreover, the Quenched Solid Density Functional Theory (QSDFT) method was chosen to determine pore size distributions. The results showed a significant influence of the primary structure of Pine cones on the formation of the porous structure of the developed ACs. Among others, it was evidenced by a very high degree of surface heterogeneity of all the obtained ACs, irrespective of the degree of impregnation with potassium hydroxide and the activation temperature. Moreover, the analysis of carbon dioxide adsorption isotherms showed, that the porous structure of the studied ACs samples contains micropores accessible only to carbon dioxide molecules. The results also showed a significant advantage of the LBET method over those conventionally used for porous structure analysis based on Brunauer–Emmett–Teller (BET) and Dubinin–Raduskevich (DR) equations, because it takes into account surface heterogeneities. The novel analyses methods were more fully validated as a reliable characterization tool, by extending their application to the isotherms for ACs developed from the same precursor by phosphoric acid activation, and for samples arising from these ACs, further subjected to additional post-treatments. The effect of the raw material used as precursor was moreover analysed by comparison with previous reported results for other ACs. The complementarity of the results obtained with the LBET and QSDFT methods is also noteworthy, resulting in a more complete and reliable picture of the analyzed porous structures.
Materials and methods
The paper by Gomez-Delgado et al. 41 presents the results of an analysis of the influence of preparation conditions, on the formation of the porous structure of activated carbons developed from Pine cones. The method used to prepare the activated carbons was described in detail in 41 . Briefly, the preparation process was carried out in two stages: the first consisted of the carbonization of the precursor material at 773 K under a N 2 flow, while the second stage consisted of the thermal treatment of the carbonized precursor after being impregnated with KOH. In particular, the effects of the degree of impregnation, i.e., the ratio R of KOH to the carbonization product of the cones (R = 1, 2, and 3), and the activation temperature T (T = 873, 973, and 1073 K) on the development of the porous structure of the obtained activated carbons (ACLR, ACMR and ACHR, respectively), were analyzed 41 .
Porous structure analysis was performed based on nitrogen (N 2 ) adsorption isotherms determined at 77 K, and carbon dioxide (CO 2 ) adsorption isotherms determined at 273 K using Micromeritics ASAP 2020 HV for individual activated carbon samples. The morphological characterization of the precursor and the resulting activated carbons was performed by scanning electron microscopy (SEM), using a Zeiss Supra 40 electron cannon field emission microscope (Carl Zeiss AG, Oberkochen, Germany).
On the basis of adsorption isotherms determined, the values of specific surface area S BET from the Brunauer-Emmett-Teller (BET) equation 42 , the total pore volume V t from the volume of N 2 adsorbed at maximum relative pressure P/P 0 = 0.99, and the micropore volume V micro from the Dubinin-Raduskevich (DR) equation were calculated 43 . Bearing in mind the well-known limitations of the BET and DR methods, especially the lack of consideration for the surface heterogeneity, a concept has been developed to extend the conducted studies with a comprehensive analysis of the porous structure based on nitrogen adsorption isotherms using the LBET [44][45][46][47][48] and QSDFT [49][50][51][52][53][54][55] methods.
The LBET method, the theoretical foundations of the LBET models and their derivations, as well as the numerical fast multivariate procedure for adsorption system identification are described in detail in earlier publications [44][45][46][47][48] . Therefore, only the most important information about the LBET method necessary for the correct interpretation of the results will be briefly presented, including a detailed discussion of the parameters of the implemented LBET models. www.nature.com/scientificreports/ The LBET method considers the surface heterogeneity, the possibility of adsorbate molecule cluster branching, and the geometrical and energy limitations of the formation of clusters of adsorbate molecules. In this method, the process of adsorption on a heterogeneous surface is viewed as the clusterisation of adsorbate molecules in highly dispersed space limited by the geometry of micropores. The clusters are constructed by adding consecutive layers being in equilibrium with the volatile phase. The set of adsorbate molecules, which were adsorbed mainly due to adhesive interactions with the adsorbent matter, is treated as the first layer of adsorption. Joining of further adsorbate molecules is viewed as the second layer of adsorption [44][45][46][47][48] .
The adsorbate clusters considered in this theory are assumed to be constructed in configurationally independent ways, each starting with a unique primary site. In the LBET method, there exists a distinction between the two types of models [44][45][46][47][48] . The first model type refers to the adsorption system in which the limitation in the number of layers results from competing the physical adsorption. The second model type describes the systems in which limitations of cluster size result from pores size.
In addition, the LBET method enables the determination of the shape of the adsorption energy distribution on the surface of the material, thus giving much more information on the porous structure of activated carbons being analysed. In particular, the following porous structure parameters were determined using the LBET method and a method for fast multivariant identification of adsorption systems [44][45][46][47][48] : the dimensionless energy parameters Q A /RT and B C , corresponding to the maximum value of the adsorption energy on the first and subsequent layers, respectively; the volume of the first adsorbed layer V hA (cm 3 /g) interpreted as the volume of space accessible for the first adsorption layer; the geometrical parameter of the porous structure α determining the height of the adsorbate molecule clusters, α∈ (0-1) and α = 0 meaning that in pores of the analysed material only individual molecules are present; the geometrical parameter of the porous structure β determining the width of the adsorbate molecule clusters; the surface heterogeneity parameter, h, expressing the energetical heterogeneity of the surface.
The energetical heterogeneity of the microporous adsorption systems significantly and negatively impacts the numerical conditioning of the system identification tasks. To solve this problem, a unique fast multivariate method for fitting the LBET class models to the adsorption isotherms was proposed, which is also employed to define the value of the surface heterogeneity indicator h and the shape of the distribution of adsorption energy on the first layer [44][45][46][47][48] .
Additionally, it is calculated the dispersion of fitting error, σ e , expressing the quality of fitting the theoretical adsorption isotherm to empirical data. To make possible application of the LBET method for near and supercritical temperatures, an original fluid state model was elaborated.
Ethical approval. The pine cones samples were supplied by a logging company. They constitute a by-product of the activity. The forestry activity in Argentina is regulated by the Forest Law No. 26331, whose enforcement authorities are the Ministry of Agriculture, Livestock and Fisheries, and the Ministry of Environment and Sustainable Development of the Nation. The regulations of logging and forest care are included in the framework of the recommendations of International Union for Conservation of Nature (IUCN) Policy Statement on Research Involving Species at Risk of Extinction. Pinus canariensis has been categorized by IUCN, as a least-concern species. Besides, guidelines of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) are carefully taken in account for the forest activity.
E. Gomez Delgado, G.V. Nunell, P.R. Bonelli, and A.L. Cukierman carried out the formal characterization of the Pine cones used in the present study, according to the keys to Pinus canariensis species as thoroughly outlined and illustrated in "A Handbook of the World's Conifers" by Aljos Farjon (Volumes I and II, 2nd Revised edition, Brill, Leiden-Boston, pages 663-665, 2017).
No voucher specimen has been deposited in a publicly herbarium since there are only few pine species in South America, all being introduced, that can be easily identified.
Discussion of the results
On the basis of previous results of nitrogen adsorption isotherms analyses carried out by means of BET and DR methods, it was shown that with the increase in both the degree of impregnation as well as the activation process temperature, both the specific surface area and the volume of micropores of the obtained activated carbons increased significantly (Table 1).
Activated carbons obtained from Pine cones at impregnation ratio R = 3, were characterized by very large specific surface areas S BET as well as micropore volumes V micro ; the highest values of these parameters were determined for activated carbon ACH3 obtained at the highest activation temperature i.e. 1073 K (see Table 1).
The comparison of the micropore volume V micro and total pore volume V t also provides very interesting conclusions, which indicates that only micropores are present in the activated carbons analysed. However, the presence of micropores alone in the activated carbons analyzed, despite the fact that it is an extremely desirable feature in adsorption technologies, undermines the reliability of the results obtained using the BET method.
Application of the LBET method, also showed that increasing both the degree of impregnation R and the activation temperature, led to increase the values of V hA parameters determined for individual activated carbon samples. On the other hand, the value of the energy parameter Q A /RT decreases significantly with increasing the value of the impregnation degree R.
The values of the heterogeneity parameter h determined for the analysed samples indicate that the surface of the obtained activated carbons from Pine cones is strongly heterogeneous. The fact that such a value was obtained for all the analysed activated carbon samples indicates that the morphological structure of the original raw material and of the char obtained from it had a significant impact on the porous structure. The values of www.nature.com/scientificreports/ geometrical parameters α and β indicate that only single nitrogen molecules adsorb in the micropores of the analysed material. Nitrogen adsorption isotherms and adsorption energy distributions (AED) obtained by applying the LBET method to the experimental data for the activated carbons are presented in Figs. 1, 2 and 3.
The shapes of adsorption energy distribution (AED) for the first adsorbed layer determined for the analysed activated carbon samples based on the nitrogen adsorption isotherms indicate the occurrence of a wide range of adsorption energy on the first adsorbed layer.
As part of the research, pore size distributions (PSD) and cumulative pore volumes (CPV) were also determined based on the nitrogen adsorption isotherms using the QSDFT method [49][50][51][52][53][54][55] . They are presented in Fig. 4. The results show that both experimental variables investigated, namely the activation temperature and impregnation ratio, had influence on the development of pores and their characteristics. At constant temperature, increasing the impregnation ratio led to a greater amount of pores in the matrix of the activated carbons, as shown in the corresponding cumulative pore volume plots. Table 1. Parameters characterizing the microporous structure of the activated carbons developed by chemical activation of Pine cones with KOH, obtained with different impregnation ratios and process temperatures, based on the analysis of N 2 adsorption isotherms using the BET, DR, LBET, and DFT methods. Where: S BET -the specific surface area; V micro -the micropore volume; V t -the total pore volume; V hA -the volume of the first adsorbed layer, Q A /RT-the dimensionless energy parameter for the first adsorbed layer; B C -the dimensionless energy parameter for the higher adsorbed layers; α-the geometrical parameter of the porous structure determining the height of the adsorbate molecule clusters; β-the geometrical parameter of the porous structure determining the width of the adsorbate molecule clusters; h-the surface heterogeneity parameter; S QSDFT -the micropore specific surface area, V QSDFT -the volume of micropores. www.nature.com/scientificreports/ The PSDs of the activated carbons obtained with R = 1 or R = 2 reached diameters between 1.0 and 1.4 nm, but when R rose to 3 the upper limit of the PSD extended to ~ 2.0 nm. This can be attributed to the widening of the existing pores likely due to the rupture and weakening of the walls of the porous matrix, as might be inferred from certain signs of fragmentation and disintegrated particles in the SEM images for the activated carbons obtained with the highest R, that are later shown in Fig. 9. Accordingly, it appears that, at constant temperature, there was an over-activation effect when R = 3 was used, resulting in a widening of the distributions towards larger pore sizes. www.nature.com/scientificreports/ Likewise, as can be inferred from the cumulative pore volume plots, at constant R, the presence of micropores rises with increase in the temperature, even though the upper limits of the PSDs remain unchanged, thus indicating that the PSD broadness is more sensitive to changes in R.
The PSDs plots indicate that all the samples are microporous, showing pores especially in the supermicroporous range (0.7 nm-2 nm). This result is consistent with the pore diameter calculated using the N 2 isotherm since for all samples this diameter is around 1.8 nm, being the smallest diameter that can be obtained from the information given by the N 2 isotherm. This would also be in agreement with the data presented in Table 1, showing that the total volume of pores (V t ) and the volume of micropores (V micro ) are similar for all the activated carbons.
Physical adsorption of N 2 at 77 K is widely used for porosity characterization, because it covers a wide relative pressure range (from 10 -8 to 1 P/P 0 ). However, it has the disadvantage of possible problems of N 2 diffusion inside the narrow porosity (size < 0.7 nm). To overcome this problem, the use of other adsorbates at higher temperatures has been proposed. Carbon dioxide CO 2 adsorption at 273 K is considered an easy alternative to N 2 for the assessment of such narrow microporosity 56 . Despite the larger dimension of CO 2 molecules compared to N 2 , the higher adsorption temperature, in contrast to N 2 at 77 K, results in a larger kinetic energy of the CO 2 molecules, that are able to enter into the narrow pores, thus avoiding the diffusional problems above mentioned. Therefore, in the present study, carbon dioxide adsorption isotherms determined at 273 K were additionally analyzed for all the obtained activated carbon samples from Pine cones. The DR, LBET, and QSDFT methods were used for the calculations analogously to the nitrogen adsorption isotherms, and the results are summarized in Table 2 and shown in Figs. 5, 6, 7, 8.
As can be seen from the results collected in Table 2, as both the activation temperature and the degree of impregnation increase, the value of the parameter V micro , i.e. the volume of micropores calculated from the DR equation, increases; similarly, the values of the parameter V hA , i.e. the volume of the first absorbed layer calculated using the LBET method, also increases. It is pointed out that the increase in the values of the mentioned parameters is more influenced by the degree of impregnation compared to the influence of the temperature of the activation process. www.nature.com/scientificreports/ The values of the energy parameters for the first layer as well as for the higher layers, i.e. Q A /RT and B C , respectively, indicate strong interactions of the first layer with the surface of the tested activated carbon samples and much smaller interactions between the subsequent adsorbed layers.
In turn, the values of the surface heterogeneity parameter h determined for individual activated carbon samples indicate that the surface of these materials is strongly energetically heterogeneous. However, the values of geometric parameters α and β for most samples are practically similar to each other and indicate that high and significantly branching clusters of carbon dioxide molecules are formed in the micropores of these materials.
The exceptions are samples ACL1 and ACH3 for which lower values of geometric parameters β were obtained. This is related to the fact that in the case of ACL1, the microporous structure did not develop significantly, which was influenced by both the low degree of impregnation and the relatively low temperature of the activation process. On the other hand, in the case of activated carbon marked as ACH3, a lower value of the parameter β, in comparison with the other samples indicates the formation of narrower clusters of carbon dioxide molecules, while the structure is more developed, which is shown by the highest value of the V hA parameter among all the samples analyzed.
Comparing the results obtained from the analysis of carbon dioxide adsorption isotherms with those obtained from nitrogen adsorption isotherms, an earlier suspicion that the porous structure of the studied activated carbons samples may contain pores inaccessible to nitrogen molecules, but accessible to carbon dioxide molecules, was confirmed.
The values of energy parameters and especially geometric parameters of LBET method determined for carbon dioxide adsorption isotherms are very similar, so it can be concluded that in the case of the primary raw Table 2. Parameters characterizing the microporous structure of the activated carbons developed by chemical activation of Pine cones with KOH, obtained with different impregnation ratios and process temperatures, based on the analysis of CO 2 adsorption isotherms using the DR, LBET, and QSDFT methods. www.nature.com/scientificreports/ material used, its morphological structure has a significant effect on the development of the porous structure of the obtained activated carbons. The pore size distributions PSD and cumulative pore volumes CPV, shown in Fig. 8, were also determined as part of the work conducted.
Based on these pore size distributions, it can be seen that the range from 0.3 to 0.9 nm is found for all the analyzed activated carbons, that have a bimodal structure of the smallest pores. This observation indicates the significant influence of the morphological structure of the original raw material on the formation of the microporous structure of the obtained activated carbons, as stated earlier. The mentioned feature of Pine cones as a primary material opens up many possibilities for obtaining adsorbents with unique porous structures, including those designed for carbon dioxide sequestration processes. The presence of particles of different sizes and shapes can also be observed, possibly originated from fragmentation of the cell wall. On the other hand, it may be noticed that part of the original morphology of the precursor remained preserved after the activation process. This fact is more evident for the sample obtained with the lowest temperature and impregnation ratio (ACL1). In this image, it is even possible to visualize a fibrous structure composed of long channels arranged in parallel, showing that the directional pattern of the precursor vessels remained practically intact and without signs of structural collapse. Overall, the SEM images indicate that more severe conditions led to intensify the development of the porous structures of the activated carbons.
Furthermore, the effect of the chemical nature of the activator on the formation of the porous structure of activated carbons was also analyzed. For this purpose, the results of calculations for the activated carbons obtained by KOH activation were compared with the results of analyses for activated carbons obtained from the same precursor by H 3 PO 4 activation by Nunell et al. 58 . In the mentioned work, the activated carbons were obtained by activation of Pine cones using a 50% solution of H 3 PO 4 acid, at a weight ratio of activator to precursor equal to 2, at 723 K (carbons designated as ACP).
The resulting activated carbons were also subjected to additional heat treatment at 1073 K in an inert N 2 atmosphere (activated carbons designated as ACPT) and to impregnation with urea aqueous solution and subsequent heat treatment at 623 K (activated carbons designated as ACPU) 58 . The nitrogen adsorption isotherms Table 3. From the results obtained, it can be concluded that the activated carbon designated as ACP has a relatively large S BET specific surface area and a large pore volume. It was also shown from the LBET analysis that the volume of the first adsorbed layer defined by the parameter V hA is large. The values of energy parameters Q A /RT and B C point to favorable conditions for the occurrence of the multilayer adsorption process, and the value of parameter h indicates that the surface of the activated carbon obtained with the acid is strongly heterogeneous, showing the same value than the KOH-derived carbons. Table 3. Parameters characterizing the microporous structure of the activated carbons obtained by phosphoric acid activation (ACP) and subjected to thermal (ACPT) and chemical (ACPU) post-treatments, based on the analysis of N 2 adsorption isotherms using the BET, LBET, and QSDFT methods. www.nature.com/scientificreports/ The values of h decreases markedly for the post-treated activated carbons, thus indicating that the additional treatments promote strong modifications in the surface heterogeneity of the phosphoric acid-derived activated carbon. On the other hand, the values of geometrical parameters α and β indicate that high and non-branching clusters of adsorbate molecules are formed in the pores of this adsorbent.
LBET M No. V hA (cm 3 /g) Q A /RT
ACPT activated carbon subjected to an additional heat treatment is characterized by significantly lower values of S BET , V t and V hA parameters while having a lower degree of surface heterogeneity compared to the ACP sample without additional heat treatment (see Table 3). The ACPU activated carbon obtained by impregnation of ACP with an aqueous solution of urea and successive heat treatment is characterized by significantly lower values of S BET , V t and V hA parameters while having the lowest degree of surface heterogeneity among the analyzed samples. Figure 10 shows nitrogen adsorption isotherms and adsorption energy distributions (AED) obtained by applying the LBET method to the experimental data for the activated carbons prepared by chemical activation with phosphoric acid (ACP) at pre-established experimental conditions, and for the samples arising from the thermal (ACPT) and chemical (ACPU) post-treatments. Besides, pore size distributions and cumulative pore volumes calculated for these samples are displayed in Fig. 11.
The results clearly attest the pronounced influence of the activator used on the porous structure, indicating that phosphoric acid activation of the Pine cones led to development of larger pore sizes, mostly within the mesopore range, that remained in the post-treated samples although with a reduction in the pore volumes.
Comparison of research results with those obtained in previous publications
In this section the effect of the biomass primary raw material on the formation of the porous structure of activated carbons in the process of activation with potassium hydroxide is analyzed. For this purpose, the results presented above were compared with those obtained for activated carbons made from a variety of biomass raw materials by activation with KOH, reported in earlier publications [59][60][61] . The results in 59 include an analysis of the porous structure of activated carbons made from demineralized Kraft lignin by chemical activation using, inter alia, potassium hydroxide KOH. The activation was carried out at different temperatures T, i.e. 873 K, 973 K and 1073 K, with a mass ratio R equal to 3, and with different mass ratios of R hydroxide to raw material equal to 1, 2 and 3, at an activation temperature T = 973 K 59 .
Comparing the results of the porous structure analysis for the activated carbons obtained from Kraft lignin and Pine cones, a significant influence of the primary raw material on the formation of the structure of activated carbons and their adsorption properties may be inferred. Namely, comparing in particular the parameters of the porous structure of activated carbon obtained from Kraft lignin at 873 K, with a mass ratio of R = 3, it can be noted that the said activated carbon is characterized by a significantly higher specific surface area determined from the BET equation and the volume of micropores. Likewise, the comparison indicates a higher adsorption energy for the first adsorbed layer as well as a significantly higher value of the volume of the first adsorbed layer and a significantly lower degree of surface heterogeneity with respect to the activated carbon obtained from Pine cones under identical preparation conditions.
There are also significant differences in the determined values of the geometric parameters of the LBET models. Namely, the values of the mentioned parameters calculated for the activated carbon obtained from Kraft lignin by KOH activation at 873 K, with a mass ratio of 3, indicate that very high and unbranched clusters of nitrogen molecules are formed in the micropores of this material. In contrast, the values of geometric parameters determined for activated carbon obtained from Pine cones under the same preparation conditions indicate that only single adsorbate molecules are adsorbed in the pores. In the case of activated carbons made from Kraft lignin, at the activation process temperature of T = 973 K, with a mass ratio of R = 3, a very high value of specific surface area, volume of micropores, as well as volume of the first adsorbed layer was obtained, not only in comparison with the sample of activated carbon obtained from Pine cones under the same conditions but also in comparison with activated carbon obtained from Kraft lignin at 1073 K 59 .
Analysis of the porous structure of activated carbons obtained at 1073 K also leads to interesting conclusions. As already mentioned in the case of activated carbons obtained from Kraft lignin, the sample obtained at this temperature was characterized by lower values of specific surface area, volume of micropores and volume of the first adsorbed layer, indicating the destructive effect of higher temperature on the pore structure, i.e. on the burning of walls between adjacent pores. The situation is different for activated carbons obtained from Pine cones, namely, at 1073 K, activated carbons with the largest values of specific surface area, volume of micropores and volume of the first adsorbed layer were obtained among carbons obtained from Pine cones. It is also worth noting that samples of activated carbons obtained from Kraft lignin were characterized by significantly lower degrees of surface heterogeneity compared to samples obtained from Pine cones, which, regardless of the temperature of the activation process, had the highest degree of surface heterogeneity (h = 9).
Analysis of adsorption energy distributions determined by the LBET method and pore size distributions determined by the QSDFT method also provide important information. As shown in the article 59 , the shapes of adsorption energy distributions on the first layer determined for activated carbons obtained from Kraft lignin indicate the existence of a wide spectrum of medium-energy sites. In contrast, activated carbons obtained from Pine cones showed the existence of a significant proportion of high-energy sites.
Equally important information about the analysed materials was provided by pore size distributions determined by the QSDFT method, namely, it was shown that activated carbons obtained from Kraft lignin were characterized by a wide pore size distribution covering a range up to about 4 nm, while activated carbons obtained from Pine cones, covered a range limited only to micropores, i.e. up to 2 nm.
Interesting results are also provided by a comparison of the obtained structure parameters for activated carbons obtained from Kraft lignin and Pine cones at the activation process temperature T = 973 K and at mass ratios of activator to activated substance R = 2 and 3. As shown, the activated carbon prepared from Kraft lignin at 973 K and at a mass ratio R equal to 2 was characterized by a very high value of the specific surface area parameter-more than two and a half times that of the activated carbon prepared from Pine cones under the same preparation conditions. Activated carbon obtained from Kraft lignin, moreover, was characterized by twice the pore volume compared to activated carbon obtained from Pine cones at virtually identical volume of the first adsorbed layer. However, it is noteworthy that adsorbent obtained at 973 K from Kraft lignin at a mass ratio R equal to 2 was characterized by a very high degree of surface heterogeneity (h = 9), identical to those obtained from Pine cones. However, activated carbon obtained from Kraft lignin at the same temperature and mass ratio R = 3, was characterized by a much lower degree of surface heterogeneity.
Significant differences were also observed in the shapes of the adsorption energy distribution on the first layer for activated carbons prepared from Kraft lignin obtained at different mass ratios, in contrast to activated carbons obtained from Pine cones, for which the energy adsorption distributions (AED) are characterized by virtually identical shapes. A comparison of the pore size distributions (PSD) determined for the analysed activated carbons also provided interesting observations, namely, activated carbons obtained at R = 2 were characterized by a much narrower range of pore sizes, compared to activated carbons prepared at a mass ratio of R = 3.
Another article 60 presents the results of analysis of the structure of activated carbons produced from Pistachio nut, Hazelnuts and Pecan nuts shells. These adsorbents were obtained by a two-step process, i.e. carbonization at 773 K and KOH activation with mass ratios R = 1, 2, 3 and 4 at an activation process temperature of 1073 K. Comparing the results of the analysis of the structure of activated carbons prepared from the shells of the three www.nature.com/scientificreports/ kind of nuts with the results obtained for Pine cones activated carbons, it is noteworthy that the values of specific surface area and volume of the first adsorbed layer are significantly smaller, as well as the greater heights of clusters of adsorbate molecules in the case of activated carbons made from Pine cones. It is noteworthy that activated carbon with the largest volume of micropores, however, was obtained from Pine cones at a mass ratio of R = 3. Activated carbons obtained from the shells of the three kind of nuts were characterized by a high or very high degree of surface heterogeneity 60 but lower than for the activated carbons produced from Pine cones under the same preparation conditions. However, the exception was the activated carbon obtained from pecan shells at a mass ratio R of 3 and at an activation process temperature of 1073 K. This sample was characterized by the lowest degree of surface heterogeneity 60 .
The analysis of the shapes of adsorption energy distributions (AED) on the surface of the tested adsorbents and the determined pore size distributions also revealed differences in the porous structure of the obtained activated carbons related to the different structure of the primary raw material. Namely, the adsorption energy distributions determined for activated carbons obtained from Nut shells showed a predominant share of sites with high adsorption energy, while the aforementioned distributions determined for carbons obtained from Pine cones were characterized by a homogeneous distribution of sites with different energies. Some analogies between the porous structure of activated carbons obtained from Walnut shells and Pine cones were observed for the shape of the pore size distributions PSD. Namely, for lower mass ratios, much narrower PSDs were determined; however, the distributions determined for activated carbons prepared from Nut shells were bimodal, i.e., there were two peaks on the distributions corresponding to the proportions of dominant micropores and with an increase in the mass ratio of the activator to the activated substance, the proportion of larger micropores and mesopores increased, in contrast to activated carbons obtained from Pine cones, in which only micropores are present. However, if one compares activated carbons obtained from Nut shells at R = 1 and 2 with adsorbents obtained from Pine cones at mass ratios of 1, 2, and 3 respectively, one can see similarities in the pore size distribution. These similarities indicate not only similarities in the structure of the biomass material, but also the significant influence of the activator on the formation of the porous structure.
The article by Kwiatkowski and Broniek 61 presents the results of a study of the structure of activated carbons prepared from Mahogany, Ebony and Hornbeam wood, through a carbonization process at 773 K and a chemical activation process with potassium hydroxide at 1073 K, with mass ratios R = 1, 2 and 3. The aforementioned activated carbons obtained from high-density biomass materials were characterized by very high values of specific surface area, volume of micropores, and volume of the first adsorbed layer, which is not surprising given the properties of the primary raw material 61 . However, an interesting observation made in the aforementioned article was that as the mass ratio R increased from 1 to 2, the degree of surface heterogeneity increased, while already at a mass ratio R equal to 3, the surface heterogeneity decreased significantly. In comparison, in the case of activated carbons obtained from Pine cones, the degree of heterogeneity was constant and independent of the preparation conditions, which clearly indicates the specific properties of the original raw material.
Another feature of activated carbons resulting from the characteristics of the primary raw material is the shape and size of the clusters of adsorbate molecules are formed. In the case of activated carbons obtained from hard wood species at R = 1, medium-height clusters of adsorbate molecules were formed, and at a mass ratio of R = 2, significantly higher clusters of nitrogen molecules were already formed, while already at R = 3 in the case of activated carbons obtained from Ebony wood and Hornbeam wood, only single nitrogen molecules were adsorbed in the pores 61 . There are also significant differences in the shapes of adsorption energy distributions (AED) determined for activated carbons obtained from different woods and Pine cones, namely, in the case of activated carbons obtained from Mahogany, Ebony and Hornbeam woods, with a mass ratio of R = 1, the shapes of the AED plot indicate the dominant contribution of high-energy sites. On the other hand, for activated carbons obtained from Mahogany and Ebony wood at a mass ratio of R = 2, the shape of the adsorption energy distribution shows a dominant share of medium adsorption energy sites. While the shapes of the AEDs plots for activated carbons obtained from different types of wood at R = 3 indicate the presence of a wide range of adsorption sites with different adsorption energies 61 . In contrast, in the case of activated carbons obtained from Pine cone, the shapes of the AEDs plots point to the presence of a medium-width spectrum of sites with different adsorption energies on the first layer. Note that the shapes of the adsorption energy distributions for activated carbons obtained from Pine cones at different R mass ratios are very close to each other, indicating the dominant influence of the primary raw material on the formation of their adsorption properties.
A comparative analysis of the shapes of the PSDs plots determined for activated carbons obtained from different types of wood and Pine cones indicates both some analogies and differences due to the different structure of the primary raw material from which the said carbons were obtained. In the compared activated carbons, as the mass ratio of activator to activated substance increased, an increase in distribution width was observed, covering an increasingly wide range of pore sizes. However, the distributions determined for activated carbons obtained from Mahogany, Ebony and Hornbeam wood, have a bimodal structure of micropores, and with an increase in the mass ratio R, there is a successive increase in the proportion of larger micropores and small mesopores, i.e., up to about 4 nm 61 . In the case of activated carbons obtained from Pine cones, analogously, as the mass ratio value increased, the width of the PSD plot increased, covering, however, at R = 3 the maximum only the range of micropores, i.e., up to 2 nm.
Based on the analysis of the various experimental results presented above, it is shown that using identical preparation conditions, activated carbons with different adsorption properties are obtained from different biomass materials, which indicates that the nature of the primary raw material has a dominant influence on the formation of the porous structure of the activated carbons obtained from it. Therefore, it is reasonable to search for new materials for the production of activated carbons, especially of biomass origin, which is the subject of many research works. However, it should be emphasized that in order to properly select preparation conditions taking into account the nature of the primary raw material to obtain a carbon adsorbent with specific adsorption www.nature.com/scientificreports/ properties, a detailed study of the effect of preparation methods and conditions on the formation of the porous structure is required. Such studies require suitably precise and reliable methods for assessing adsorption properties and the forming porous structure, especially the complementary analysis by LBET and QSDFT methods. As part of the analysis, the unique characteristics of activated carbons obtained from Pine cones were demonstrated, including the presence of only micropores in their structure, thanks to which they can be used in advanced adsorption techniques and technologies, including in particular carbon dioxide sequestration.
Conclusion
A thorough analysis of the porous structures of activated carbons developed by chemical activation of Pine cones with KOH has been carried out by applying novel advanced numerical methods to N 2 and CO 2 adsorption isotherms obtained for their characterization. The LBET analysis applied showed that the activated carbons were characterized by a very high degree of energy heterogeneity, very large values of the volume of the first adsorbed layer as well as specific surface areas. The analysis of nitrogen adsorption isotherms showed that single adsorbate molecules adsorb in the pores of the studied activated carbons. They also exhibited huge amounts of narrow micropores, as characterized in depth by the analysis applied to CO 2 adsorption isotherms. This feature is of significant importance for the potential use of the analysed samples in carbon dioxide sequestration and other processes.
The analyses also showed a significant advantage of the LBET method over popular methods of porous structure analysis based on BET, and DR methods. The LBET method made it possible to determine the size of micropores and the degree of surface heterogeneity. Nevertheless, it should be kept in mind that it is only an approximation of the real porous structure based to a large extent on the adsorption of its specific mathematical model as well as an approximate model of the mechanism of adsorption processes occurring on the surface of adsorbents. There are very complicated mechanisms occurring during the processes of chemical activation, and thus practically no possibility to predict them. Therefore, in order to obtain adsorbents with appropriate parameters of the porous structure, it is necessary to perform a series of experiments supplemented by a reliable analysis of the structure, using advanced methods.
Data availability
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222209444 | pes2o/s2orc | v3-fos-license | Diagnostic Accuracy of Next Generation Sequencing Panel using Circulating Tumor DNA in Patients with Advanced Non-Small Cell Lung Cancer: A Systematic Review and Meta-Analysis
Background/Objectives Until now, no meta-analysis has been published to evaluate the diagnostic performance of next-generation sequencing (NGS) panel using circulating tumor (ctDNA) in patients with advanced non-small cell lung cancer (aNSCLC). The aim of the study was to carry out a systematic review and a meta-analysis in order to determine the accuracy of NGS of ctDNA to detect six oncogenic driver alterations: epidermal growth factor receptor (EGFR); anaplastic lymphoma kinase (ALK); ROS proto-oncogene 1, receptor tyrosine kinase (ROS-1); serine/threonine-protein kinase B-RAF (BRAF); RET proto-oncogene (RET); and MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 in patients with aNSCLC. Methods MEDLINE/PubMed, Cochrane Library, Latin American and Caribbean Health Sciences Literature (LILACS), and Centre for Reviews and Dissemination databases and articles obtained from other sources were searched for relevant studies that evaluate the accuracy (sensitivity and specificity) of NGS using ctDNA in patients with aNSCLC. The studies were eligible when NGS of ctDNA was compared with tissue tests to detect at least one of the six oncogenic driver alterations. Diagnostic measures (sensitivity and specificity) were pooled with a bivariate diagnostic random effect. All statistical analyses were performed with software R, v.4.0.0. Results Ten studies were eligible for data extraction. The overall pooled estimates of sensitivity and specificity were 0.766 (95% CI: 0.678–0.835); 0.999 (95% CI: 0.990–1.000), respectively. Conclusions The analysis has demonstrated that the NGS panel using ctDNA has a high accuracy to identify the six actionable oncogenic driver alterations in patients with aNSCLC. Therefore, it can be considered a reliable alternative to guide the patients with aNSCLC to the right treatment who cannot undergo an invasive procedure or have insufficient tissue material for molecular tests.
INTRODUCTION
Lung cancer is the cancer with the greatest incidence all over the world (11.6% of all cases) and it also represents the main cause of cancer death. 1,2 The majority of the patients with lung cancer are diagnosed in metastatic stage which has a 5-year survival rate of 4.7%. 3 Among the histological types, non-small cell lung cancer (NSCLC) is the most common, representing around 80% to 85% of all cases in which approximately 40% are adenocarcinoma, 25% to 30% are squamous carcinoma, and 10% to 15% are large cell carcinomas. [4][5][6] In the era of precision medicine, the therapeutic decisions for lung cancer are very dependent on histological and molecular characterization. 7 NSCLC is considered a heterogeneous disease with diverse molecular characteristics. 8 NSCLC has become an eminent example of precision medicine among solid tumors. 9 In personalized medicine, patients are selected for a specific treatment based on the presence of specific biomarkers which indicates a greater chance of the patient to benefit from the treatment. 10 Therapeutic options for NSCLCs have increased significantly with the emergence of targeted therapies and immunotherapies. 10 The National Comprehensive Cancer Network (NCCN) guideline recommends that patients with aNSCLC should be tested for epidermal growth factor receptor (EGFR); anaplastic lymphoma kinase (ALK); ROS proto-oncogene 1, receptor tyrosine kinase (ROS-1); serine/threonine-protein kinase B-Raf (BRAF); MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 skipping; RET proto-oncogene (RET); neurotrophic receptor tyrosine kinase (NTRK); and programmed death-ligand 1 (PD-L1). 11 The NCCN guideline strongly advises the use of broad molecular profiling in order to identify rare driver mutations for which drugs may be available. 11 However, approximately 20% to 30% of patients with NSCLC have insufficient tissue material to assess oncogenic driver mutations. 12,13 In this situation, the NCCN guideline also recommends plasma testing in NSCLC patients in order to detect EGFR, ALK, ROS-1, BRAF, MET, and RET. 11 Liquid biopsy is a less invasive procedure that can access the bloodstream through a needle stick, avoiding the risks of tissue biopsies. Circulating tumor DNA (ctDNA) can be used to provide the same genetic information as a tissue biopsy necessary to interrogate key companion diagnostics. 14 Besides, liquid biopsy can also overcome other limitations of tissue biopsies such as detecting tumor heterogeneity and the molecular changes in cancer cells after they are exposed to therapy. [14][15][16][17] The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) recommends liquid biopsy not as a replacement for tissue biopsy but in cases that there is insufficient tumor tissue specimens or in cases where tissue specimens are not feasible. 18 The CAP/IASLC/AMP considers next-generation sequencing panel using ctDNA (ctDNA NGS) a reliable platform in which it can assess single-base variants, indels, copy number changes, and translocations and it can reach acceptable sensitivity and optimal specificity. 18 Until now, no meta-analysis has been published to evaluate the diagnostic performance of ctDNA NGS in patients with advanced NSCLC. Thus, we conducted a systematic review and a meta-analysis in order to investigate the diagnostic accuracy of ctDNA NGS in detecting the six oncogenic driver mutations: EGFR, ALK, ROS-1, BRAF, RET, and MET exon 14 in patients with advanced NSCLC.
Study Design
A comprehensive electronic search was performed and included studies that were published until May 2019 in the following databases: MEDLINE/PubMed, The Cochrane Library, Latin American and Caribbean Health Sciences Literature (LILACS), and Centre for Reviews and Dissemination. A gray literature search was also performed in order to detect non-indexed publications.
Search Strategy and Study Selection
Search strategy was defined in order to answer the following question: "Is the ctDNA NGS panel an accurate test to detect oncogenic driver mutations in patients with aNSCLC when compared to tissue genotyping method?" (Table S1).
Specific keywords and terms for each database were considered. The strategies used in each database are shown in Table S2. Article language was limited to English.
Studies were eligible when ctDNA NGS was applied to detect at least one of the following biomarkers in aNSCLC patients: EGFR, ALK, ROS-1, BRAF, RET, and MET exon 14 alterations. Also, studies must use any tissue genotyping method as the gold standard. Exclusion criteria included the absence of sensitivity or specificity data, the analysis of patients with diagnoses other than aNSCLC or healthy subjects. Two reviewers (Ho and Sebastião) evaluated eligibility of studies according to these criteria.
Data Extraction
Two reviewers (Ho and Sebastião) extracted data from all eligible studies. Name of first author, year of publication, histologic type of NSCLC, clinical stage, comparator ("gold standard"), and diagnostic results for EGFR, ALK, ROS-1, BRAF, RET, and MET exon 14 alterations-true positive (TP), false positive (FP), false negative (FN), and true negative (TN)-were collected from eligible studies. EGFR T790M was also considered in our analysis. Genomic alterations in EGFR, ALK, ROS-1, BRAF, RET, and MET exon 14 evaluated by tissue genotyping were considered the "gold standard."
Quality Assessment
The quality of included studies was assessed using the standardized instrument Quality Assessment of Diagnostic Accuracy Tests (QUADAS-2). QUADAS-2 is designed to assess the quality of primary diagnostic accuracy studies. This tool evaluates the studies based on four key domains: patient selection, index test, reference standard, and flow and timing. Two reviewers (Ho and Sebastião) evaluated the quality of eligible studies. 19,20
Statistical Analysis
To assess the test accuracy, data of TP, FP, FN, and TN were tabulated and stratified by study. These diagnostic data were used to calculate the pooled sensitivity, specificity, and diagnostic odds ratio (DOR).
Statistical analysis was performed using the summary Receiver Operating Characteristic (sROC) and the bivariate approach. The sROC is the standard method for meta-analysis of diagnostic accuracy. The bivariate model jointly analyzes the sensitivity and specificity, considering any correlation between these two parameters using a random effect model. 21 The heterogeneity between studies was measured by Cochran's Q test to test the inconsistency index (I 2 ) (p < 0.05 or I 2 >50%). 22 All statistical analysis was performed with software R, v.4.0.0. The bivariate was fitted by the mada package which is based on the bivariate model of Reitsma et al, bivariate random effects model. 21
Characteristics of Eligible Studies
Searches returned 477 citations that were published until May 2019. After screening using the predefined eligibility criteria, 10 studies were included ( Figure 1). 13,[23][24][25][26][27][28][29][30][31] General characteristics of the 10 studies included in the review are reported on Table 1. A total of 2116 results from patients with histologically-confirmed diagnosis of advanced NSCLC with ctDNA NGS were evaluated for the six oncogenic driver mutations. Only data from advanced clinical stages were considered in the study. Two studies selected also reported data from patients with NSCLC in early (I-IIIA) stages, but only data from advanced (IIIB-IV) stages were considered. All studies evaluated the accuracy of NGS ctDNA with tissue genotyping, which may have included polymerase chain reaction (PCR) testing, fluorescence in situ hybridization (FISH) and/or immunohistochemical (IHC), or Sanger sequencing. Exclusion reasons for full-text excluded citations are described in Table S3.
Quality of Eligible Studies
The methodologic quality of the studies was evaluated by QUADAS-2 and they are summarized in Table 2.
Diagnostic Accuracy
The data extracted from each study regarding the six oncogenic driver mutations is described in Figure 2 shows the plots of confidence regions of each study, describing the uncertainty of the pair of sensitivity and FP rate (1-specificity).
The pooled DOR, which is the general diagnostic test performance, was 616.5 (95% CI: 263.0-1445.0). Heterogeneity investigation was performed among included studies, but they were considered homogeneous (Cochrane's Q P = 0.437 and I 2 =0%).
As nine of 10 studies included in the analysis had a specificity of 100% and one study had a specificity of 99.9%, the sROC curve could not be generated.
DISCUSSION
The NCCN guideline recommends plasma testing to evaluate EGFR, ALK, ROS-1, BRAF, RET, and MET alterations when there is insufficient tissue material to guide the use of target therapies in patients with advanced or metastatic NSCLC. 11 The results demonstrated that NGS ctDNA has a high accuracy to detect the six oncogenic driver mutations.
The meta-analysis demonstrated that NGS ctDNA reached an optimal specificity of 0.999 (95% CI: 0.990-1.000) which is a very important result to give confidence in prescribing target therapies in patients who will not be FPs for the six oncogenic driver mutations evaluated. The sensitivity reached an acceptable value of 0.766 (95% CI: 0.678-0.835).
These results support the recommendation by CAP/IASLC/AMP, which suggests that patients with positive results for EGFR, ALK, ROS-1, or BRAF with ctDNA NGS should start first-line therapy, as the results are considered reliable. However, a negative result from ctDNA NGS for oncogenic driver mutations cannot exclude therapies and further investigation is required. 18 Therefore, the CAP/IASLC/AMP considers liquid biopsy not as a replacement for tissue biopsy but as an alternative when there is insufficient tumor tissue specimens or in cases where tissue specimens are not feasible. 18 It is important to highlight
RET
Abbreviations: ALK, anaplastic lymphoma kinase; BRAF, serine/threonine-protein kinase B-Raf; EGFR, epidermal growth factor receptor; FISH, fluorescence in situ hybridization; IHC, immunohistochemical; NGS, non-genetic sequencing; NTRK, neurotrophic receptor tyrosine kinase; MET, MET proto-oncogene, receptor tyrosine kinase; PCR, polymerase chain reaction; PD-L1, programmed death-ligand 1; RET, RET proto-oncogene; ROS-1, ROS proto-oncogene 1, receptor tyrosine kinase. a These studies also included patients in early stages but only data from advanced stage were considered. b Methods of tissue genotyping were not described. c NGS, PCR "hotspot" testing, FISH and/or IHC, or Sanger sequencing. JOURNAL OF HEALTH ECONOMICS AND OUTCOMES RESEARCH that this recommendation was published before the FDA approval for MET exon 14 target therapy and RET fusion target therapy. With the MET exon 14 and RET fusion target therapies approvals in other countries, more guidelines may recommend the detection of these oncogenic driver mutations with tissue and plasma tests. The comparator in systematic review was restricted to tissue genotyping in order to assess the sensitivity of the ctDNA NGS. However, the limitations of using tissue genotyping as the "gold standard" is the tumor heterogeneity which might be missed by tissue biopsies. 32,33 Therefore, tumor heterogeneity can reduce overall concordance between plasma and tissue. 34 Jiang et al. have shown that subjects with stage II-IV NSCLC showed more somatic mutations in plasma than tissue samples. 34 One of the studies, Leighl et al, 26 had a FP case in MET exon 14 that was detected by ctDNA NGS, but it was not detected by tissue genotyping. As this FP result was not evaluated with other ctDNA methodology as a reflex test, the MET exon 14 could be due to the heterogeneity of the tumor.
The present study demonstrated the feasibility of using ctDNA NGS in detecting six oncogenic driver mutations to help guide the target therapies in patients with aNSCLC. However, ctDNA NGS has also the potential to monitor patients' response to therapies (target and immune therapies) and resistance mutations. Currently, the use of ctDNA is limited to cancer in advanced stages due to its low concentration in the early stages. | 2020-10-09T05:05:52.503Z | 2020-09-14T00:00:00.000 | {
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4686132 | pes2o/s2orc | v3-fos-license | Dose-finding designs for cumulative toxicities using multiple constraints
Abstract This article addresses the concern regarding late-onset dose-limiting toxicities (DLT), moderate toxicities below the threshold of a DLT and cumulative toxicities that may lead to a DLT, which are mostly disregarded or handled in an ad hoc manner when determining the maximum tolerated dose (MTD) in dose-finding cancer clinical trials. An extension of the Time-to-Event Continual Reassessment Method (TITE-CRM) which allows for the specification of toxicity constraints on both DLT and moderate toxicities, and can account for partial information is proposed. The method is illustrated in the context of an Erlotinib dose-finding trial with low DLT rates, but a significant number of moderate toxicities leading to treatment discontinuation in later cycles. Based on simulations, our method performs well at selecting the dose level that satisfies both the DLT and moderate-toxicity constraints. Moreover, it has similar probability of correct selection compared to the TITE-CRM when the true MTD based on DLT only and the true MTD based on grade 2 or higher toxicities alone coincide, but reduces the probability of recommending a dose above the MTD.
INTRODUCTION
Dose-finding clinical trials are typically small studies with the goal of evaluating the safety and tolerability of a new drug or combination of drugs in humans. In cancer clinical trials, this aim is achieved by 18 S. M. LEE AND OTHERS determining the maximum tolerated dose (MTD) which is defined as the maximum dose level that satisfies a constraint based on a pre-specified target probability of dose-limiting toxicity (DLT) (Storer, 1989). Numerous methods have been proposed to estimate the MTD. Among them are algorithmic designs such as the 3+3 design, and model-based designs such as the continual reassessment method (CRM, O'Quigley and others, 1990). These methods assign doses in a sequential manner after each cohort of patients based on a binary outcome, presence or absence of a DLT during a fixed pre-specified evaluation window. Even though toxicity data is collected during the entire treatment period on an ordinal scale from 0 to 5 based on the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) (National Cancer Institute, 2009), a DLT is generally defined as any grade 3 or higher toxicity, or dose reductions and discontinuations in the first one or two monthly cycles of treatment. This definition of a DLT which is based on the chemotherapy paradigm, only takes into account severe toxicities shortly after the initiation of treatment. New anticancer therapies such are targeted therapies and immunotherapies are administered for longer periods of time. Thus, they can lead to late-onset toxicities, and long lasting moderate toxicities resulting in dose reductions and discontinuations. A recent paper by Lee et al. showed a case-example using data from dose-finding trials of a targeted therapy, and reported that the DLT rates over extended cycles was 50%, higher than the conventional target DLT rate in the range of 20-33% (Lee and others, 2016). In current practice, dose reductions and discontinuations after the initial cycles are often stipulated in the protocol with no regard to the dose-allocation or dosedetermination method, and in some instances dose reductions that occur after several cycles of treatment, and late-onset toxicities, are not taken into account in the final recommendation of the MTD (Margolin and others, 2001). However, in the context of novel cancer treatments, it is important to take these into account to ensure that the dose level identified is tolerable when considering longer periods of administration.
Consider, for example the dose-finding trial of erlotinib, a targeted cancer therapy, in combination with pemetrexed in patients with non-small cell lung cancer published by Ranson and others (2010). This trial was designed to evaluate five dose levels for up to six cycles of treatment. However, a DLT was defined as a grade 3 or 4 toxicity, any treatment-related toxicity leading to dose reduction or interruption for erlotinib in cycle 1, any treatment-related toxicity leading to delayed administration of pemetrexed in cycle 2, treatment-related death and any laboratory abnormality in the first cycle leading to dose reduction. A total of 20 patients were enrolled in the first four dose levels, with DLTs being observed in three patients at dose level 4. However, 19 out of the 20 patients experienced treatment-related toxicities and 6 withdrew from the combination prior to the end of the 6 cycles (2 experienced toxicities that led to treatment withdrawal at dose level 2, 2 experienced toxicities that led to discontinuation of pemetrexed, and 2 refused further treatment). Thus, while DLTs were not observed until dose level 4, indications of intolerability were present in dose level 2 with 2 of 6 patients needing treatment withdrawal. The recommended dose selected was dose level 3 based on the first two cycles of treatment, given that the dose reductions and treatment withdrawals at dose level 2 were observed in later cycles.
Several methods have been proposed for the inclusion of late-onset DLTs and separately for including information on gradations of toxicity severity. Cheung and Chappell (2000) introduced the time-to-event continual reassessment method (TITE-CRM) which allows for the inclusion of late-onset DLTs. others (2005, 2007) proposed methods that take into account a patient's entire sequence of administrations. Liu and Braun (2009) proposed a method that constrains the probability of DLT for all patients to increase monotonically with both dose and number of administrations received. All these methods, consider toxicity as a binary outcome. Separately, several methods have been proposed to include information on toxicity severity in the estimation of the MTD based on complete observations. Van Meter and others (2011,2012) proposed incorporating toxicity severity using proportional odds or continuation ratio models. Lee et al. proposed the continual reassessment method with multiple constraints (CRM-MC) which allows for the specification of various toxicity thresholds (Lee and others, 2011;Cheng and Lee, 2015). Another method has been proposed using longitudinal ordinal outcomes where the MTD is defined as the dose level associated to a DLT rate per cycle (Paoletti and others, 2015;Doussau and others, 2013). Furthermore, methods have been proposed for summarizing grades and types of toxicity into a continuous or ordinal toxicity severity score (Bekele and Thall, 2004;Yuan and others, 2007;Lee and others, 2012;Ezzalfani and others, 2013), and methods have been proposed to estimate the dose level associated with a target level of a toxicity score (Bekele and Thall, 2004;Yuan and others, 2007;Ivanova and Kim, 2009;Ezzalfani and others, 2013).
The aim of our work is to propose a method that can take into account partial information on both DLTs, and moderate toxicities, in order to avoid dose reduction or temporary treatment discontinuation over an extended number of cycles. In this setting, MTD is defined as the dose that satisfies not only a pre-specified DLT constraint, but also a pre-specified moderate-toxicity constraint, given a specified toxicity evaluation window that can encompass various cycles to account for late-onset and cumulative toxicities. To achieve this, we have extended both the TITE-CRM (Cheung and Chappell, 2000) and the CRM-MC (Lee and others, 2011;Cheng and Lee, 2015), introducing the notion of time to first moderate toxicity, as well as, time to first DLT. The new model is described in Section 2. We present the simulation studies based on the motivating example in Section 3 and end with a discussion in Section 4.
Model specification
Suppose we are interested in estimating the dose level associated with a target DLT probability p DLT , where D = {d 1 , d 2 , . . . , d K } are the K doses of interest. However, we are also interested in controlling the rate of moderate toxicities, and thus we specify a target grade 2 or higher rate of p MT . Let Z be an ordinal toxicity outcome, where Z = 0 if the patient does not experience a toxicity, Z = 1 if the patient experiences moderate toxicity without a DLT and Z = 2 if the patient experiences a DLT. Note that these categories are mutually exclusive. If in some instances, the definition of DLT (Z = 2) includes some grade 2 toxicities then those grade 2 toxicities would not be included in the definition of moderate toxicity (Z = 1). Moreover, let T MT be the time to the manifestation of the first moderate toxicity, T DLT be the time to the manifestation of the first DLT and c be the patient's follow-up time. Then, the toxicity outcome, can be redefined as follows, That is, Y = 0 if a toxicity has not been observed yet, Y = 1 if only a moderate grade toxicity has been observed thus far, but no DLTs and Y = 2 if a DLT has been observed.
Let Pr(Z ≥ t|x) denote the tail probability of Z given dose x, the MTD, θ , can be defined as the maximum dose that satisfies both pre-specified toxicity constraints, where t 1 < t 2 are the pre-specified toxicity thresholds of 1 and 2, respectively and p 1 = p MT > p 2 = p DLT > 0 are their respective target probabilities. To estimate the MTD, consider a generic working model,
S. M. LEE AND OTHERS
Assuming that the maximum toxicity assessment time frame is M and taking into account the censoring information, . Thus, the likelihood of the observed data at time c after n patients are enrolled is:
Dose assignment
The model parameter β can be estimated using likelihood or Bayesian methods. If the maximum likelihood estimateβ n exists, operationally the recommended dose for the (n + 1)-th patient is the minimum of the dose levels that minimizes the distance to each pre-specified target, that is, For example, if after n patients, the dose level such that F 1 (x;β n ) is closest to p MT is dose level 3 and the dose level such that F 2 (x;β n ) is closest to p DLT is dose level 4, the (n + 1)-th patient will be assigned dose level 3. It is worth noting thatβ n does not exist until all possible values of Z are observed. Thus, to estimate β using the maximum likelihood estimate, we take a staged approach which has been previously described in Cheng and Lee (2015) and is also illustrated in detail in Section 2.3.
If Bayesian methods are used for the estimation of β, the marginal posterior distribution of the dose levels that minimizes the distance to each pre-specified target can be obtained and the dose given to the next patient is, wherex MT indicates the marginal posterior median dose level based on grade 2 or higher toxicities and x DLT the marginal posterior median dose level based on DLTs. For example, if after n patients, the marginal posterior median dose level based on grade 2 or higher toxicities is dose level 3 and the posterior median dose level based on DLTs is dose level 4, the n + 1 patient will be assigned dose level 3. This is the same dose assignment approach used for the CRM with multiple constraints (Lee and others, 2011). It should be noted that if Bayesian methods are used, it can be used from the beginning of the trial.
Dose-finding for cumulative toxicities 21
Simulation study
Using the setting of the erlotinib and pemetrexed trial, we assume five dose levels (K = 5). Given that, we are interested in controlling for both the rate of DLT and moderate toxicities, two toxicity constraints are imposed. The first constraint is that the probability of grade 2 or higher toxicities is less than 0.50, that is, P(Z ≥ 1|x) ≤ p MT = 0.50. The second constraint is that the probability of DLT is less than 0.25, P(Z = 2|x) ≤ p DLT = 0.25. Even though the sample size for the study was 20, we consider a sample size of 24 given that only four dose levels were evaluated in the original trial. We also consider a sample size of 40 to evaluate the impact of sample size. The evaluation window is six cycles of treatment. We assume an empiric working model as proposed in Cheng and Lee (2015) and in line with the conventional empiric model often used for the CRM. Thus, F 1 (x; β) = x β 1 and F 2 (x; β) = x β 1 +β 2 . The skeleton was selected based solely on the DLT constraint using the indifference interval (δ) calibration approach by Lee and Cheung (2009). Given five dose levels, a target DLT rate of 0.25, and assuming the MTD is dose level 3 and an empiric dose-toxicity working model, the δ value for sample sizes of 24 and 40 are 0.07 and 0.06, respectively. Given the minor differences in the δ values, a δ of 0.06 which corresponds to 0.06, 0.14, 0.25, 0.38, 0.50, was used for both sample sizes. Moreover, in line with the original TITE-CRM which showed that the linear weight function yielded similar operating characteristics compared to more complicated weight functions, we assume that the weight functions are linear, that is, w T MT (c) = w T DLT (c) = c/6. Dose skipping was not allowed.
Two-thousand simulations were performed using both likelihood and Bayesian approaches. For the likelihood approach a three-stage design was implemented whereby cohorts of three were used in the first-stage until the occurrence of a grade 2 or higher toxicity. If only grade 2 or lower toxicities were observed, the TITE-CRM using the first constraint, P(Z ≥ 1|x) ≤ 0.50, was applied. If the occurrence of DLTs and no toxicities were observed, but no grade 2 toxicities, the TITE-CRM using DLT as outcome was applied. Once full heterogeneity was achieved, that is, at least one patient was observed with each possible value of the outcome at the end of six cycles, the proposed method was invoked. The second stage was skipped if full heterogeneity was achieved in the first-stage. Moreover, for our simulations, if in the first cohort grade 2 toxicities (but no DLT) were observed for all patients, we assigned the same dose for the next cohort. If in the first cohort DLTs were observed for all patients, we stopped the trial.
For the Bayesian approach, we considered two distinct parametrizations and associated prior distributions. In the first case each parameter β l , where l = 1, 2, is parametrized in the exponential form, that is β l = e γ l , where each γ l is assumed to have independent normal distribution with zero mean and standard deviation of 1.34 as prior distribution. This is the conventional parametrization and prior used for the empiric working function in the dfcrm package in R (Cheung, 2013;R Core Team, 2013). In the second case, we use the parametrization proposed for the CRM with multiple constraints (Lee and others, 2011), and assume independent exponential distributions with a rate of 1 as the prior distribution for each β l in line with the CRM convention (O'Quigley and others, 1990;Yuan and others, 2007). Based on these priors, we can use Markov Chain Monte Carlo (MCMC) to obtain the marginal posterior distribution of the maximum dose level which satisfies the individual constraints P(Z ≥ 1 |x MT ) ≤ p MT and P(Z ≥ 2|x DLT ) ≤ p DLT and in both cases assign doses based on the approach previously mentioned in Section 2.2. The MCMC were performed using R 3.2.1 (R Core Team, 2013) with rstan version 2.6.0. The same skeletons were used for both likelihood and Bayesian approaches.
To simulate cumulative toxicities over time we utilized a discrete-time Markov process whereby a transition matrix P was specified. The transition matrix is upper triangular given that it represents the maximal toxicity grade which can only worsen with time. All patients start at baseline without any toxicities and transition to states with maximal toxicities of grade 2 (moderate toxicity) or DLTs. Each step of the Markov process represents a toxicity assessment visit at the end of a cycle of treatment. Given that we are interested in three categories of outcomes a 3 × 3 transition matrix is specified for each dose 22 S. M. LEE AND OTHERS level. The maximum toxicity assessment time frame of six cycles is used to define the MTD, that is, the probability of maximal grades at state M, P 6 , is used for the definition of the MTD. Various different simulation scenarios are presented. The transition matrices used for generating the various scenarios are displayed in supplementary material available at Biostatistics online. In addition, for each data set patients are assumed to arrive either at a fixed rate of six patients in six cycles (i.e. one patient per cycle) or assuming that the number of patients follows a Poisson distribution with the same rate of the fixed accrual rate. In these cases, patient responses remained unchanged and only the entry time into the trial is altered. For each dose assignment, when using the proposed method, patients who have not reached the maximum time frame of six cycles are included in the likelihood based on the simulated outcome at a time c less than six with a respective linear weight of c/6 based on their observed follow-up.
Given that there are no comparison methods, we compared our results to the benchmark for the CRM with multiple constraints using complete observations after six cycles to assess the performance our method (Cheung, 2014). The benchmark is a theoretical design which provides an accuracy upper-bound given our design objective. It is only theoretical because it requires complete toxicity profiles which are not observed in practice. For the benchmark, the complete outcome distribution for all dose levels was specified based on the various scenarios, complete toxicity profiles were simulated based on the sample size of the trial, and the sample proportions for each of the dose levels were calculated using the simulated toxicity profiles for each constraint. The recommended dose level based on the benchmark for the CRM-MC with complete observations is the minimum of the dose levels that minimizes the distance between the sample proportions and each pre-specified target. Moreover, for the scenarios in which the two constraints coincide, we compared our results to those of the TITE-CRM using only the DLT constraint. Under those scenarios, the MTD for the proposed method and the TITE-CRM is the same. The Bayesian TITE-CRM was used with an empiric working function, a N (0, 1.34) prior for the model parameter, and the same skeleton mentioned above, 0.06, 0.14, 0.25, 0.38, 0.50. This is the conventional prior used for the empiric working function in the dfcrm package in R (Cheung, 2013;R Core Team, 2013) 3. RESULTS
Simulation trial
We illustrate the proposed method using a single simulated trial with 24 patients and the design parameters specified in Section 2.3. Figure 1 displays the dose levels assigned and the toxicity outcomes for the 24 patients in the trial. The x-axis is the study time in months. The y-axis is the dose level at which the patient was treated. Each number represents a patient's entry time into the study using a fixed accrual of one patient per month. A circled number indicates the time to first moderate toxicity and a squared number indicates the time to first DLT. This example is generated based on scenario 10 where the true MTD is dose level 2 with the constraint for grade 2 or higher toxicities being the limiting one. The parameters are estimated using likelihood estimation with a three-staged design with cohort sizes of three in the first stage. Thus, the first three patients were assigned dose level 1. The dose was escalated to dose level 2 for the next cohort (patients 4, 5, and 6) with no toxicities being observed by the third month. The second and third patient both experienced their first moderate toxicity by the fifth month and the second stage was invoked whereby the seventh patient was assigned using the TITE-CRM to dose level 1. The dose was escalated again to dose level 2 for patients 8 and 9. By the ninth month, full heterogeneity was present with the sixth patient having had their first DLT, using the proposed method, patient 10 was treated at dose level 2. With a new occurrence of moderate toxicity for patient 9 in the 10th month, the dose was still escalated to dose level 3 for patient 11 who had a moderate toxicity the first month. Thus, the dose was de-escalated to dose level 1 for patient 12. By the 12th month, patient 10 had their first moderate toxicity in dose level 2, and thus patients 13 and 14 remained at dose level 1. The remaining 10 patients were all assigned dose level 2 with two occurrences of moderate toxicity, one for patient 12 in dose level 1 and one for patient 19 in dose level 2. Two additional moderate toxicities were observed during follow-up for patients 22 and 24. At the end of the trial, the method recommended dose level 2 as the MTD, with grade 2 or higher toxicities reported in 6 out of 16 patients (38%) and one DLT reported.
Simulation results
The simulation results for a sample sizes of 24 are displayed in Figures 2 and 3 when data are generated using a fixed accrual rate of 1 patient per cycle. Figure 2 displays the probability of recommending the true MTD, and Figure 3 displays the probability of recommending a dose above the true MTD. The top five scenarios in each figure display scenarios where the true MTD based on grade 2 or higher toxicity and DLT constraints coincide. The bottom six scenarios display scenarios were the two constraints do not coincide. In scenarios 7-11, the true MTD based on grade 2 or higher toxicities is a lower dose level than that based on DLT. The white bars represent the proposed method, referred to as the TITE-CRMMC, using likelihood estimation. The stripe bars represent the proposed method using Bayesian estimation. The gray bars represent the results for the benchmark and the solid black bars the results using the TITE-CRM. For the scenarios when the two constraints coincided, the TITE-CRM with multiple constraints had a much higher probability of correct selection (PCS) compared to the TITE-CRM when the true MTD was dose level one (0.86 and 0.83 for the likelihood and Bayesian approaches, respectively versus 0.71) and similar performance, within 0.05, when the true MTD was higher. Moreover, in all of these scenarios having multiple constraints substantially reduced the probability of selecting a dose above the true MTD compared to the TITE-CRM. The difference depended on the true MTD and, as expected, decreased when the true MTD was a higher dose level. Thus, when the primary and secondary constraints coincide, the TITE-CRM with multiple constraints has similar PCS compared to the TITE-CRM, but is more conservative. The PCS for the TITE-CRM with multiple constraints is slightly worse than the 24 S. M. LEE AND OTHERS Fig. 2. Probability of correct selection (PCS) of the proposed method, referred as TITE-CRMMC, 24 patients. L stands for likelihood estimation using staged approach. B stands for Bayesian. Grade 2+ indicates the probability of grade 2 or higher toxicity. DLT indicates the probability of DLT. The true MTD based on each constraint is specified in bold.
PCS of its benchmark, but still within 0.01 to 0.08 across all scenarios. When the limiting constraint was the DLT constraint, suggesting that the additional constraint on moderate toxicities was unnecessary, the performance of the proposed method using likelihood estimation was slightly worse compared to the Dose-finding for cumulative toxicities 25 Fig. 3. Probability of recommending a dose above the MTD for the proposed method, referred to as TITE-CRMMC, TITE-CRM, but using Bayesian estimation the performance was slightly better in terms of PCS. However, the methods were not as conservative and recommended doses above the true MTD more frequently than the TITE-CRM (differences within 0.04 and 0.07). The TITE-CRM with multiple constraints performed well at selecting the true MTD when the constraint regarding grade 2 or higher toxicities was the limiting constraint. While it had lower PCS compared to when both constraints coincided (within 0.10 for the likelihood approach and within 0.07 for the Bayesian approach for all scenarios), in some instances the benchmark also had lower PCS, indicating that these are harder scenarios. The performance was also not affected by the number of dose levels between the true MTD based on the DLT constraint and the constraint based on grade 2 or higher toxicities as indicated by scenarios 7-9. Generally, the performance of the likelihood staged approach was similar to that using Bayesian estimation starting from the first patient. The likelihood approach performed slightly worse when the true MTD did not coincide and it was one of the higher dose levels (scenarios 6 and 11).
The PCS and the probability of recommending a dose above the true MTD improved for all methods using a sample size of 40. The relative performance of the methods was similar compared to using a sample size of 24. With the larger sample size, the results for the proposed method using likelihood and Bayesian estimation were more similar, especially in the few scenarios were there were slight differences using a sample size of 24 such as scenarios 6 and 11. The results based on a fixed rate of entry and assuming that the number of patients follows a Poisson distribution with the same accrual rate were very similar. The tables displaying the simulation results, as well as, the results for simulations assuming the number of patients enrolled in the study follows a Poisson distribution and for a sample size of 40 are available as supplementary material at Biostatistics online. The tables also display information on the number of patients that were assigned before full heterogeneity was observed to invoke both constraints. Using likelihood estimation and the staged approach, the median number of patients before full heterogeneity was observed ranged between 10 and 22 depending on the true probabilities of toxicities and the entry time. This could explain why under some scenarios the performance of the TITE-CRM with multiple constraints is similar to the TITE-CRM. Moreover, the two parametrizations used along with the Bayesian estimation yielded identical results (data not shown). Thus, only the results using the normal prior are displayed.
DISCUSSION
In this article, we propose a method for including partial information when imposing multiple constraints. This method is an extension of the TITE-CRM that includes in the model both time to first DLT and time to a first secondary event, such as grade 2 or higher toxicity. By including outcome and corresponding follow-up time prior to reaching the maximum time evaluation window in the modeling framework using a weighted likelihood during the conduct of the trial the method allows for the toxicity assessment window to be extended to evaluate late-onset as well as cumulative moderate and DLT which are disregarded when using current methods with assessment windows of one or two cycles. Thus, dose recommendations during the trial are based on partial information at a given time point. However, the final recommendation is based on complete observations and only depends on the failure time distribution through in-trial dose assignments, similar to the TITE-CRM.
Both moderate and late-onset toxicities are important to consider when estimating the MTD because they can lead to dose-reductions, dose-discontinuations, and intolerability over time. Indeed, the general assumption for cytostatic agents, such as erlotinib, is that the more cycles a patient is treated before progression the better it is. Moreover, unless a patient progresses, treatment should be given until a desired success outcome. This is why controlling both acute and cumulative toxicity is highly important. In this work, we have tried to achieve this goal by proposing a method that not only takes into account DLTs that occur during the first one or two cycles, but also DLTs and moderate toxicities occurring after long-term administration. This is illustrated in our motivating example where there was a need to control for the rates of both DLT and moderate toxicities, which did not necessarily occur in the first couple of treatment cycles. While a simple solution would be to redefine DLT based on a longer evaluation window and including moderate toxicities, extending the evaluation window can substantially increase the duration of a trial when using complete observations. Furthermore, including moderate toxicity in the definition of DLT and using TITE-CRM may identify an overly conservative dose when using conventional target DLT rates of 0.25% to 0.33%.
The method has good performance characteristics with sample sizes similar to those used for a single DLT constraint and performs better than the TITE-CRM with a single DLT constraint in the case that both the primary and secondary constraints for the individual toxicity constraints coincide. Moreover, as observed with the CRM with multiple constraints, having multiple constraints can help reduce the probability of selecting doses above the MTD while yielding a similar PCS. The method can be easily implemented in practice using either Bayesian or likelihood estimation. While the performance of the two are similar, the likelihood estimation may be preferred by clinicians given that the specification of a prior is not required and it is easier computationally. However, it also requires a staged approach based on the heterogeneity of responses.
Moreover, the proposed method can easily be extended to account for three constraints, a constraint for grade 2 or higher toxicities, a constraint for DLT, and potentially a constraint for life-threatening and deadly toxicities, or using a continuous or ordinal toxicity severity outcome with three relevant severity thresholds. The MTD is then defined as the maximum dose that satisfies all three constraints. From the practical point of view, there seems to be little use in applying more than three constraints. Given that the definition of mild toxicities is much more subjective and mild toxicities are very prevalent, imposing a constraint for mild toxicities should be carefully considered. Future dose-finding clinical trials should take into account cumulative toxicity at least in the final recommendation of the MTD if it cannot be done during the dose allocation process. Currently, there is a big gap between dose recommendation and everyday practice in which dose adjustment and modifications are routinely done according to toxicities observed after the first cycle. Therefore, early phase clinical trials should align with clinical practice, and stop focusing on the first cycle only, especially in the context of new anticancer treatments.
SOFTWARE
Software in the form of R code is available on GitHub and can be requested from the corresponding author (sml2114@cumc.columbia.edu).
SUPPLEMENTARY MATERIAL
Supplementary material is available online at http://biostatistics.oxfordjournals.org. | 2018-04-03T00:16:20.802Z | 2017-11-13T00:00:00.000 | {
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125388481 | pes2o/s2orc | v3-fos-license | Emulsion Flow Analysis of a Sensor Probe for Sustainable Machine Operation
Working fluids possess several applications in manufacturing processes, for instance lubricants in metals machining. Typical metal working fluids are formulated as oil-in-water emulsions. The maintenance of the physical stability of the working fluid during operation is a key factor for the sustainability of the relevant process. Therefore, continuous control of the working fluids stability and performance during machine operation is an essential tool for maintenance of the process performance. Turbidity measurement (TM) is a process technique for emulsion stability and quality assessment, where light transmission and absorption of an emulsion system is analyzed. However, for in-process measurement and fluid quality detection during the machine operation by TM, it is necessary to implement a transmission inline sensor into the working fluid flow line. The continuous flow measurement may cause problems for long-term sensor operation regarding, e.g., biofouling of the sensor optical glasses or erroneous measurements due to emulsion droplets segregation effects. In the present investigation, computational fluid dynamic (CFD) simulations have been adapted to obtain the emulsion flow conditions within a typical TM sensor probe, thereby allowing an assessment of the adhesion probability of microorganisms as well as droplet segregation effects. The simulation results indicate some temporal changes of the dispersed phase concentration in the detected emulsion flow. Due to droplet segregation in the emulsion, the flow velocity needs to exceed a certain value for reliable operation. It is shown here that in this flow regime microbiological attachments on the probe surfaces may be sufficiently avoided. A minimum critical flow velocity is derived to avoid biomolecule adhesion and thus durable operation of the sensor.
Introduction
Emulsions as dispersed multiphase systems are used as metal working fluids (MWFs) in material processing operations for lubrication and heat removal purposes [1].For a typical oil-in-water formulation of an MWF emulsion, the dispersed oil concentration amounts to 2-10 v/v% where the droplets have mean sizes of 0.1-2.0µm [2].MWF typically consist of different (mineral) oil mixtures and chemical additives to stabilize the emulsion, inhibit corrosion, kill bacteria via biocides, and avoid foam formation.These ingredients increase the performance of the MWF.The use of MWF in machining operation decreases mechanical stresses caused by shearing and friction in the contact zone between the tool and work piece.MWF further washes away chips and fines from the nascent metal Fluids 2017, 2, 9 2 of 10 surface.This way, rewelding of the newly formed surface is a preventative measure and the wetted surface is protected [3].MWF operation cycles within machining processes may last over several weeks and months.Due to metal working machining operation stresses, the MWF composition and morphology may change with time, resulting, e.g., in increased mean droplet size and broader droplet size distributions (DSDs), which may impact the MWF performance and, therefore, the machine operation efficiency and sustainability ( [1,4]).
In-situ and in-process emulsion quality measurements are to be applied for monitoring the change of the physical stability of the working fluid, where turbidimetry measurements (TM) has recently been successfully applied in different emulsion systems ( [5][6][7]).The turbidity spectrum of a MWF emulsion is influenced by the chemical composition, droplet size (distribution), internal structure, and concentration of the dispersed phase ( [5,8]).Measurement of the turbidity of an emulsion system requires contacting the fluid flow and optical access to the fluid.Therefore, during machine operation, sensor probes need to be dipped into the stream of the flowing MWF emulsion.The optical path through the liquid necessary for the measurement principle may be realized by an internal reflection arrangement.Thereby, the emulsion flows through a small channel within the sensor probe where a focused light beam passes the stream twice, forward and backward (see Figure 1).
MWF composition and morphology may change with time, resulting, e.g., in increased mean droplet size and broader droplet size distributions (DSDs), which may impact the MWF performance and, therefore, the machine operation efficiency and sustainability ( [1,4]).
In-situ and in-process emulsion quality measurements are to be applied for monitoring the change of the physical stability of the working fluid, where turbidimetry measurements (TM) has recently been successfully applied in different emulsion systems ( [5][6][7]).The turbidity spectrum of a MWF emulsion is influenced by the chemical composition, droplet size (distribution), internal structure, and concentration of the dispersed phase ( [5,8]).Measurement of the turbidity of an emulsion system requires contacting the fluid flow and optical access to the fluid.Therefore, during machine operation, sensor probes need to be dipped into the stream of the flowing MWF emulsion.The optical path through the liquid necessary for the measurement principle may be realized by an internal reflection arrangement.Thereby, the emulsion flows through a small channel within the sensor probe where a focused light beam passes the stream twice, forward and backward (see Figure 1).
Typical flow velocities of an MWF inside pumping lines of metal working machines are in the range 1 < < 2.5 m/s [9].Segregation effects for the different emulsion droplet sizes for flow around the sensor probe may impact the measurement performance and hence need to be evaluated.Additionally, due to microorganisms in the water-based emulsion, biological films inside the probe that will interfere with the measurement may form.To avoid adhesion of microorganisms to sensor surfaces, it has been shown that minimum wall shear stresses τwall > 3-10 Pa are necessary ( [10,11]), where wall shear stress levels τwall > 20-100 Pa are found to even completely remove existing bio-films from surfaces ( [12]).Thus, in the present investigation, the multiphase emulsion flow of a working fluid around a TM sensor probe will be analyzed for the derivation of flow regimes of sustainable process conditions.
Multiphase Flow Analysis
Multiphase computational fluid dynamic (M-CFD) simulations are utilized to evaluate the flow patterns within and around typical sensor probes used in turbidity measurements with a rectangular slit channel as shown in Figure 1 [7].Multiphase flow around cylindrical objects has already been intensively investigated ( [13,14]).Liquid droplets tend to segregate in regions with low fluid velocities, leading to an increased local droplet concentration which may cause a decrease of the physical stability of the droplets [15].
For low Reynolds numbers (Re = 5-40), recirculating vortices may arise downstream of a cylinder.As the Reynolds number increases (Re > 40-350), these eddies periodically detach from the rear surface of the cylinder, carrying the droplets away from the probe [16].The authors of [17] have shown that, for low Stokes numbers Typical flow velocities of an MWF inside pumping lines of metal working machines are in the range 1 < u < 2.5 m/s [9].Segregation effects for the different emulsion droplet sizes for flow around the sensor probe may impact the measurement performance and hence need to be evaluated.Additionally, due to microorganisms in the water-based emulsion, biological films inside the probe that will interfere with the measurement may form.To avoid adhesion of microorganisms to sensor surfaces, it has been shown that minimum wall shear stresses τ wall > 3-10 Pa are necessary ( [10,11]), where wall shear stress levels τ wall > 20-100 Pa are found to even completely remove existing bio-films from surfaces ( [12]).Thus, in the present investigation, the multiphase emulsion flow of a working fluid around a TM sensor probe will be analyzed for the derivation of flow regimes of sustainable process conditions.
Multiphase Flow Analysis
Multiphase computational fluid dynamic (M-CFD) simulations are utilized to evaluate the flow patterns within and around typical sensor probes used in turbidity measurements with a rectangular slit channel as shown in Figure 1 [7].Multiphase flow around cylindrical objects has already been intensively investigated ( [13,14]).Liquid droplets tend to segregate in regions with low fluid velocities, leading to an increased local droplet concentration which may cause a decrease of the physical stability of the droplets [15].
For low Reynolds numbers (Re = 5-40), recirculating vortices may arise downstream of a cylinder.As the Reynolds number increases (Re > 40-350), these eddies periodically detach from the rear surface of the cylinder, carrying the droplets away from the probe [16].The authors of [17] have shown that, for low Stokes numbers the droplets will recirculate behind the cylinder and then entrain to the wall.At higher Stokes numbers, this effect vanishes.In this paper, simulations were carried out assuming various mean-sized monodispersed droplets (no distribution).Typical droplet sizes of metalworking fluid emulsions for machine operation are in the order of 100 nm for fresh mixed emulsions up to several microns for aged fluids [18].This leads to Stokes numbers in the order of 5 × 10 −9 for minimal droplet size and inlet velocity to 5 × 10 −3 in the case of maximal droplet size and velocity, thus at minimum three orders of magnitude less than unity.
Measurement Facilities
The numerical flow simulation results are evaluated by experiments in a test flow channel and a typical metal working machine.The fluid velocity in the flow channel varied between 0.04 m/s and 2.5 m/s and in the metal working machine between 0.3 m/s and 3.5 m/s; therefore, the range of Reynolds numbers investigated is Re cyl = u inlet d cyl ν = 260-22,750.The emulsion concentration was varied in the range of 1-10 v/v% in the flow channel, whereas the concentration in the machine was constant at 5 v/v%.The MWF under investigation is a commercial emulsion system with a narrow mono-modal droplet size distribution and a mean droplet diameter of d mean = 150 nm.
Numerical Model of Multiphase Flow
The Eulerian-Eulerian approach has been utilized for the numerical investigation of the multiphase flow around the turbidity sensor, where continuous and dispersed phases are both treated by solving separate momentum, mass, and continuity equations.The governing conservation equations are numerically closed with the Reynolds averaged Navier-Stokes method (RANS).The non-conservative form of the momentum equation in an incompressible two-component multiphase flow [19] is used as where α is the phase fraction, u is the velocity, p is the pressure, g is the gravity constant, Ik c,p is the combined Reynolds and viscous stress, and S includes all momentum source terms ( [20,21]).The combined turbulent stress ν t and viscous stress ν equal the effective viscosity ν eff c = ν + ν t .As shown by [22], the viscosity of emulsions can change significantly, especially for nanodroplets smaller than ca.200 nm and high volumetric fractions.In the present case, however, since the simulations were carried out for droplets with mean sizes of 0.1-10.0µm, and a volumetric fraction of 0.05, which results in a relatively small interaction area between dispersed and continuous phases, the emulsion viscosity was assumed to be constant.This assumption is consistent with the observed experimental values, and predictions by emulsion viscosity models [22].Furthermore, the Eulerian-Eulerian model approach is a two-fluid model and is based on the individual properties of each fluid.Therefore, the viscosity of the emulsion is not pertinent to the model.The ratio of viscosities is not pertinent, either.As the turbulent viscosity also depends on the phase fraction, the equation can be solved using an empirical correlation, e.g., by using a turbulent dispersion response coefficient C t .This approach for a turbulent flow description is governed by the fact that the discrete phase turbulent momentum fluxes are directly linked to their continuous phase counterparts [20].This leads to an equation for the dispersed phase: where the indices p and c correspond to the particulate dispersed and continuous phase, respectively.The value for the response coefficient is usually set to a constant value of 1, which is adequate for phase fractions above approximately 5% according to [23].Note that this way particle turbulent dispersion is modeled while turbophoresis is neglected.The continuity equation is applied to fulfill the assumption of an incompressible flow.Three main forces act on the dispersed droplets and define the source term for the momentum transfer where V is the cell volume.The drag F d , lift F l and virtual mass force F vm are derived from empirical correlations and theoretical models [24].The drag force depends on the relative velocity u rel = u p − u c and the shape of the particle, given by its area towards the flow A and its drag coefficient C D .Multiple correlations identify the value of the coefficient C D as a function of the Reynolds number.
The standard model for rigid spherical particles is an expanded version of the derivation by [25] to ensure the correct limiting behavior in the inertial regime and valid for small emulsion particles with d p < 10 µm in liquid/liquid flows as Re p < 30, Eo < O(10 −3 ) and µ p /µ c >> 1 [26].In addition, the emulsifier builds a layer around the droplets, thus preventing internal circulation and, therefore, changes in C D .The swarm influence may be neglected for volume concentrations below 10 v/v%.M-CFD simulations were carried out with the OpenFOAM [27] 2.1.0solver "twoPhaseEulerFoam" using a hexahedron structured mesh as shown in Figure 2. The region around the cylindrical probe is refined by generating an o-grid type mesh structure that allows perpendicular cell adjustment towards the probe walls (compare Figure 2, left, top).Hence, numerical diffusion can be minimized, and convergence and mesh performance are improved.The first grid point wall distance is a critical factor due to the small dimension of the measurement slit in the probe and varied depending on the flow speed.Low distance correlation [28] was utilized, where the y + wall distance is kept between 1 and 4 for the k-ω-model.The grid structure has been checked for proper resolution and grid size dependency.The mesh is validated regarding to the dispersed mass flow through the slit and the pressure drop.A mesh with about 850 thousand hexahedron cells finally is used that offers reasonable computational times while deviating less than 10% from the results obtained when using 1.8 million cells.
Fluids 2017, 2, 9 4 of 10 adequate for phase fractions above approximately 5% according to [23].Note that this way particle turbulent dispersion is modeled while turbophoresis is neglected.The continuity equation is applied to fulfill the assumption of an incompressible flow.Three main forces act on the dispersed droplets and define the source term for the momentum transfer where V is the cell volume.The drag , lift and virtual mass force vm are derived from empirical correlations and theoretical models [24].The drag force depends on the relative velocity rel = − and the shape of the particle, given by its area towards the flow A and its drag coefficient .Multiple correlations identify the value of the coefficient as a function of the Reynolds number.
The standard model for rigid spherical particles is an expanded version of the derivation by [25] to ensure the correct limiting behavior in the inertial regime and valid for small emulsion particles with dp < 10 μm in liquid/liquid flows as Rep < 30, Eo < O(10 −3 ) and μp/μc >> 1 [26].In addition, the emulsifier builds a layer around the droplets, thus preventing internal circulation and, therefore, changes in CD.The swarm influence may be neglected for volume concentrations below 10 v/v%.M-CFD simulations were carried out with the OpenFOAM [27] 2.1.0solver "twoPhaseEulerFoam" using a hexahedron structured mesh as shown in Figure 2. The region around the cylindrical probe is refined by generating an o-grid type mesh structure that allows perpendicular cell adjustment towards the probe walls (compare Figure 2, left, top).Hence, numerical diffusion can be minimized, and convergence and mesh performance are improved.The first grid point wall distance is a critical factor due to the small dimension of the measurement slit in the probe and varied depending on the flow speed.Low distance correlation [28] was utilized, where the y + wall distance is kept between 1 and 4 for the k-ω-model.The grid structure has been checked for proper resolution and grid size dependency.The mesh is validated regarding to the dispersed mass flow through the slit and the pressure drop.A mesh with about 850 thousand hexahedron cells finally is used that offers reasonable computational times while deviating less than 10% from the results obtained when using 1.8 million cells.The physical properties of the dispersed phase (oil) were taken from [29] (a) and (b) and the continuous phase was water.The flow inlet into the channel is characterized by a plug flow with a uniform velocity, a constant particle concentration, and a zero pressure gradient, whereas velocity and concentration at the outlet are represented by zero gradient boundary conditions.
Results and Discussion
Figure 3 illustrates the time averaged mean flow field approaching the probe and through the slit channel in the probe by streamlines.The splitting behavior of the flow is obvious.The flow around the cylinder dominates the flow structure and subsequently that behind the small slit.The emulsion is partly sucked into the slit and around the probe.
Fluids 2017, 2, 9 5 of 10 uniform velocity, a constant particle concentration, and a zero pressure gradient, whereas velocity and concentration at the outlet are represented by zero gradient boundary conditions.
Results and Discussion
Figure 3 illustrates the time averaged mean flow field approaching the probe and through the slit channel in the probe by streamlines.The splitting behavior of the flow is obvious.The flow around the cylinder dominates the flow structure and subsequently that behind the small slit.The emulsion is partly sucked into the slit and around the probe.Thus, high gradients occur at the slit inlet and the velocity pattern is close to a block profile.This pattern cannot develop over the probe slit length of 4.5 mm, resulting in a high velocity gradient throughout the entire slit.Due to vortex shedding in the flow field behind the cylinder, the mean droplet volume fraction inside the sensor slit obtains a time-dependent behavior with a variation of about ∆α ≈ 1%.
Figure 4 illustrates a certain droplet concentration fluctuation in the probe channel for a free stream velocity of 0.03 m/s ( cyl ≈ 195).The droplet concentration in the initial phase < 5 s converges and develops into a time-dependent state for > 5 s to be described by a sinusoidal function.The cylinder of the probe defines the droplet movement because of the detaching eddies.This temporal effect correlates with the Strouhal number = • inlet (7) where the frequency f of drop concentration fluctuations is derived from the simulation with the characteristic length L as the diameter of the cylinder.The calculated Strouhal number calc = 0.227 for the concentration fluctuation is related to the Strouhal number of a cylinder cyl = 0.2 for the onset of the vortex shedding process at a Reynolds number cyl ≈ 140-260 [17].Thus, high gradients occur at the slit inlet and the velocity pattern is close to a block profile.This pattern cannot develop the probe slit length of 4.5 mm, resulting in a high velocity gradient throughout the entire slit.Due to vortex shedding in the flow field behind the cylinder, the mean droplet volume fraction inside the sensor slit obtains a time-dependent behavior with a variation of about ∆α ≈ 1%.
Figure 4 illustrates a certain droplet concentration fluctuation in the probe channel for a free stream velocity of 0.03 m/s (Re cyl ≈ 195).The droplet concentration in the initial phase t < 5 s converges and develops into a time-dependent state for t > 5 s to be described by a sinusoidal function.The cylinder of the probe defines the droplet movement because of the detaching eddies.This temporal effect correlates with the Strouhal number where the frequency f of drop concentration fluctuations is derived from the simulation with the characteristic length L as the diameter of the cylinder.The calculated Strouhal number Str calc = 0.227 for the concentration fluctuation is related to the Strouhal number of a cylinder Str cyl = 0.2 for the onset of the vortex shedding process at a Reynolds number Re cyl ≈ 140-260 [17].
Fluids 2017, 2, 9 5 of 10 uniform velocity, a constant particle concentration, and a zero pressure gradient, whereas velocity and concentration at the outlet are represented by zero gradient boundary conditions.
Results and Discussion
Figure 3 illustrates the time averaged mean flow field approaching the probe and through the slit channel in the probe by streamlines.The splitting behavior of the flow is obvious.The flow around the cylinder dominates the flow structure and subsequently that behind the small slit.The emulsion is partly sucked into the slit and around the probe.Thus, high gradients occur at the slit inlet and the velocity pattern is close to a block profile.This pattern cannot develop over the probe slit length of 4.5 mm, resulting in a high velocity gradient throughout the entire slit.Due to vortex shedding in the flow field behind the cylinder, the mean droplet volume fraction inside the sensor slit obtains a time-dependent behavior with a variation of about ∆α ≈ 1%.The normalized time and volume averaged droplet concentration in the probe slit for various mean droplet sizes in the range 0.1-10 µm are illustrated in Figure 5.For each mean droplet size, a separate simulation run has been performed.An increase of the mean droplet concentration with increasing droplet size is found with a maximum of the concentration variations for d p ≈ 7 µm.The normalized time and volume averaged droplet concentration in the probe slit for various mean droplet sizes in the range 0.1-10 µ m are illustrated in Figure 5.For each mean droplet size, a separate simulation run has been performed.An increase of the mean droplet concentration with increasing droplet size is found with a maximum of the concentration variations for ≈ 7 μm.During continuous MWF operation not only bacteria, but also fungi and yeast may appear that affects the machine operation and performance as well as the measurement reliability and accuracy [30].Even though in MWF operation the settling of monocultures of microorganisms is reported [12], their influence on each other is scarcely evaluated.Hence, microbes can act differently under certain circumstances while travelling through the sensor probe slit.Various types of microbes yield critical shear stress values to overcome adhesion ( [10,11,31]).Thus, the criteria of minimum wall shear stress can predict contamination of surfaces.
The calculated wall shear stress distribution in the probe slit channel of the sensor at a fluid velocity of 0.03 m/s is illustrated in Figure 6 as color plot on the slit surfaces.The cylindrical probe configuration can be seen from these results.The flow is separated to a flow around and through the slit.The fluid velocity can be kept high at the inner slit walls, where at the edges of the sensor the velocity gradients are very low.Even at high inlet velocities, the wall shear stress only increases slightly in these areas.Round edges might improve this part of the sensor probe to avoid bacterial contamination and strong differences in the maximum and minimum stresses.During continuous MWF operation not only bacteria, but also fungi and yeast may appear that affects the machine operation and performance as well as the measurement reliability and accuracy [30].Even though in MWF operation the settling of monocultures of microorganisms is reported [12], their influence on each other is scarcely evaluated.Hence, microbes can act differently under certain circumstances while travelling through the sensor probe slit.Various types of microbes yield critical shear stress values to overcome adhesion ( [10,11,31]).Thus, the criteria of minimum wall shear stress can predict contamination of surfaces.
The calculated wall shear stress distribution in the probe slit channel of the sensor at a fluid velocity of 0.03 m/s is illustrated in Figure 6 as color plot on the slit surfaces.The cylindrical probe configuration can be seen from these results.The flow is separated to a flow around and through the slit.The fluid velocity can be kept high at the inner slit walls, where at the edges of the sensor the velocity gradients are very low.Even at high inlet velocities, the wall shear stress only increases slightly in these areas.Round edges might improve this part of the sensor probe to avoid bacterial contamination and strong differences in the maximum and minimum stresses.The normalized time and volume averaged droplet concentration in the probe slit for various mean droplet sizes in the range 0.1-10 µ m are illustrated in Figure 5.For each mean droplet size, a separate simulation run has been performed.An increase of the mean droplet concentration with increasing droplet size is found with a maximum of the concentration variations for ≈ 7 μm.During continuous MWF operation not only bacteria, but also fungi and yeast may appear that affects the machine operation and performance as well as the measurement reliability and accuracy [30].Even though in MWF operation the settling of monocultures of microorganisms is reported [12], their influence on each other is scarcely evaluated.Hence, microbes can act differently under certain circumstances while travelling through the sensor probe slit.Various types of microbes yield critical shear stress values to overcome adhesion ( [10,11,31]).Thus, the criteria of minimum wall shear stress can predict contamination of surfaces.
The calculated wall shear stress distribution in the probe slit channel of the sensor at a fluid velocity of 0.03 m/s is illustrated in Figure 6 as color plot on the slit surfaces.The cylindrical probe configuration can be seen from these results.The flow is separated to a flow around and through the slit.The fluid velocity can be kept high at the inner slit walls, where at the edges of the sensor the velocity gradients are very low.Even at high inlet velocities, the wall shear stress only increases slightly in these areas.Round edges might improve this part of the sensor probe to avoid bacterial contamination and strong differences in the maximum and minimum stresses.Figure 7 illustrates the calculated occurring minimum and maximum wall shear stress τ wall inside the slit of the sensor, which increases exponentially with increasing inlet velocities u inlet .The minimal wall shear stress τ wall,min is typically located next to the edges of the slit, and the maximal wall shear stress τ wall,max is located at the verge of the slit.Thus, microbiological adhesion to the walls will more likely take place at the middle and edges of the slit channel.Even at high inlet velocities, the wall shear stress stays low in these areas.Therefore, short slits are superior to longer ones.Flow velocities in the range of 1-3 m/s of MWF emulsions in metal working machines will lead to wall shear stress of τ wall = 10-200 Pa (see Figure 7).For the examined sensor geometry, a correlation for the minimum and maximum wall shear stress dependent on the inlet velocity is ln(τ max ) = 1.152• ln(u inlet ) + 4.798 and ln(τ min ) = 1.402• ln(u inlet ) + 2.504. ( Fluids 2017, 2, 9 7 of 10 wall shear stress τ wall,max is located at the verge of the slit.Thus, microbiological adhesion to the walls will more likely take place at the middle and edges of the slit channel.Even at high inlet velocities, the wall shear stress stays low in these areas.Therefore, short slits are superior to longer ones.Flow velocities in the range of 1-3 m/s of MWF emulsions in metal working machines will lead to wall shear stress of τ wall = 10-200 Pa (see Figure 7).For the examined sensor geometry, a correlation for the minimum and maximum wall shear stress dependent on the inlet velocity is An increase of the fluid velocity directly leads to higher velocity gradients and therefore wall shear stresses.The calculated minimum wall shear stress extending the literature values of 3 Pa for avoiding, respectively, the resolution of biomolecules from surfaces is achieved at mean inlet velocities crit > 0.4 m/s.This threshold should be exceeded to avoid the biological impact on the turbidity measurements.
In order to study the impact of the MWF multiphase flow conditions around the sensor on the measurements, MWF turbidity spectra were detected in a test channel as well as in a machine during operation.The influence of the flow velocity and the droplet concentration on the detected spectra were investigated.Figure 8 illustrates the standard deviation σ between the turbidity spectra measured at a MWF test channel ( =0 ) and the in-machine operation turbidity spectra measured τ( = inlet ) for a concentration range of 1-10 /%: where m is the number of turbidity measurements in the spectra τ(λ).The standard deviation σ for all concentrations and velocities is about 10 −1 for the test channel and less than 10 −1 for the measurement in the machine.The lower standard deviation for the machine is caused by the somewhat aged MWF compared to the fresh MWF used in the flow channel.Figure 8 illustrates the spectra for MWF emulsion with 5 /%.The spectra do not change with flow velocity, indicating that there is no significant change of the concentration inside the slit for velocities up to 3.5 m/s in the metal working machine.The experimental data validate the CFD simulations, as no segregation effects have been detected in the velocity range typically occurring in metal working machines.An increase of the fluid velocity directly leads to higher velocity gradients and therefore wall shear stresses.The calculated minimum wall shear stress extending the literature values of 3 Pa for avoiding, respectively, the resolution of biomolecules from surfaces is achieved at mean inlet velocities u crit > 0.4 m/s.This threshold should be exceeded to avoid the biological impact on the turbidity measurements.
In order to study the impact of the MWF multiphase flow conditions around the sensor on the measurements, MWF turbidity spectra were detected in a test channel as well as in a machine during operation.The influence of the flow velocity and the droplet concentration on the detected spectra were investigated.Figure 8 illustrates the standard deviation σ between the turbidity spectra measured at a MWF test channel τ(λ u=0 ) and the in-machine operation turbidity spectra measured τ λ u=u inlet for a concentration range of 1-10 v/v%: where m is the number of turbidity measurements in the spectra τ(λ).The standard deviation σ for all concentrations and velocities is about 10 −1 for the test channel and less than 10 −1 for the measurement in the machine.The lower standard deviation for the machine is caused by the somewhat aged MWF compared to the fresh MWF used in the flow channel.Figure 8 illustrates the spectra for MWF emulsion with 5 v/v%.The spectra do not change with flow velocity, indicating that there is no significant change of the concentration inside the slit for velocities up to 3.5 m/s in the metal working machine.The experimental data validate the CFD simulations, as no segregation effects have been detected in the velocity range typically occurring in metal working machines.
Figure 1 .
Figure 1.Measurement arrangement of inline light transmission sensor probe with cylindrical shaft in a flow channel of machining process.
Figure 1 .
Figure 1.Measurement arrangement of inline light transmission sensor probe with cylindrical shaft in a flow channel of machining process.
Figure 2 .
Figure 2. Setup of the sensor probe arrangement and display of the main features of the mesh.The physical properties of the dispersed phase (oil) were taken from [29] (a) and (b) and the continuous phase was water.The flow inlet into the channel is characterized by a plug flow with a
Figure 2 .
Figure 2. Setup of the sensor probe arrangement and display of the main features of the mesh.
Figure 4 .
Figure 4. Time-dependent droplet concentration inside the sensor probe slit for a droplet size of 150 nm.
Figure 4
illustrates a certain droplet concentration fluctuation in the probe channel for a free stream velocity of 0.03 m/s ( cyl ≈ 195).The droplet concentration in the initial phase < 5 s converges and develops into a time-dependent state for > 5 s to be described by a sinusoidal function.The cylinder of the probe defines the droplet movement because of the detaching eddies.This temporal effect correlates with the Strouhal number = • inlet(7)where the frequency f of drop concentration fluctuations is derived from the simulation with the characteristic length L as the diameter of the cylinder.The calculated Strouhal number calc = 0.227 for the concentration fluctuation is related to the Strouhal number of a cylinder cyl = 0.2 for the onset of the vortex shedding process at a Reynolds number cyl ≈ 140-260[17].
Figure 4 .
Figure 4. Time-dependent droplet concentration inside the sensor probe slit for a droplet size of 150 nm.
Figure 4 .
Figure 4. Time-dependent droplet concentration inside the sensor probe slit for a droplet size of 150 nm.
Figure 5 .
Figure 5. Dependency of the droplet concentration in the sensor probe slit on the droplet size.
Figure 6 .
Figure 6.Mean wall shear stress distribution inside the probe slit for an inlet velocity of inlet = 0.03 m/s and a drop size of 150 nm.
Figure 7
Figure 7 illustrates the calculated occurring minimum and maximum wall shear stress τ wall inside the slit of the sensor, which increases exponentially with increasing inlet velocities inlet .The minimal wall shear stress τ wall,min is typically located next to the edges of the slit, and the maximal
Figure 5 .
Figure 5. Dependency of the droplet concentration in the sensor probe slit on the droplet size.
Figure 5 .
Figure 5. Dependency of the droplet concentration in the sensor probe slit on the droplet size.
Figure 6 .
Figure 6.Mean wall shear stress distribution inside the probe slit for an inlet velocity of inlet = 0.03 m/s and a drop size of 150 nm.
Figure 7
Figure 7 illustrates the calculated occurring minimum and maximum wall shear stress τ wall inside the slit of the sensor, which increases exponentially with increasing inlet velocities inlet .The minimal wall shear stress τ wall,min is typically located next to the edges of the slit, and the maximal
Figure 6 .
Figure 6.Mean wall shear stress distribution inside the probe slit for an inlet velocity of u inlet = 0.03 m/s and a drop size of 150 nm.
8 )Figure 7 .
Figure 7. Maximal and minimal wall shear stress within the sensor probe in dependence of the inlet velocity inlet and a drop size of 150 nm.
Figure 7 .
Figure 7. Maximal and minimal wall shear stress within the sensor probe in dependence of the inlet velocity u inlet and a drop size of 150 nm. | 2019-04-22T13:05:08.199Z | 2017-02-23T00:00:00.000 | {
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213832270 | pes2o/s2orc | v3-fos-license | MANDEVILLE AND BERKELEY – A MISSING DIALOGUE
The goal of the present paper is to point out a peculiar style of debate between two well-known philosophers, Bernard Mandeville and George Berkeley, carried out in The Fable of the Bees, Alciphron, and The Letter to Dion. While philosophers often fall short of trying to understand each other in their literary exchanges, they usually try to convince the opponent. This is hardly the case in the Berkeley – Mandeville debate. Here the exchange is not confined to private letters addressing directly the views of the other philosopher. Nor does it aim to address a few experts in the field of moral and political philosophy. Instead, the debate is carried out in public and with the aim of convincing the general reader. This shapes the discussion under consideration here, which is exemplified by the struggle for different evaluation of the normative concept of luxury. For Berkeley, this is a strongly negative word anchored in tradition and ethics, but Mandeville is one of the first thinkers to argue for a positive evaluation of this concept, since it encourages trade and production, and thus prosperity of a nation.
Berkeley, however, had very specific reasons for writing as he did. He took himself to be unmasking an opponent who was hiding his real views and intentions and engaging in subterfuge. As early as 1713 Berkeley was meeting London freethinkers in their fashionable coffee-houses and debating them. He realized there is a gap 1 between what they say in private and what they are prepared to write publicly, so his task is not an easy one: to attempt to draw conclusions from principles that their authors would disavow if challenged, opening himself to the charge of wilful misinterpretation: "a gentleman in private conversation may be supposed to speak plainer than others write, to improve on their hints, and draw conclusions from their principles" (Alc III, 23). 2 So his target is not The Fable of the Bees as a book, and that is why he does not quote it once in Alciphron, except for the slogan "private vices, public benefits". For the same reason it is problematic to view Lysicles as Mandeville's "spokesman", 3 and reproach him (and Berkeley) with misstating Mandeville's case.
So if Berkeley is not trying to refute Mandeville's position directly, 4 but wants to show the consequences that flow from it, how does he go about this task and how far is he successful? First of all, he attempts to provide a translation of Mandeville's de-moralizing 5 talk of vice into plain English, for as Lysicles explains: "Thus in our dialect a vicious man is a man of pleasure, a sharper is one that plays the whole game, a lady is said to have an affair, a gentleman to be a gallant, a rogue in business to be one that knows the world. By this means we have no such things as sots, debauchees, whores or rogues in the beau monde" and Euphranor sums the position up: "Vice then is, it seems, a fine thing with an ugly name" (Alc II, 69). But Berkeley's second, more outspoken spokesman Crito is not very fond of this linguistic innovation, and translates it into plain English: "... to cheat, whore, betray, get drunk..." (Alc II, 84). 1 Berman calls the crypto-atheistic messages in some early modern writers their "esoteric" doctrine, whose purpose was to insinuate denials of God to like minded readers, while not being explicit enough to warrant prosecution. Berman (1988). 2 When quoting Berkeley, Alc stands for Alciphron, the Roman number is the number of the dialogue in it -Mandeville is dealt with in the second one -and the last number stands for the page in Luce's edition. PC stands for Philosophical Commentaries, the number is the number of the entry. Address to Magistrates has page numbers from the online version. With Mandeville, the number refers to the page in Kaye's edition. 3 "Lysicles and Alciphron are not meant as caricatures of Mandeville and Shaftesbury, but men of fashion who, enamoured of the notions and argumentative style of those writers, have made them part of their conversation" (Walmsley 1990, 125). 4 The only direct argument could be Euphranor's suggestion that a person who drinks in moderation may be more beneficial to a society and its economy in the long run than a drunkard who, one can expect, will not live very long. So this private vice is not a public benefit. But this argument was in fact first made by Francis Hutcheson and Berkeley merely repeats it. 5 For a useful summary of the process of stripping the concept of luxury of its moralizing connotations, and Mandeville's role in it, see Berry (1994, 101 -139).
We may see how such coarse language might give rise to the charge of uncharitable reading of Mandeville in those commentators not attentive enough to Berkeley's rhetorical aim.
The second strategy of resisting the de-moralizing of the language of vice goes deeper and strikes at the heart of the Mandevillian paradox "private vices, public benefits". The semantic tension between the private negativity and public positivity of something which had been understood to be negative both for the individual and the community, namely vice, forms the basis of Mandeville's project and success as a writer. Berkeley rejects the paradox and attempts to reaffirm the traditional dual perniciousness of vice through a series of exempla 6 reacting to Lysicles' examples. For instance, Berkeley has Lysicles (Alc II, 68) reproduce Mandeville's exemplum of the highway-man (Fable,(88)(89). Mandeville eschews moral words in his little story to the point of euphemism "A Highway-man having met with considerable Booty...", in which he is matched by the diplomatic Lysicles "when his time is come, a poor family may be relieved by fifty or a hundred pounds set upon his head", tactfully not mentioning the robber's victims and his just execution. Both want to draw our attention away from condemning the criminal and display only the supposed good that comes from his actions. But the forthright Crito does not accept a sanitization of these concepts, he provides many stories where vice does not injure solely the perpetrator, but also the people around him. For instance, "Callicles ... before he became of age … killed his old covetous father with vexation, and ruined the estate he left behind him, or, in other words, made a present of it to the public, spreading the dunghill collected by his ancestors over the face of the nation" (Alc II, 70). Crito's little stories contrast with Lysicles' callous examples and remind the reader that no man is an island while decrying the purely economic calculations of the public good.
These two aspects of Berkeley's rhetorical strategy, the resistance to the sanitizing jargon of the freethinkers and the stress on the vicious person's injury to other people, have been much misunderstood by commentators 7 and wilfully misrepresented by Mandeville himself. However, they were successful at least in the sense that they elicited a reply, for Mandeville was not wont to engage in dialogue.
Why did then Mandeville reply at all? We could identify two possible reasons for that. One is that Mandeville felt Alciphron did real damage to his reputation through the quality of its writing: "... Berkeley alone made effective use against him of his own weapons of satire and ridicule" (Viner 1). The second reason is that Berkeley's critique came from the outside, the two positions did not overlap and could hardly be reconciled, so Mandeville, instead of incorporating it and strengthening his own position through it, as was the case with Butler, could only demarcate it and fortify it against the rival.
However, Mandeville responded in a curious way, for he claims that Berkeley had not read The Fable before he criticised it, that he was reacting to hearsay and to the bad reputation the book already had. Now this rhetorical strategy is symptomatic of Mandeville's broader dismissive attitude to "philosophical debate", and it also allows him to assume the higher moral ground of one who is being condemned without getting a fair hearing. But as a consequence, the whole Letter to Dion is spent repeating what Mandeville had said in his earlier works and denying the unpalatable consequences that Berkeley, according to Mandeville, had drawn from them.
Some commentators value the Letter as the last word of Mandeville, and they seek a development of his position in it. That may be the case, but the Letter's content does not shed any new light on the matter of dispute between both thinkers. And it is positively harmful when taken at face value together with its rhetorical ploy as a proof that Berkeley never read The Fable before criticising it. 8 So, as we have seen, there is no dialogue between our two thinkers. But why are they talking at all, if they do not want to convince their opponent? Well, as we have said, they want to convince the general public.
The results of our discussion so far have been rather disappointing. Given the nature of the philosophical systems of Berkeley and Mandeville, their confrontation, had they seriously attempted one, would have been complex and probably more interesting. In the following we will attempt to supply what is missing in their actual dialogue.
The Two positions Regarding Virtue and Vice
Let us proceed now to the two different philosophical positions themselves and let us set them against each other to view the more sharply their differences. "Man is an Animal, formidable both from his Passions, and his Reason; his Passions often urging him to great Evils, and his Reason furnishing Means to achieve them" (Berkeley 1738, 5). This bleak picture of human nature is actually Berkeley's, not Mandeville's. The driving force of human action is the passions, and reason is surprisingly relegated to the subservient role of finding means of gratifying those passions, very much like the role of reason later in Hume, for example. Such an animal is not sociable, and must be tamed to render it fit for society, and that is the job of "civil and religious Institutions". They carry out this task through educating the animal and imbibing it with proper notions and principles. What are these notions and how can they influence human conduct? They do it through influencing judgement, which can sometimes overrule the passions, because the notions have a passion-like dimension. An example of a notion is: "...to believe that God scans all his Actions, and will reward or punish them..." (Berkeley 1738, 9). Now, believing in an omniscient God who rewards good acts and punishes evil ones is not a purely theoretical assent to a proposition for Berkeley, for reward and punishment are pleasure and pain, the primary motivators of any animal, man included: "Punishments and Rewards have always had, and always will have the greatest Weight with Men" (Berkeley 1738, 15).
These motivating notions, psychologically present as fear of punishment and desire for reward, are expressed in the language of vice and virtue in the English of Berkeley's time. The state takes care to educate the population in them: "... in a wellordered State, (there is) a certain System of Salutary Notions, a prevailing Set of Opinions ... taught and instilled by the general Reason of the Public; that is, by the Law of the Land" (Berkeley 1738, 8). Now Berkeley takes himself to be a propagator of these notions, it is in fact his job as a clergyman of the established church.
The notions are also culture specific and we shall look for them in vain in other societies. Of three important vices, luxury, avarice and ambition, Berkeley says that they are absent in savages, but not because the savage is himself virtuous and unspoiled by civilisation, "but because the Opportunities and Faculties for such Vices are wanting" (Berkeley 1738, 14). And he gives the example of "savages" who, when given the opportunity, will be drunk for several days and nights together, echoing probably his experience with American natives during his stay in Rhode Island. Moreover, as societal virtues and vices, they are not to be found in the other animals.
The step from pleasure and pain to virtue and vice deserves to be explicated in more detail. 9 Berkeley is a sophisticated hedonist from the very beginning, from the time of his Philosophical Commentaries (1707 -1709), these are the basic motivators of action: "No agent can be conceiv'd indifferent as to pain and pleasure" (PC 143), and the content of pleasant and painful ideas is ultimately determined by God, since it is Him who puts the ideas of real things into our minds: "We do not properly speaking in a strict philosophical sense make objects more or less pleasant, but the laws of Nature do that" (PC 144).
If God puts these ideas into our minds, how come we go wrong sometimes and do the wrong thing? How can we misunderstand the divine language so badly? We err because there are two types of pleasures, natural true pleasures with universal validity, and fantastical particular pleasures made up by the mind itself. The latter ones "presuppose some particular whim or taste accidentally prevailing in a sett of people, to which it is owing that they please" (Olscamp 1970, 49). An example here would be fashionable clothes, houses, and luxury in general. Now pursuit of these fantastical pleasures leads to excess, is irrational and brings only misery, while in the search for the natural pleasures consists true happiness, for they were intended by God to reward us for the right use of our faculties. There is also the traditional scale of pleasures, with sensual delights being the lowest, but everything starts with them: "Sensual Pleasure is the Summum Bonum. This the Great Principle of Morality. This once rightly understood all the Doctrines even the severest of the Gospels may cleerly be Demonstrated" (PC 769). So the right notion of pleasures will help us decipher the difficult precepts of the New Testament and remove the trap of ascetic rigorism that Mandeville will so ably exploit.
Surprisingly, there is no distinction between our pleasure and profit: "I allow not of the Distinction there is made twix't Profit and Pleasure" (PC 541) and the very next entry ties ignoring of this happy coincidence with fundamental mistakes in ethics: "I'd never blame a Man for acting upon Interest. he's a fool that acts on any other Principle. the not understanding these things has been of ill consequence in Morality" (PC 542). So Berkeley sees two pitfalls in the study of ethics: the role of pleasure in the severe strictures of Christianity and the relationship between pleasure and the actor's interest. It is these two flashpoints that Mandeville will attack. For Berkeley, there is no space to drive in the Mandevillian wedge of dividing private good from the public good. Virtuous conduct is what supports the common good, vicious the opposite. Indeed, the very words "virtue" and "vice" are used to excite men to useful action that will be rewarded and warn them against a dangerous course that will be punished. They are not descriptions of actions, they serve rather as recommendations. Of "virtuous" and "honourable" Berkeley says: "those words should excite in the mind of the hearer an esteem of that particular action and stirr him up to the performance of it..." (Olscamp 1970, 150).
With Mandeville's views on virtue and vice we enter a different universe: "what I call Vices are the Fashionable Ways of Living, the Manners of the Age, that are often practis'd and preach'd against by the same People" (Letter, 48). Two aspects of Mandeville's position on vice stand out here: vice is, in fact, a positive phenomenon, and people who say otherwise are hypocrites. Economically speaking, vice as an excess of consumption creates needs that were not there before, and consequently trades and jobs to satisfy those needs, employing multitudes of people. The increased circulation of money benefits everybody. But it is dirty money, at least according to Mandeville's official position and protestations. For he still wants to claim that vice is evil and virtue good; his definition is very strict, he confines "the Name of Virtue to every Performance, by which Man, contrary to the impulse of Nature, should endeavour the Benefit of others, or the Conquest of his own Passions out of a Rational Ambition of being good" (Fable, 48 -49). Self-denial forms a necessary part of virtue, and whatever is not virtue is vice. This superficially orthodox position of moral rigorism, modelled on the Calvinists and the Jansenists of the sixteenth century, helps Mandeville achieve his rhetorical purposes of presenting his paradox "private vices, public benefits", of in fact eulogizing vices while claiming, tongue-in-cheek, that his book is a book of "exalted morality" (Letter, 41). (Recall Berkeley's warning against divorcing selfinterest, pleasure and profit from good action.) Mandeville's rhetorical ploy was not lost on his readers, 10 most enjoyed his writings, some did not, and took him and his book to court, and lost, twice -a testament more to the power of his political protection than to his innocence. Now perhaps we begin to appreciate the difficulty Berkeley was facing of trying to pin down this eel of a writer. Not only does Mandeville evaluate vice and virtue differently to Berkeley, they also play a different and rather fundamental role in his story of the establishing of human society. Man is an animal of conflicting passions for Mandeville as well, who comes into conflict with other men because of desiring the same things and their scarcity. To mitigate the inevitable conflict, "Lawgivers and other wise Men" tried to "to make the People ... believe, that it was more beneficial for every Body to conquer than indulge his Appetites, and much better to mind the Publick than what seem'd his private Interest" (Fable,42). This extolment of self-denial serves a peace-making function and the social fiction works only because men were led to believe that they were better than animals and should not behave like them. For refraining from their instincts men are rewarded by flattery, "the Aerial Coin of Praise" (Fable, 55) of being called virtuous. So moral virtues are a pro-social fiction, they are "the Political Offspring which Flattery begot upon Pride" (Fable, 51). Berkeley, on the other hand, calls this central principle of Mandeville "a horrible Thing" (Address, 31) -and he is not happy with the reduction of man to a mere animal either.
Mandeville thinks he can explain Berkeley's endeavours: "Thus Sagacious Moralists draw Men like Angels, in hopes that the Pride at least of Some will put 'em upon copying after the beautiful Originals which they are represented to be" (Fable, 52). So Berkeley would be the traditional moralist working within the reigning and socially useful conception of vice and virtue to prevent conflict and strengthen society, a sagacious moralist. And he explicitly envisages Berkeley in this role in the Letter: "Men may differ in Opinion, and both mean well. You, Sir, think it for the Good of Society, that human Nature should be extoll'd as much as possible: I think, the real Meanness and Deformity of it to be more instructive" (Letter, 63 -64, my italics). This is the first of two points of contact between our two thinkers, the question of their respective roles, so let us explore it in a more detail.
Berkeley, not surprisingly, does not accept this categorisation of his position by Mandeville and, on the contrary, has grave doubts about the integrity of the opposing position: "Euph. Virtue then, in your account, is a trick of statesmen. Lys. It is. Euph. Why then do your sagacious sect betray and divulge that trick or secret of state, which wise men have judged necessary for the good government of the world?" (Alc II, 80) From Berkeley's point of view it is a dangerous statement that virtue is a trick of the politicians, although it claims to be the result of theoretical scientific analysis, for it cannot fail to have political repercussions. If virtue is a trick, why not get rid of it and base society on a true, scientific foundation? That danger is latent in all socially scientific theories, and Berkeley's prescience was borne out by Mandeville's posthumous role in the radical French enlightenment 11 and its political issue, the French Revolution.
So Mandeville sees Berkeley's account of virtue and vice as the traditional moralist's story that he can scientifically explain away, while Berkeley sees Mandeville's theory as a mere attack on the existing social order in general and the role of Christianity in it in particular. Both are partly right and partly wrong. Mandeville seems to have won in the long run, the Christian concepts of "vice" and "virtue" no longer have the same normative function they had in his time, and so could be deconstructed and subverted by his analysis. But with his rigorist caricature of Christianity he was breaking in an open door, because for Berkeley Christianity and individual profit do not exclude each other. Berkeley, on the other hand, sees no merit in Mandeville's tendency to build a naturalist account of ethics. He reads his story of the origin of society literally, and we cannot really fault him with that. Statements like "the first Rudiments of Morality, broach'd by skilful Politicians, to render Men useful to each other as well as tractable, were chiefly contrived that the Ambitious might reap the more Benefit from, and govern vast Numbers of them with the greater Ease and Security" (Fable, 47, my italics) sound quite provocative and revolutionary. Apparently, this story acquired the meaning of allegory only retroactively in the second volume of the Fable, 12 published in 1728 as Berkeley was sailing for America. 11 To which, rather ironically, Alciphron greatly contributed, having been translated as early as 1734, while the Fable itself only in 1740: "This advance reputation excited interest in the Fable in radical philosophical circles in France, where an established tradition of libertine and social argument was firmly in place" (Hundert 1994, 102). 12 "But it is in Part II, which he wrote largely to correct misconceptions caused by the deliberately paradoxical Part I, that Mandeville most stressed the gradualness of evolution. A great part of the volume is devoted to tracing the growth of society in a surprisingly scientific manner, and completely contradicts the literal interpretation of the allegory in the earlier portion of Part I" (Kaye 1924, lxv -lxvi). For a convincing naturalist interpretation of the Fable, see Hundert (1994, 35 -61).
Berkeley probably did not read the second volume before writing -1485215030 Alciphron. This second volume contains Mandeville's considered final position, and so Berkeley could not appreciate its compactness and novelty.
So Mandeville sees himself meta-philosophically to be providing a theory of human social behaviour, while seeing Berkeley attempt to work within that scheme to, perhaps, strengthen the bond of society. But Mandeville's theory is couched in terms that are value-laden and not neutral, and, frankly, borrowed from the moralist's vocabulary, such as "vice" and "virtue", and this fact is both a weakness of the theory as well as a strength of its presentation. Mandevillian paradoxes read well, but he then feels the need to sanitize his use of such emotive words. He does so, for instance, in the parable of dirty streets of London in the Letter (10 -12), where he contrasts the subjective yearning of London pedestrians (who "regard Nothing but their own Cloaths and private Conveniency") for cleaner streets with the general explanation that the waste and excrement on the streets is the inescapable result of the "greatness" of the city: "it is impossible London should be more cleanly before it is less flourishing". Private inconvenience once again turns into a public benefit, and Mandeville hopes his pointing out this paradox will stop people wishing for cleaner streets: "if they have any Concern in (London's) Welfare, they will hardly ever wish to see the Streets of it less dirty". The moral seems to be that the scientific analysis leaves everything as it is, it only unmasks some people's confused conceptual thinking and wishes.
With the benefit of three centuries of hindsight we now know that the parable fails, people continue to wish for cleaner streets, that was why they built the London sewage system in the Victorian era, when later they began to choke on smog they switched to cleaner ways of heating their houses, and when they began to choke on car fumes, they introduced the London congestion charge. The parable fails on another, less trivial level, too. There is no catharsis in the explanation and the words, expressing the speakers' wishes, still continue to resonate and be evaluative. It is a constant complaint of Berkeley that Mandeville divorces the words not only from their evaluative function, but from their normative and political function: "Can any thing be more inconsistent than to condemn in practice what is approved in speculation? Truth is one and the same, it being impossible a thing should be practically wrong and speculatively right" (Alc II, 74).
The obstinate words refuse to relinquish their value-laden connotations, and in the Fable's second volume and the Origin of Honour (1732) Mandeville attempts to devise new terms, purely technical ones this time. "Self-love" is common to all animals, but "selfliking" 13 belongs only to man, and it is the morally acceptable expression of pride.
According to Jeremy Bentham, Mandeville, by devising such new terms had "broken the chains of ordinary language" (Hundert 1994, 61) and Hundert even speaks of "establishing a science of unsocial yet socialized man on the ruins of an exhausted language of morals" (Hundert 1994, 61). Thus Mandeville can be portrayed as discarding the old vocabulary and groping for a new one, but the question remains whether he in fact finds it. After all, his "self-liking" has not stood the test of time.
For his part, Berkeley is a rather conservative speaker of English, and does not approve of the free-thinkers' conceptual innovations. 14 From his perspective of reading the Fable's first volume, the sweeping generalizations 15 and semantic paradoxes of pitting familiar words into unfamiliar positions turn it into a tale of twisting of old words with a smattering of anti-clericalism. Mandeville himself is disarmingly forthright about his preferred bombastic style and intention: "the true reason why I made use of the title Private Vices, Publick Benefits ... was to raise Attention: As it is generally counted to be a Paradox, I pitch'd upon it in Hopes that those who might hear or see it, would have the Curiosity to know, what could be said to maintain it; and perhaps sooner buy the Book, than they would have done otherwise" (Letter, 54). A stronger contrast in styles we could hardly devise: when Berkeley wrote a book refuting the material substance, quite a paradoxical topic, he purposefully concealed it in the text without mentioning it in the subtitle to his Principles.
Our two thinkers do not differ much in their pessimistic anthropology of man, but their attitudes towards the two main normative ethical notions of the time, vice and virtue, do not have much in common: Berkeley provides a new explanation and role for them by his innovative metaphysics and the idea that God speaks to us directly, telling us how to behave, thus strengthening these two traditional concepts and working within the prevailing Christian paradigm, while Mandeville attempts a deconstruction of them, revealing them as a sociological fiction and explaining their emergence in a naturalistic and evolutionary 16 framework. But still the question remains: how is such a radical divergence concerning these two frequented words even possible? The problem of the traditional moralist words and their ingrained positive and negative connotations, together with their normative function, will be further analysed in the next chapter.
The Case Study of "Luxury"
To account for this semantic dissonance between our two thinkers, we descend from the level of vice/virtue to the level of one particular vice, namely "luxury". Now, luxury 17 is one of few terms that have changed their polarity over time: it started as a negative term and is now largely positive, hence it can appear in advertisement, for example. Mandeville and Berkeley were writing their books during the time of this momentous transformation.
In Plato's Republic II we find a description of a society where the human needs for food, clothing and dwelling are served by strictly functional items, the dishes are vegetarian, clothing worn only in winter and the houses are without decoration. Glaucon is not happy with this "city of pigs" and wants some furniture and civilized food. Socrates explains that if the wish for more luxurious items is granted, the society becomes fevered and inflamed, and it will have to wage war to obtain these luxuries which go beyond necessity. Moreover, upon acquiring these luxuries, the warriors wallow in them and become soft and weak, being in turn defeated by other, less softened warriors. In the Roman context, virtue is the property of man, vir, and luxuria works the other way, to weaken the warrior and make him woman-like, it is thus the archetypal vice. In the Greco-Roman histories, luxury enters a healthy city from the East and weakens it so that it is easily defeated. The Romans enact laws to fight it and limit spending on banquets and clothes. The sumptuary laws testify to luxury becoming one of the central and normative concepts of the culture. Christian Europe takes over the pagan concepts of vice and virtue and reads it retroactively into the Bible, identifying vices with sins. Luxury became a sin in opposition to sobriety and chastity. Medieval sumptuary laws were enacted prescribing dress and food to subjects, in England the last passing in 1604.
Berkeley uses the classical concept of luxury with all of its connotations. On the theoretical level, he accepts the explanatory common-place of Roman historians that luxury is the cause of the downfall of societies: "Sallust observes, that a little before the downfall of the Roman greatness, avarice (the effect of luxury) had erased the good old principles of probity and justice... consult any historian, look into any writer. See, for instance, what Xenophon and Livy say of Sparta and Rome..." (Alc II, 75 -76, my italics). He proves a faithful reader of Plato: "luxury ... makes a nation, like a diseased, pampered body, look full and fat with one foot in the grave" (Alc II, 105, my italics) when he claims that luxury is the main programme of the free-thinkers' campaign. On the practical level he gives his wife a spinning wheel as a wedding gift and encourages her to wear home spun clothes, refusing to buy imported fashionable cloths. 18 On the political level, when making his proposal to set up an Irish national bank in Dublin, Berkeley suggests to raise the original capital by taxing imported wine and "foreign silks, linens, and laces" (Hight 2013, 399). And he even lobbies for re-introducing sumptuary laws, writing in 1742 to John Percival, MP for Westminster: "As luxury seems the real original root of those evils under which we groan ... must it not seem at the same time that ... sumptuary laws are highly expedient if we would cut out the core of the national evil. To attempt or even mention such things now would be madness ..." (Hight 2013, 448). He called such plans "utopian schemes" and admitted taking Plato's Republic "if not for a rule, yet for an incentive", knowing well there was much political opposition to the old concept of luxury with its legal ramifications.
Indeed, the fact that his wife wore home spun was held against him during the crucial deliberations in 1729, when it was finally decided that the government grant to fund his Bermuda college would not be paid. 19 On the other side, there was a new meaning of the word "luxury" being advocated by proponents of trade, shorn of its negative connotations. Nicholas Barbon in his Discourse of Trade (1690) argued that too much frugality hurts trade, and that prodigality is "a Vice that is prejudicial to the Man but not to Trade" (Berry 1994, 111), a contrast that will form the basis of Mandeville's position. A new modern anthropology of Hobbesian mould provides the support here, a view of man and his infinite desires, which can never be satisfied and thus cannot be excessive, i.e. luxurious in the traditional sense. Barbon commends expenditure on buildings and clothes as promoting trade, fashion is seen as beneficial, for man is naturally ambitious and wants to outdo others in the way he looks. The Enlightenment thinkers eventually divide into two camps, the traditional moralists condemning luxury and fashion, including Locke, Shaftesbury, Berkeley, Rousseau and Lord Kames, and the progressive, permissive wing with Mandeville, Voltaire, Hume and Smith. Berry fittingly calls their divorcing questions of commerce and politics from those of morality "de-moralization of luxury" (Berry 1994, 138). The word "luxury" itself is torn between them, being ambivalent in the eighteenth century and the subject of a fierce culture war.
Mandeville's take on the debate is original. There is nothing in his position that is not already at least implicitly in Barbon, but whereas Barbon expresses his commendation of luxury in straight, objective terms, Mandeville pretends to still condemn it. He uses the moralist's negative words to force them into semantic paradoxes. He defines luxury strictly as "that (which) is not immediately necessary to make Man subsist as he is a living Creature" (Fable, 107). The justification for the definition's strictness is that what is luxury for one person may not be luxury for a person of a higher rank and that therefore a looser definition would make the word so vague as to be useless. This little definitional trick enables Mandeville to stretch the meaning of luxury to cover everything, at the expense of emptying it of any sensible descriptive content. Berry puts the point succinctly: "It enables him to claim (strictly) that everything is a 'luxury' but, of course, this is also to say equally that nothing is a luxury" (Berry 1994, 129). Now that the concept of "luxury" is untied from its surrounding concepts he can play with it as he wishes, he can claim that luxury is bad but it is so pervasive that if you want to have a "decent" standard of living, you have to embrace it, and he fills hundreds of pages to that effect.
One consequence of Mandeville's definitional approach, and also a point Berkeley will attack, is the destruction of the concept of "moderation". Now these two concepts are defined against each other and hold each other in their respective positions, if you enlarge "luxury" to include everything, then you empty the concept of "moderation". Mandeville recognises the intimate connection between these two concepts and the danger it poses to his definition of luxury: "If you tell me, that Men may make use of all these Things with Moderation, and consequently that the Desire after them is no Vice, then I answer, that either no Degree of Luxury ought to be call'd a Vice, or, that it is impossible to give a Definition of Luxury..." (Letter,58,my italics). He is at his most passionate and bellicose when describing moderation: "Oh rare Doctrine! Oh easy Christianity! To be moderate in numberless Extravagancies ... But if we grant the Possibility of it, how shall we know and be convinced that they are sincere ... when we have Nothing but their bare Word for it ...?" (Letter, 41, my italics) So he dispatches moderation by claiming it is hypocrisy.
Berkeley, on the other hand, finds room for moderation, and blocks this pre-emptive move: "Lys. ... what moves men to build and plant but vanity, and what is vanity but vice? Euph. But if a man should do those things for his convenience or pleasure, and in proportion to his fortune, without a foolish ostentation or over-rating them beyond their due value, they would not then be the effect of vice; and how do you know but this may be the case?" (Alc II, 73, my italics) By allowing at least the possibility of sincerity, Berkeley attempts to save moderation and consequently, luxury. Mandeville is keen to deny even this possibility, rejecting all external and objective criteria such as ostentation, and limiting himself to the verbal assurance of moderation. But by doing this he does to hypocrisy what he did to luxury, stretch it so much that he squeezes all meaning out of it.
When it comes to the struggle for the word luxury, Berkeley seems to be on stronger ground for he defends the way it is used, while Mandeville has to twist it and other words, too, to get the result he wants. But such as analysis would underestimate the acuteness of Mandeville's ear, for he hears changes to it that Berkeley is unwilling to countenance. "Luxury" often collocated with verbs "enervate" and "effeminate", both were negative descriptions of its negative influence. Mandeville mentions both and of the first one he says: "the great Dread I had more particularly against the Word, to enervate, and some consequent Thoughts on the Etymology of it, did me Abundance of Good when I was a Schoolboy: But since I have seen something of the World, the Consequences of Luxury to a Nation seem not so dreadful to me as they did" (Fable,118). He probably describes a general decline in the efficacy of these words, for "luxury" was a contested word at the time. But to claim that the changes were driven or effected by Mandeville or any other writer, as Bentham did, would be too optimistic. Such a momentous change as the reverse in the polarity of a word is rarely caused by one speaker of the language, or even a group of speakers. The fact that no sumptuary laws had been passed for more than a century before our two writers, not even during the fundamentalist Commonwealth era, may have something to do with these broad changes, of which their writings form merely a record.
To sum up, we have identified two points of contact between our writers, which went beyond their misrepresentations of each other: the question of the status of Mandeville's doctrine and its potential political consequences, and the role of moderation and consequently of hypocrisy in it. And what about the dispute itself between Mandeville and Berkeley? In the short term, Berkeley, together with other divines, was successful in preventing the freethinkers from subverting the morals, religion and political establishment of the United Kingdom. The happy marriage of the new science and old piety saw off the deist threat, 20 with Berkeley's optical metaphor of God speaking directly to His creatures being of particular importance. In the long term, Mandeville's opinions prevailed, with the word "luxury" losing the remnants of its normative function and opprobrium as well as becoming a positive term. But its ambivalence prevented both thinkers from engaging in a meaningful discussion, for they were pulling it in opposite directions. | 2020-01-09T09:14:52.768Z | 2019-12-30T00:00:00.000 | {
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244171483 | pes2o/s2orc | v3-fos-license | Synthesis of Lapachol-Based Glycosides and Glycosyl Triazoles with Antiproliferative Activity Against Several Cancer Cell Lines
Lapachol ( 1 ), a natural naphthoquinone, presents several biological activities including antitumor activity, used as anticancer coadjuvant whose use was abandoned because of adverse effects. Herein, we reported the synthesis and cytotoxicity evaluation against cancer cell lines of a series of O glycosides and glycosyl triazoles derived from lapachol. In addition to the determination of IC 50 , the DNA fragmentation and clonogenicity were also evaluated. The glycoside derived from D -glucose ( 5 ) was far more active than lapachol ( 1 ) and more active in tumor cell lines HL60, Jurkat, THP-1 and MDA-MB-231 than to the non-tumoral PBMC cell line, indicating an improvement in activity and selectivity as compared with lapachol ( 1 ). Compound 5 and the glycosides derived from D -galactose ( 14 ) , D - N - acetylglucosamine ( 15 ) and L -fucose ( 16 ) showed good results in the DNA fragmentation and clonogenicity assays in the studies of subdiploid DNA content, indicating a pro-apoptotic potential and a good antiproliferative activity of these glycosides.
Introduction
Cancer is one of the principal causes of death worldwide and its incidence is expected to increase in the next years. According to WHO, about 30 million cases of cancer are expected to occur until 2040, with almost 50% taking place in developing countries [1]. Despite the great efforts in prevention and treatment of cancer there is a continuous need for new options. The lack of selective action towards the cancer cells of most current existing anticancer drugs results in toxicity to host tissues. Thus, the search for new potent and safer drugs of synthetic and natural origin is being pursued by several groups around the world. Lapachol (1), 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthalenedione is a natural 1,4-naphthoquinone isolated from plants of the Bignoniaceae family, mainly Handroanthus impetiginosus. It presents several biological activities including antitumor activity [4,5]. This compound has been used as coadjuvant in the chemotherapy of certain tumors but its use was abandoned because of adverse effects, mainly related to blood clotting. [5,6] Some synthetic routes were established to get lapachol (1), firstly by Fieser [7], being obtained in low yields. Recently, lapachol was synthetized from lawsone in better yields [8]. Several derivatives of 1 were prepared with antifungal, antibacterial, antiviral and antitumor activity [5]. Eyong et al. described the synthesis of atovaquone, which was approved for treatment of pneumonia (Pneumocystis pneumonia), toxoplasmosis and malaria [4,9]. Atovaquone is a naphthoquinone as well as lapachol and studies carried out in the last years have shown that atovaquone has a potent antitumor activity [10]. The chemical structure of l and some of its derivatives with antitumor activity are shown below (Fig. 2). [4,5].
The quinones are able to inhibit the mitochondrial oxidation and phosphorylation, as well to inhibit the enzyme succinate oxidase [11] which plays an important role in the citric acid cycle and the electron transport chain. Other mechanisms seem to be related to the intercalation of the naphthoquinones between the DNA base pairs [5] and inhibition of topoisomerases [12]. The main mechanism of action is related to the formation of reactive oxygen species (ROS), through semiquinone radicals. Both cause damage to cell macro molecules and consequently cell death [12].
The major problem for the clinical use of 1 is its low bioavailability, due to low water solubility, which implicated in the use of large doses for attaining plasmatic levels, causing severe side effects. The first attempt to enhance water solubility of lapachol was reported by Linardi and co-workers who described the synthesis of the β-D-glucoside of 1 (compound 6) and the corresponding peracetylated derivative 5 (Fig. 3) [13]. These two compounds were evaluated in vivo in mice bearing P-388 lymphocytic leukemia. The peracetylated glucoside 5 was active, enhancing the lifespan of mice by 80%, while the deacetylated derivative was inactive. According to the authors, the peracetylated glucoside was possibly acting as prodrug that could be absorbed by the cancer cells due to its lipophilic character. The unprotected derivative 6, being more hydrophilic, was possibly unable to cross the cell membranes being, therefore, inactive. [13] Several anticancer drugs possess a carbohydrate moiety in their structures, as shown in Fig. 1 for etoposide and doxorubicin. The work of Linardi and co-workers [13] showed that the attachment of a glucosyl moiety to lapachol can be a good approach to obtain new anticancer compounds. Glycosidic derivatives of lawsone, another naphthoquinone, has been obtained and assayed for antitumor activity. The glycosides were cytotoxic against HL-60 (acute promyelocytic leukemia), SKBR-3, MCF-7 and MDA-MB-231 (breast cancer) cells indicating that the variation of the carbohydrate moiety and the anomer type (α or β-glycoside) influence the cytotoxicity [14,15]. Some these lawsone glycosides with antitumor activity are shown in Besides classical glycosides, obtained by direct glycosylation, one strategy widely used to link a carbohydrate moiety to a natural or synthetic compound is the Cu(I)-catalyzed cycloaddition reaction between an alkyne derivative of the compound with a glycosyl azide, to get glycosyl triazoles [16]. Based on this, several glycosyl triazoles derived from naphthoquinones with antitumor activity are described in the literature (Fig. 5) [17][18][19]. Recently we described the synthesis and cytotoxic evaluation against HL-60 human leukemia cells of lapachol glycosides 5 and 15. These compounds showed low IC50 values, circa 5.0 µM. The mechanism of cytotoxic seems to involve the activation apoptosis signaling pathways, such as the DNA fragmentation, chromatin condensation and decrease of the mitochondrial transmembrane potential [20].
In the present work we describe the synthesis and cytotoxicity evaluation against cancer cell lines of a series of O-glycosides and glycosyl triazoles derived from lapachol. The structures of the synthesized compounds are shown in Fig. 6. The presence and orientation of groups (OH and NHAc) that can modulate the physico-chemical properties of the compounds was considered, taking into account that the parent carbohydrates have different solubility and that the O-acetyl groups confer lipophilic properties to the peracetylated derivatives. We also considered the presence of specific carbohydrate transporters in the cell surface [21], that should facilitate the transport of the deacetylated glycosides across the cancer cell membrane. 7 2 Results and discussion
Scheme 2.
Reaction conditions for the obtention of 2-O-propargyllapachol and the glycosyl azides.
11
The infrared spectra of the deprotected glycosyl triazoles 21-24 showed, as expected, absorption bands in the region of 3357-3281 cm -1 due to OH stretching of the carbohydrate moiety. The 1 H NMR and 13 C NMR spectra of deprotected glycosyl triazoles 21-24 agree with their chemical structures. The mass spectra of all lapachol derivatives showed molecular weight compatible with the proposed structures (supplementary data available).
Biological activities 2.2.1 Cytotoxic activity
Lapachol (1), its classical glycosides 5 and 14-16 and glycosyl triazoles derivatives 17-24 were evaluated for their cytotoxicity against six human cancer cell lines: HL60 (acute promyelocytic leukemia), Jurkat (acute T-cell leukemia), THP-1 (acute monocytic leukemia), MCF-7 (breast adenocarcinoma), MDA-MB-231 (triple-negative breast cancer) and HCT-116 (colorectal carcinoma). Cell viability was evaluated using the MTT method to evaluate cell viability as previously described [38][39][40]. As model of non-tumoral lineages, compounds were tested against human peripheral blood mononuclear cells (PBMC) and viability measured by resazurin assay [41]. Etoposide and lapachol were used as positive controls. Compared with lapachol (1), the majority of its glycosides were more cytotoxic towards one or more tumor cell lines, lapachol (1) being cytotoxic only against HL60, with poor activity when compared to its derivatives. To evaluate the toxicity to non-tumor cells, selected compounds were tested on peripheral blood mononuclear cells (PBMC) cells. The results are shown in Table 1. Table 1. Cytotoxicity of lapachol (1) and lapachol-based glycosyl triazoles 17-24 against four cancer cell lines and against human peripheral blood mononuclear cells ( a IC50, μM).
Evaluation of DNA fragmentation assay as indicative of cell death by apoptosis
The subdiploid DNA quantification accomplished in this work was used as strategy to measure the DNA fragmentation, being an indicative of cell death activation by apoptosis, according described by Nicoletti et al [51,52]. According to the protocol used, cells with DNA fragments by death apoptosis process can be evaluated by quantification of subdiploid DNA content. The classical glycosides (compounds 5 and 14-16) presented higher cytotoxicity against the majority of tumor cell lines and the best selectivity index (SI) for at least one tumor cell lines, so they were selected for the quantification of subdiploid DNA content as indicative of the pro-apoptotic potential [51]. There was an increase in subdiploid DNA content in the tumor cell lines treated with classical lapachol glycosides 5 and 14-16, but not with lapachol. The compounds were evaluated in the concentration of 50 µM and glycosides induced DNA fragmentation in all tumor cell line (Fig. 7). A common characteristic of cell lines used in this work is related to presence or absence of checkpoint p53 protein activity. The leukemic cell lines HL60, Jurkat and THP-1 lack p53 protein [53][54][55]. cell line showed p53 wild type protein [59] and the activity of chemotherapeutics may be mediated by p53 and Bax pro-apoptotic proteins which activate the apoptosis mitochondrial pathway and can activate caspase-3 [58]. On this way, the MCF-7 cells are the only ones used in this work that lack caspase-3 [60].
For MCF-7 cells other mechanisms of apoptosis induced by different chemotherapeutic agents may occur independently of caspase-3 so that DNA fragmentation can be observed despite the absence of caspase-3 [60]. Therefore, different pathways should be involved in the pro-apoptotic potential observed for new lapachol glycosides.
Effect of lapachol glycosides in the clonogenic survival of solid tumor lineages
Is well known that the DNA damage inducing agents induce cell cycle arrest at checkpoints. This is a cell survival response that allows them to repair damaged DNA and is not directly related to cell death. Cells performed in triplicate. Colonies of more than 50 cells were counted, and the surviving fraction was calculated relative to control (DMSO, 0.5%) to account for basal plating efficiencies 2. 3 Influences of the chemical structure of lapachol glycosides on bioactivity The cytotoxicity against the evaluated cancer cell lines was in general higher for lapachol glycosides (5,
14-16, 17-20, 22
and 24) than that of lapachol (1), indicating that carbohydrate moieties influenced in the cytotoxicity against tumoral and non-tumoral cells. The higher activity of peracetylated lapachol glycosides can be explained due the presence of the O-acetyl groups on saccharidic moiety, which impair appropriate liposolubility to the peracetylated glycosides enabling these compounds to cross the cell membrane [13].
The presence of hexose transporters on cell surface [21] may have facilitated the entry of the deacetylated lapachol glycosides that were active.
The nature of the carbohydrate moieties seems to influence on activity, wherein the variation of sugar improves or reduces the cytotoxic activity. For example, the peracetylated lapachol glycosyl triazole derived from L-fucose (20) was the most active against HCT-116 while its deacetylated analogue
Conclusions
The synthesis of 12 lapachol-based glycosides and glycosyl triazoes and evaluation against several cancer cell lines is described in the present work.
4.2.3
General procedure for the synthesis of lapachol glycosyl triazoles (17)(18)(19)(20)(21)(22)(23)(24) To a 50 mL round bottom flask was added 28 (0.30 mmol) dissolved in 1 mL of tetrahydrofuran, followed by the appropriate glycosyl azide (0.27 mmol), dissolved in 0.5 mL of tetrahydrofuran. Then, Cu(OAc)2.H2O50% mol, dissolved in 0.5 mL of water and sodium ascorbate 60% mol, dissolved in 1 mL of water were added in a stepwise manner. The reaction mixture was stirred at room temperature for 4 h and monitored by TLC analysis. The tetrahydrofuran was removed by distillation at reduced pressure. For 22 peracetylated glycosyl triazoles the reaction residues were solubilized in 50 mL CH2Cl2 and washed with 2 x 50 mL H2O and subsequently washed with 3 x 50 mL alkaline EDTA 20% w/v. The organic phase was dried over Na2SO4 and filtered. The organic phase was removed by distillation at reduced pressure. The deacetylated glycosyl triazoles (21)(22)(23)(24) were purified directly. The derivatives 17-20 were added to Florisil and purified with silica column with following mobile phase (CH2Cl2: ethyl acetate/4:6) and the deacetylated using ethyl acetate: MeOH/9:1 as mobile phase.
Evaluation of cell viability of human PBMC by resazurin assay
The cell viability assay was performed according to O'Brien et al. (2000), with modifications. [40] Resazurin is a blue dye and is weakly fluorescent until it is irreversibly reduced to pink and red fluorescent resorufin. It is used as an oxidation-reduction indicator in cell viability assays and its intensity is A blank of each sample was performed to avoid unspecified reactions of the compounds with resazurin (blank) and the results were analyzed using Prism 7.0 (GraphPad Software Inc).
Selectivity index (SI) determination
After determining the IC50 values for tumor (HL60, Jurkat, THP-1, MCF-7, MDA-MB-231 and HCT-116 cells) and non-tumor cells (PBMC), the selectivity index was calculated. Determination of the SI was performed by the ratio between IC50 of PBMC and IC50 tumor cell [65].
Clonogenic assay
The MCF-7, MDA-MB-231 and HCT-116 cells were seed in 6-well plates at a density of 400 cells/well. After 6 hours of incubation, cells were treated with the compounds (at their IC50 and IC80) or control (DMSO 0.5%). The cells were incubated with the compounds for 24 hours and then the medium was removed and replaced by supplemented DMEM medium without the compounds [66]. The cells were incubated for another 14 days and after incubation, the colonies were fixed in 70% alcohol for 15 minutes, stained with crystal violet (30% in ethanol) for 30 minutes and kept at room temperature overnight for drying. Colonies with 50 or more cells were counted. The survival fraction (ratio between the number of colonies treated with the compounds and the number of colonies counted in the control) were calculated and the results were analyzed by GraphPad Prism 7.0.
Statistical analysis
Data are expressed as the means± SD (standard deviation). Statistical analysis was conducted using the Prism 7.0 statistical package (GraphPad Software, USA). To ascertain significance, we used a one-way ANOVA with Bonferroni post-test. Statistical significance was considered at a limit of p<0.05 from three independent experiments conducted in triplicate. | 2021-10-19T15:26:41.507Z | 2021-09-27T00:00:00.000 | {
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255547459 | pes2o/s2orc | v3-fos-license | Scalable Apparatus to Measure Posture and Locomotion (SAMPL): a high-throughput solution to study unconstrained vertical behavior in small animals
Balance and movement are impaired in a wide variety of neurological disorders. Recent advances in behavioral monitoring provide unprecedented access to posture and locomotor kinematics, but without the throughput and scalability necessary to screen candidate genes / potential therapeutics. We present a powerful solution: a Scalable Apparatus to Measure Posture and Locomotion (SAMPL). SAMPL includes extensible imaging hardware and low-cost open-source acquisition software with real-time processing. We first demonstrate that SAMPL’s hardware and acquisition software can acquire data from D. melanogaster, C.elegans, and D. rerio as they move vertically. Next, we leverage SAMPL’s throughput to rapidly (two weeks) gather a new zebrafish dataset. We use SAMPL’s analysis and visualization tools to replicate and extend our current understanding of how zebrafish balance as they navigate through a vertical environment. Next, we discover (1) that key kinematic parameters vary systematically with genetic background, and (2) that such background variation is small relative to the changes that accompany early development. Finally, we simulate SAMPL’s ability to resolve differences in posture or vertical navigation as a function of effect size and data gathered – key data for screens. Taken together, our apparatus, data, and analysis provide a powerful solution for laboratories using small animals to investigate balance and locomotor disorders at scale. More broadly, SAMPL is both an adaptable resource for laboratories looking process videographic measures of behavior in real-time, and an exemplar of how to scale hardware to enable the throughput necessary for screening.
INTRODUCTION
Measuring posture and locomotion is key to understand nervous system function and evalu- 23 ate potential treatments for disease -particularly neurological disorders 1 . Behavioral screen- 24 ing is a fundamental part of both basic and translational approaches to disease 2,3 . For screens, 25 measuring behavior from large numbers of animals is necessary to differentiate individual vari- 26 ation 4 from changes seen in disease models and/or improvement following treatment 5,6 . The 27 demand for such high-throughput measurements comes at a cost: often, measurements that 28 require high resolution -such as posture -are limited. Modern machine learning algorithms 29 and inexpensive videographic / computing hardware have automated measurements of pos- 30 ture and kinematics [7][8][9] and illuminated our understanding of animal behavior [10][11][12] . We sought 31 to combine videographic analysis of posture and vertical locomotion with the scalability amenable 32 to screening. 33 Over the past decade, we have studied posture and locomotion using the larval zebrafish as a 34 model. Neural architecture is highly conserved across vertebrates, making larval zebrafish an 35 excellent model to understand the underpinnings of locomotion 13,14 and balance 15 . For our 36 studies, we developed a new apparatus/analysis pipeline to measure the statistics of posture 37 in the pitch (nose-up/nose-down) axis and locomotion as larvae swam freely in depth. We dis- 38 covered that larvae learn to time their movements to facilitate balance 16 , that larvae modulate 39 the kinematics of swimming to correct posture 17 , and that larvae engage their pectoral fins to 40 climb efficiently 18 , and implicated different neuronal circuits in each of these behaviors. While 41 informative, data collection was slow (months) on small numbers (<5) of apparatus. Increasing 42 throughput remains a challenge common to laboratories that develop new tools to measure 43 behavior. 44 To meet the needs of scalability, resolution, and extensibility we developed SAMPL: a low-cost, 45 open-source solution that measures posture and vertical locomotion in real-time in small an- 46 imals. Further, we provide a turn-key analysis pipeline to measure larval zebrafish balance be- 47 navigate (i.e. climb/dive) in the water column. Our new dataset represents two weeks worth 54 of data collection, and allowed us to detail variation in postural/locomotor behaviors. By mea- 55 suring behavior across different genetic backgrounds and development, we report two new 56 findings. First, variation in posture/locomotion is systematic across genotype and second, the 57 scale of variation in behavior across development is much larger than background genetic 58 variation. We use these new data to simulate the resolving power for each behavioral param- 59 eter as a function of data gathered -foundational information to rigorously assay the effects of 60 candidate genes or small molecules on posture or locomotion. SAMPL thus offers a straight- 61 forward way to gather data from small animals, and a turn-key solution to screen for balance 62 and vertical locomotion in larval zebrafish. More broadly, SAMPL offers a template for labora- 63 tories looking to scale their own behavioral apparatus to achieve the throughput necessary for 64 screens. SAMPL will thus facilitate reproducible studies of postural and locomotor behaviors in 65 both health and disease, addressing unmet needs in treating neurological disorders, particu- 66 larly with balance symptoms 19 . 67
68
SAMPL hardware & software overview 69 To overcome measure posture with the throughput necessary for genetic and drug screens, 70 we deployed SAMPL, a real-time videographic system ( Figure 1A) that records small animal 71 behavior in the vertical axis. Below we briefly describe the hardware and software that com- 72 prise SAMPL. SAMPL's hardware consists of three simple modules: an infrared (IR) illumination 73 module ( Figure 1B), a camera-lens module ( Figure 1C), and two clamps to hold fish chambers 74 ( Figure 1D). All three modules are mounted directly ( Figure 1A) onto an aluminum breadboard 75 ( Figure S1) and a light-tight enclosure covers the entire apparatus to permit individual control 76 of lighting ( Figures 1F and 1G). Details of hardware and software design can be found in Ap- 77 pendices 1&2. A complete parts list is in Table 1, hardware assembly instructions in Appendix The second module captures videographic data. It consists of a camera and lens optimized for 86 speed, resolution, compactness, and affordability. The camera hardware satisfies the follow- 87 ing demands: (1) large pixel size with low noise allowing for high dynamic range / signal-to- 88 noise ratio; (2) sufficient resolution to resolve subtle changes to animal posture; (3) an interface 89 with sufficient bandwidth for data transfer; (4) availability. The lens achieves (1) close focus; (2) 90 sufficient depth-of-field to cover the entire depth of the imaging arena; (3) high image qual- 91 ity; (4) compact size; (5) high IR transmission rate; (6) ease of integrating an IR-pass filter. We 92 adapted a 50 mm IR-optimized lens by placing a 0.3" extension tube between the lens and 93 the camera to achieve higher magnification ratio with minimum working distance. The space 94 between camera adapters and the extension tube allows us to fit a 25 mm IR-pass filter; the 95 extension tube gives a mount point to connect the module to the base ( Figure 1A). Using this 96 camera-lens module, we image an area~400 mm 2 ( Figure 1E, pink square) at 166 Hz with 97 1200×1216 pixels at a focal distance of~24 cm. 98 The final module is a rectangular arena optimized for vertical locomotion (i.e. parallel to the 99 focal plane). By design, the chamber size is larger than the imaging area, allowing stochastic 100 sampling of freely behaving animals in a large enough arena. The bottom of the chamber is 101 below the field of view so that animals sitting at the bottom will not be recorded. We assem- 102 bled custom-fabricated chambers from laser-cut acrylic by cementing transparent front and 103 back sides to a U-shaped piece that forms the narrower sides ( Figure 1E). We designed two 104 types of chambers with different inner widths to adapt to the needs of different experiments: 105 a wider standard chamber optimized for larger groups of animals and a narrower chamber for 106 1-3 animals ( Figure 1E). Chambers can be easily dropped into the holders ( Figure 1D) from the 107 top of the behavior box and secured in place for recording. 108 SAMPL includes a complete suite of open-source software for acquisition/real-time extraction saved as an AVI file. 118 SAMPL's modules and software were designed to scale, minimizing footprint and experimenter 119 time. We multiplex apparatus, providing three distinct compiled applications designed to run 120 simultaneously on one computer to reduce cost/footprint. A set of three SAMPL apparatus and 121 a computer case fit on one 24"x36" shelf ( Figure 1H). One SAMPL "rack" consists of four such 122 shelves (81.5" high) and costs~$40,000-$45,000 (December 2022, before volume discounts). 123 In our laboratory, trained experimenters can load such a rack for a typical 48 hr experiment in 124 30 minutes. Taken together, SAMPL's design is ideal to efficiently gather data describing pos- 125 ture and vertical locomotion. 126 127 SAMPL is well-suited to collect data from a wide range of small animals. We demonstrate the 128 flexibility of SAMPL's acquisition suite using three common model organisms. By changing 129 SAMPL's thresholds (Table 2), we could acquire data from three different organisms: Drosophila 130 melanogaster climbing behavior (Figures 2A and 2B), continuous locomotion in Caenorhabdi- 131 tis elegans ( Figures 2C and 2D), and swimming in Danio rerio ( Figures 2E and 2F). We present 132 raw video from the epochs in Figure 2 together with plots of real-time image processing (fly & 133 worms, Movie 2; fish, Movie 3). These results demonstrate SAMPL's excellent flexibility and ro- 134 bustness in real-time recording and analysis of vertical locomotion of small animals. 135 SAMPL validation: measuring postural and locomotor kinematics in real-time 136 Next, to demonstrate how SAMPL facilitates efficient collection of high-quality kinematic data, 137 we gathered a new dataset from larval zebrafish (7-9 days post-fertilization, dpf) that swam 138 freely in the dark. A typical experimental repeat consisted of two sequential 24-hour sessions 139 using 3 SAMPL boxes. Data were pooled across 27 repeats for subsequent analysis of kine- 140 matics. Each 24-hour behavior session yielded on average 1223±481 bouts per day for the 141 standard chamber (6-8 fish) and 1251±518 bouts per day for the narrow chamber (1-3 fish). 142 While not analyzed, running a single fish in the narrow chamber yielded 891±903 bouts over 143 24hrs. Based on the number of apparatus used, we estimate that a similar dataset (total n=121,979 144 bouts) could be collected in two weeks using a single SAMPL rack. 145 We first used our data to establish basic distributions of locomotion and posture. We used SAMPL's 146 processing algorithm to extract the following information in real-time: (1) pitch, defined as the 147 angle between the long axis of the fish's body and the horizon ( Figure 2E); (2) x (azimuth), z 148 (elevation) coordinates of the center of the pixels that correspond to the fish. After collection, we used SAMPL's processing suite to extract basic postural kinematics during swimming. Ze-150 brafish larvae swim in discrete periods of translation called "swim bouts" ( Figure 2F) 16,20 . We 151 defined swim bouts as periods where the instantaneous speed exceeds 5 mm/sec ( Figure 2F, 152 dashed line). The time of the peak speed was defined as t = 0 ms ( Figure 2F, cyan lines). Swim 153 bouts were aligned to peak speed for extraction of kinematic parameters; the period 250 ms 154 before and 200 ms after peak speed was reserved for future analysis. We observed that ze-155 brafish larvae swim predominantly at slower speeds with mean and standard deviation mea-156 sured 12.90±4.91 mm/s, on par with previous reports 16,[20][21][22] . Larvae showed a broad distri-157 bution of postures evaluated at peak speed (8.48°±15.23°) with a positive (nose-up) average, 158 suggesting that SAMPL detected a variation of nose-up and nose-down swim bouts. SAMPL 159 can thus rapidly acquire a rich dataset of spontaneous locomotor behavior and a wide range of 160 "natural" postures. 162 SAMPL includes data analysis and visualization code (Python source and sample datasets pro-163 vided) optimized to extract key parameters of balance and locomotion from larval zebrafish. 164 We use our "two-week" dataset to demonstrate that SAMPL can resolve these four parameters: We conclude that SAMPL's resolution and throughput allows rapid and deep insight into each 170 parameter, detailed below. Data analysis using the provided scripts on the provided dataset 171 runs in 30 minutes on a typical analysis computer (M1 processor, 16GB RAM). Full details of 172 analysis/visualization is provided in Appendix 4, and a step-by-step guide to set up the relevant 173 environment and to run experiments provided in Appendix 5. 174 Proper balance requires active stabilization. Zebrafish larvae are front-heavy and therefore sub-175 ject to destabilizing torques in the pitch (nose-up/nose-down) axis. Swim bouts counteract the 176 resultant forces, stabilizing the fish. Zebrafish larvae learn to initiate swim bouts when unsta-177 ble 16 . We first defined movement rate as the reciprocal of the inter-bout interval (Figures 3A 178 and 3B). More extreme postures were associated with higher movement rate ( Figure 3C), with 179 a parabolic relationship ( Figure 3D, R 2 = 0.14). We expect that the majority of the residual vari-180 ance reflects a previously-reported dependence of movement timing on angular velocity 16 .
181
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 27, 2023. The three coefficients of the parabola represent the baseline posture, the basal rate of move-182 ment, and -key to our analysis -the degree to which postural eccentricity relates to movement 183 rate, or "sensitivity," ( Figure 3D). SAMPL therefore permits efficient quantification of a crucial 184 posture-stabilizing behavior: the relationship between perceived instability and corrective be-185 havior. 186 Like most animals, larval zebrafish go where their head points. To adjust their vertical trajec-187 tory (i.e. to climb or dive) larvae must rotate their bodies away from their initial posture, point-188 ing in the direction they will travel ( Figures 4A and 4B) 17,23 . Previous work 17 established that 189 steering rotation in larvae swimming spontaneously occurs mostly before they reach the peak 190 speed ( Figure 4C). A larva's steering ability reflects the relationship between the change in pos-191 ture before the peak speed and the resultant deviation in trajectory ( Figure 4D). We parame-192 terized steering as the slope (gain) of the best-fit line between posture and trajectory evaluated 193 at the time of peak speed ( Figure 4E). A gain of 1 indicates that the observed trajectory could 194 be explained entirely by the posture at the time of peak speed ( Figure 4F). SAMPL revealed 195 that 7 dpf larvae exhibit an average steering gain at 0.67, suggesting an offset between pos-196 ture and trajectory at the time of peak speed ( Figure 4E, R 2 = 0.92). SAMPL allows us to infer 197 how effectively larvae steer using axial (trunk) musculature to navigate the water column. 198 To climb (Figures 5A and 5B) fish generate lift with their pectoral fins, assisting steering rota-199 tions and subsequent axial undulation 24,25 . Larval zebrafish learn to climb efficiently by coor-200 dinating their trunk and fins 18 . We defined the attack angle, or the additional lift associated 201 with each climb, as the difference between the steering-related changes and the resulting tra-202 jectory ( Figure 5C). We evaluated attack angle after pectoral fin loss, revealing a clear contribu-203 tion to climbs ( Figure 5D). Next, we demonstrate a positive correlation (with rectification and 204 asymptote) between steering-related rotations and fin-based attack angle ( Figure 5E, left). No-205 tably, after peak angular velocity, rotations are poorly correlated with attack angles (r = -0.17) 206 ( Figure 5E, right). These residuals reflect the initial angular deceleration as fish reach their peak 207 speed ( Figure 5A). We parameterize the relationship between the initial rotation and the attack 208 angle using logistic regression ( Figure 5F, R 2 = 0.31). The regression reveals the maximal slope 209 of the sigmoid relating steering and lift ( Figure 5G). We named this slope "fin-body ratio" as it 210 describes how larvae distribute labor between axial and appendicular muscles, i.e. between Larvae must actively maintain their preferred posture in the pitch axis. To do so, they rotate 214 partially towards their preferred orientation as they decelerate ( Figures 6A to 6C). The magni-215 tude of these rotations scales with the eccentricity of their posture before a swim bout 17 . We 216 estimated the slope (-0.17) of the line that related initial posture and the amount the fish ro-217 tated back toward the horizontal ( Figure 6D), R 2 = 0.56. As the behavior is corrective, the re-218 lationship is negative; we therefore define the gain of righting as the inverse of the slope (Fig-219 ure 6E). We further define the "set point" as the point where an initial posture would be ex-220 pected to produce a righting rotation of zero ( Figures 6E and 6F). SAMPL facilitates quantifi-221 cation of corrective reflex abilities (gain) and associated internal variables (set point). 222 Taken together, our estimates of key posture and locomotor parameters establish that SAMPL 223 can rapidly generate datasets that permit rich insight into the mechanisms of balance and ver-224 tical navigation. 225 SAMPL can resolve slight variations in posture control strategies across genetic backgrounds 226 To be useful SAMPL must resolve small but systematic differences in key measures of posture 227 and vertical locomotion. Even among isogenic animals reared in controlled environments, ge-228 netic differences contribute to behavioral variability [26][27][28][29][30][31][32][33] . The "two-week" dataset analyzed Figure 7C). Correspondingly, 244 and righting gain ( Figure 7K), all of which contributes positively to their higher posture stability. 246 These results demonstrate that SAMPL is capable of detecting inter-strain variations in locomo-247 tion and balance behavior. 248 In contrast, larvae of different ages adopt different strategies to stabilize posture and navigate 249 in depth [16][17][18] To contextualize the magnitude of strain-related differences we gathered a lon-250 gitudinal dataset by measuring behavior from the same siblings of the AB genotype at three 251 timepoints: 4-6, 7-9, and 14-16 dpf (Table 3). We observed that the standard deviation of IBI 252 pitch for 4 and 14 dpf larvae was 38.1% higher and 11.3% lower, respectively, than the aver-253 age result of 7 dpf larvae (Table 3). Across strains at 7 dpf, the variation was much smaller: from 254 11.8% higher to 11.2% lower. Similarly, relative to 7 dpf larvae, sensitivity of 4 dpf larvae was 255 considerably lower (-42.5%), and increased to 23.6% higher by 14 dpf (Table 3); variations among 256 7 dpf strains were up to 10.0% lower and 15.4% higher. 257 Our analysis of new data supports three key conclusions. First, SAMPL can uncover small, sys-258 tematic differences in the way fish swim and stabilize posture. Second, SAMPL can make lon-259 gitudinal measures of the same complement of animals as they develop. Third, relative to de-260 velopment, the effect of genetic background is small. We conclude that SAMPL's capacity to 261 resolve small differences supports its usefulness as a tool screen for modifiers of postural con-262 trol and vertical locomotor strategies.
263
Estimating SAMPL's resolution 264 Our dataset establishes SAMPL's ability to resolve small kinematic differences between cohorts. 265 How does SAMPL's power change as a function of the size of the dataset? We used resampling 266 statistics to estimate SAMPL's resolution as a function of the number of the bouts (Methods). 267 To ensure our most conservative estimate, we resampled data combined across AB, SAT and 268 WT genotypes at 7dpf. 269 As expected, the width of the confidence interval for any estimated parameter decreased with 270 the number of bouts ( Figure 8A). The most challenging parameter to estimate is coordination 271 between fin and trunk (fin-body ratio) The steepness with which the confidence interval width 272 decreases follows the number of regression coefficients necessary for each measure: fin-body 273 ratio (4 parameters); bout timing (3 parameters); and steering or righting (2 parameters). We 274 therefore propose that these particular measures can serve as a general guide for the chal-275 lenge of estimating parameters within a SAMPL dataset. 276 reject the null hypothesis 34 . We address this question by asking how much data one would 278 need to gather in order to detect meaningful effects. We simulated difference of particular 279 magnitudes by imposing an offset on each parameter (sensitivity, steering gain, fin-body ra-280 tio, and righting gain) while preserving the original variance (Methods). Offsets were expressed 281 as a fractional difference, and resampling was used to estimate the effect size one would see 282 as a function of the number of bouts/IBIs when comparing kinematic parameters between the 283 original dataset and the dataset with an imposed effect (Methods). 284 Broadly, we find that for all kinematic parameters, the smaller the percent change, the larger 285 the required sample size ( Figure 8B). Steering and righting gains require the fewest bouts to 286 detect a 1-2% change with an effect size > 0.5 ( Figure 8B, green and red). However, sensitivity 287 and fin-body ratio require relatively larger datasets to confidently discriminate small changes 288 ( Figure 8B, brown and magenta). We conclude that the full "two-week" dataset we generated 289 using SAMPL (n = 121,979 bouts) is sufficient to reveal any biologically-relevant differences be-290 tween two conditions. 291 In summary, these simulations demonstrate that a single SAMPL rack divided into two condi-292 tions (6 apparatus / each) could, in two standard 48-hour runs, generate sufficient data to re-293 solve meaningful differences in postural and locomotor kinematics between two conditions. 294 We provide detailed instructions in Appendix 5 addressing experimental design strategies to 295 maximize SAMPL's resolution. 297 We present SAMPL, a scalable solution to measure posture and locomotion in small, freely-298 moving animals. We start with a brief overview of the hardware and software, with compre-299 hensive guides to every aspect of SAMPL's hardware and software included in the Appendices. Danio rerio (zebrafish). To illustrate the depth of insight accessible using SAMPL we explored a 303 new dataset -consisting of two weeks worth of data -that illuminates four key parameters of 304 zebrafish navigation in depth: bout timing, steering, fin-body coordination, and righting. We 305 made two discoveries using SAMPL's analysis suite: (1) systematic changes to zebrafish pos-306 ture and locomotion across genetic backgrounds and (2) that these changes were small rela-307 tive to variation across developmental time. Finally, we use our new dataset to define SAMPL's 308 resolution: how much data an experimenter would need to collect to detect meaningful ef-309 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made SAMPL was designed to scale efficiently. Data is gathered by a compiled executable, allowing 333 SAMPL to run three apparatus off a single computer, reducing costs and space. A SAMPL rack 334 consists of 12 apparatus running off four computers with a footprint of 24"x36"x81.5" (LxWxH). 335 The key components such as the camera are readily available from multiple suppliers. Taken 336 together, SAMPL can be used immediately to screen and/or to provide videographic data from 337 freely moving animals at scale. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
DISCUSSION
The copyright holder for this preprint this version posted March 27, 2023. ; https://doi.org/10.1101/2023.01.07.523102 doi: bioRxiv preprint between the body and the horizon. As we demonstrate here, this small set of parameters de-342 fines behaviors larval zebrafish use to swim and balance in depth: bout timing (Figure 3), steer-343 ing ( Figure 4), fin-body coordination ( Figure 5), and righting ( Figure 6). While each parameter 344 has been previously defined [16][17][18] , the new data we present here illustrates differences across 345 genetic backgrounds and development and allows granular estimation of statistical sensitivity. 346 Taken together, SAMPL's focus facilitates exploration of unconstrained vertical behavior.
347
Comparisons with other approaches 348 Here, we discuss SAMPL's advantages by comparing it with other available tools for measuring 349 Drosophila, C. elegans, and zebrafish behavior. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The simple nervous system of C. elegans is a powerful model to study neural circuits that con-373 trol posture and movement. C. elegans possess a rich and tractable repertoire of motor con-374 trol 53 . For example a pattern generator creates sinusoidal waves of muscle contraction that 375 propel C. elegans on a solid substrate, and these sinusoidal movements are sculpted by pro-376 prioceptive feedback 54 . Proprioceptive feedback also controls transitions between sinusoidal 377 crawling and non-sinusoidal bending that can propel animals in a liquid environment 55-57 . 378 Other sensory stimuli elicit coordinated motor responses that are critical for navigation. De-379 creasing concentrations of attractive odorants and gustants trigger reversals followed by a pirou-380 ette or omega bend, which results in a large-angle turn that reorients animals 58,59 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Further information and requests for resources and reagents should be directed to and will be 477 fulfilled by the lead contact, Dr. David Schoppik ( schoppik@gmail.com ).
Materials availability 479
This study did not generate new unique reagents. at densities under 20 larvae per 10 cm petri dish and were fed cultured rotifers (Reed Mari-494 culture) daily. Larvae that had their behavior measured at 14 dpf were raised as stated above 495 before being moved to 2 L tanks with 300 ml of cultured rotifers at 9 dpf. At 13 dpf, they were 496 transferred back to petri dishes with E3 medium for adaptation. 497 Larvae with different strains were achieved by crossing Schoppik lab strain with a mixed AB, TU, 498 and WIK background to three different wild-type strains: AB (Zebrafish International Resource 499 Center), mixed background of AB/WIK/TU, or SAT (Zebrafish International Resource Center). 500 Reference parameter values in Table 3 for 4, 7, 14 dpf fish were gathered using the AB strain 501 fish. 502 Drosophila melanogaster (w 1118 ) were raised at 23°on standard cornmeal-agar food under a 503 12/12 light/dark cycle.
504
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Caenorhabditis elegans (C. elegans) were grown at 20°on nematode growth medium agar 505 plates seeded with Escherichia coli OP50 as previously described 129 . For Drosophila recording, four flies were transfered to a narrow chamber. A small piece of 520 water-dampened kimwipe was put at the bottom of the chamber to maintain humidity. A n 521 acrylic plug was secured at the top to prevent them from escaping the chamber. We secured 522 the chamber with the flies in the SAMPL apparatus and performed the standard SAMPL exper-523 iment using recording parameters provided in Table 2. 524 To image swimming C. elegans, eight starved N2 adult hermaphrodites were transferred to a 525 narrow chamber filled with 15 ml M9 buffer (3 g/l KH 2 PO 4 ; 6 g/l Na 2 HPO 4 ; 0.5 g/l NaCl; 1 g/l 526 NH 4 Cl) which was secured in the SAMPL apparatus as described above. Behavior recording 527 was started immediately afterwards. Refer to Table 2 for SAMPL thresholds for C. elegans de-528 tection.
529 Fin amputation 530 6 dpf zebrafish larvae were anesthetized in 0.02% tricaine methanesulfonate (Syndel) and 531 transferred to 3% Methylcellulose (Sigma). Fin amputation was done by removing pectoral 532 fins using fine forceps (FST). Specifically, one pair of forceps was used to stabilize the head of 533 the fish and a second pair was used to grab the joint and pull off the fins. Finless larvae were 534 washed three-times in E3 and fed with cultured rotifers before behavior assessment at 7 dpf. 535 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Behavior data was analyzed using the Python analysis pipeline SAMPL_analysis_visualization.
543
SAMPL_analysis() function was used to calculate swim parameters, extract bouts and inter-bout 544 intervals (IBIs) from the raw data, and align swim bouts by the time of the peak speed. 545 Each run of the experiment (recording from "start" to "stop") generates one data file ( * .dlm) 546 containing recorded raw parameters including time stamp, fish body coordinates, fish head co- To extract bouts from the raw data, first, swim features, such as speed, distance, trajectory, an-551 gular velocity, etc., were calculated using basic parameters and time interval. Next, epochs that 552 were longer than 2.5 s, contain maximum swim speed greater than 5 mm/s, and pass various 553 quality-control filters were selected for bout extraction. Epochs containing multiple bouts were 554 segmented and truncated so that each detected bout contains data from 500 ms before to 555 300 ms after the time of the peak speed. Then, bouts containing 800 ms of swim data were 556 aligned by the time of the peak speed and saved for further analysis. 557 All further quantification was performed on data during zeitgeber day, namely the 14 h light 558 time for fish raising under 14/10 h light/dark cycle. 559 To calculate IBIs, epochs with multiple bouts are selected and the duration of swim speed be-560 low the 5 mm/s threshold between two consecutive bouts is calculated. A 100 ms buffer win-561 dow is then deducted from each end of the duration to account for errors of swim detection 562 ( Figure 3A). Pitch angles during each IBI were averaged to generate an IBI pitch ( Figure 3B). 563 Definition of other bout parameters can be found in Table 3. All bout parameters (except for ki-564 netic parameters explained in the next section) reported in the main text and Table 3 are mean 565 values across swim bouts collected from multiple experimental repeats. One experimental re-566 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made To calculate larvae sensitivity to pitch changes (Figure 3), we plotted bout frequency as a func-570 tion of IBI pitch. The data was modeled using a quadratic polynomial regression (least squares) 571 defined by function: where E k and E h are the mean of k and h with V k and V h being their respective variance. Next, 619 the standard errors of the fin-body ratio were calculated and used to estimate CI widths at 0.95 620 significance level. The primary differences that we've encountered are whether a particular model implements 665 binning or other on-camera computations, heat management, and different manufacturer- 666 provided APIs. When we use multiple cameras from the same manufacturer on the same 667 computer, we have also noticed that certain cameras will throw timeout errors on some USB 668 ports but not others; shuffling cameras and ports has worked to solve this problem. At the 669 time of writing, supply chain issues mean that most major camera companies quote long lead 670 times, but cameras ordered directly through alibaba.com all shipped within two weeks.
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Illumination 672 Image quality is proportional to available light. Further, the size of the illuminated area defines 673 the size of the field that can be imaged. Finally it is imperative for our experiments that from 674 the fish's perspective that the "dark" period is completely dark. We therefore chose 940nm 675 LEDs as our source of infrared illumination. This left us with three options to build our illumina-676 tion source: LEDs mounted on adhesive strips, "star" style LEDs with 1-4 dies on a single PCB, 677 and a high-power LED array. The LED strips had too little illuminance for our purposes due pri-678 marily to the spacing of the LEDs. The high-power LED array had ample illuminance but gen-679 erated so much heat that it required active cooling. 680 We developed a simple illumination module to provide diffuse IR light across a 50mm circle 681 An LED mounted on a "star" PCB (Opulent LST-01F09-IR04-00, Mouser) provided ample light. 682 We mount each "star" LED with thermal adhesive to a small heat sink (Ohmite SV-LED-325E) 683 which in turn is glued to a Thorlabs adapter (SM1A6FW) to allows the wires to exit and the 684 LED/heatsink to connect to collimation and diffusion optics. The heat sink is machined (either 685 with a Dremel hand-held tool or a mill) on one side to allow the wires that power the LED to lie 686 flat against the heatsink. We power multiple illumination modules in series using a constant 687 current LED driver (LuxDrive BuckBlock 1000mA). Our illumination setup generates negligible 688 heat and our modules run continuously for years. 689 Our imaging parameters are fixed across experiments and optimized to give the highest qual-690 ity data we can achieve with our hardware. The gain of the camera is set either to its lowest 691 value or just above to minimize noise. Our exposure time is either 750 µsec or 1msec, allowing 692 for a crisp image in the face of the fastest movements that fish can make. The illuminated area 693 is circular, but the image sensor size is rectangular. We therefore crop the sides of the image to 694 produce a square that fits within the illuminated area.
695
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Our choice of lens was guided by the need to balance different demands: 697 1. The longer the working distance, the greater the space needed between the sample and 698 the lens. We wanted our apparatus to fit length-wise on a 24 inch shelf, and so we needed 699 to minimize the working distance. 700 2. The entire depth of the tank needs to be in focus, but not beyond that because we'd like to 701 blur our LED. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made top is a steel tray fabricated to order by MetalsCut4U (Avon Lake OH). Our current enclosure 728 took roughly three months to prototype before settling on the final design. 729 Shelving and fleet organization 730 We have organized our fleet of apparatus to sit on mobile wire shelving. Currently, we use 731 36"x24"x81.5" adjustable wire shelving units (McMaster Carr, Robbinsville NJ). We prefer to 732 have the shelving on casters as it makes accessing the back of the units considerably easier. 733 Shelving is organized such that one computer and three apparatus sit on a single shelf. En-734 closures on a given shelf are color-coded (blue, gold, and red) so that each apparatus can be 735 uniquely identified by a color/shelf/module combination; this also facilitates wire labeling. Each 736 shelf has its own power strip that controls the computer, the IR lights, and the white LEDs; all 737 strips plug into a single uninterruptible power supply (APC SmartUPS 1000C). 738 Our aim in specifying module size was to ensure that multiple investigators could set up ex-739 periments simultaneously, and to minimize the cost One unit has four shelves so that a sin- What we don't measure 758 To extract the maximum amount of useful information about posture and locomotion with 759 the minimum amount of overhead we had to be selective about what we measure. Our imag-760 ing field is located in the center of the arena; fish that swim at the bottom, top, or sides of the 761 tank where there is a boundary are excluded from tracking. While multiple fish swim in the 762 same arena, we do not take data when more than one fish is in the imaging field to sidestep 763 the need to track fish identity. Our arena is sized to allow fish to swim freely but its shape (a 764 rectangular solid) encourages fish to swim in line with the imaging plane; we exclude frames 765 where fish turn away from the field of view (i.e. are swimming towards/away from the camera). 766 Finally, capturing the full range of rapid propulsive undulations of the fish tail requires a frame 767 rate of 500Hz-1kHz 130,131 . As changes to posture and locomotion are much slower, we opted 768 to record at 160Hz. Together, these choices allowed us to optimize our algorithms to achieve 769 the speed necessary to process video in real-time. image. 775 2. Threshold the difference image such that all small differences are set to zero. 776 3. Dilate the image three times in succession to remove any larger clumps that are still smaller 777 than a fish. 778 4. Extract and quantify all particles in the image. 779 Real-time video processing allows efficient data extraction during video acquisition. Our design 780 of the architecture is further discussed in the section: Optimizations for speed. 781 Below we detail a number of additional processing and optimization steps to ensure that we 782 maximize useful data. 783 Measuring the pitch of the fish 784 To extract the pitch (the angle of the fish with respect to the horizon), we perform the following 785 steps to ensure that the sign and magnitude of the angle is correctly assigned: 786 1. Fit the particle with an ellipse and extract the angle of the long axis with respect to the hori-787 zon.
788
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made These steps ensure that the data saved follows a simple and intuitive convention for posture. 795 Optimizations for speed 796 To optimize our code for speed, we use a set of thresholds to rapidly evaluate and reject frames 797 1. Before any processing, we sum the pixel values in the frame. If it is too low (no fish in frame) 798 or too high (more than one fish in the frame) we reject the frame. 799 2. After the particles are identified we reject the frame if a particle is touching the edge (fish 800 partially out of frame), if there is more than one particle (multiple fish) or if the length of the 801 particle is too short (fish bending in/out of the field of view). We define an epoch as a set of 802 continuous frames that pass all our exclusion criteria (i.e. that contain a single fish in frame). 803 Epoch duration is tracked and, when too short, can be rejected. 804 In addition to optimizing the algorithm, we adopted a producer-consumer architecture to de-805 couple video acquisition from video processing and saving data. Our software runs two rou-806 tines: the "producer," which acquires frames from the camera and places them in a queue 807 in memory, and the "consumer" that extracts each frame from the queue and processes it in 808 turn. Our program monitors the size of the consumer buffer and, if it has less that 10% free, 809 pauses the producer routine for 15 seconds to allow the buffer to clear. In this configuration, 810 the performance ceiling shifts from CPU speed (i.e. how quickly can a frame be processed) to 811 the amount of RAM available (i.e. how many frames can be queued). At the time of writing this, 812 doubling the amount of RAM is considerably less expensive than doubling CPU performance. 813 The choice of architecture thus brings down the cost of the computer. 814 Saving raw video 815 While the bulk of our experiments rely on real-time processing of video it is often useful to save 816 the actual data. Further, we wanted to be able to set user-defined criteria to determine in real-817 time which videos were worth saving. Leveraging the producer-consumer architecture, our 818 software contains a routine that independently buffers the frames being analyzed and, if, the 819 video to be saved meets user-defined criteria, will pass the frames to an independent program 820 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Our algorithm relies on common and mature image processing routines and could be instan-828 tiated in any modern programming language. Since we had run this algorithm for the better 829 part of a decade we were confident that it was sufficiently stable to compile into a distributed User interface 838 We designed the interface to enable easy initialization of experiments, rapid graphical and 839 quantitative visualization of video processing and performance, and to minimize error. Launch-840 ing the executable starts the program, which allows the user to fill out various text, numeric, 841 and drop-down fields that describe the experiment. The user then monitors the video feed un-842 til no fish are in frame and then selects that image as the background. We have found that this 843 initial bit of monitoring both compensates for slight day-to-day differences in arena placement. 844 More importantly, it forces the user to monitor the live feed at the beginning of each experi-845 ment, a useful bit of mindfulness that minimizes lost data. Once running, the user can: mon-846 itor the output of each step in the processing algorithm graphically, monitor the number of 847 times the consumer buffer has overflowed (usually zero), update the text fields, and stop the 848 program. Hardware parameters are stored in a text file that can be easily edited directly. Experi-849 ment parameters are similarly saved to text files and can be reloaded to save time. 850 We have implemented a number of user interface items to minimize confusion in the face of a 851 fleet of instruments. First, we have color-coded versions of the executable (blue, gold, and red) 852 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made where the background clearly differentiates the version. Each version has its own configura-853 tion file that, during setup, is coded to a particular apparatus. Thus the user is always aware of 854 which apparatus they are interfacing with based on color cues. Next, we added a "debug" but-855 ton to the front panel that allows for direct monitoring and editing of all program variables. In 856 "debug" mode the user has the option to save raw video.
858
In this Appendix, we walk through box assembly and recording settings. Refer to Appendix 5 859 for executing experiments and SAMPL data analysis. Solder 2x 9" wires onto the IR LED "star." Attach IR LEDs to heatsinks using HexaTherm tape. 901 Note that in order to pass the wires through the heatsinks and the SM1A6FW adapter on the 902 opposite end, the ears of the Ohmite heatsink need to be trimmed down a little. When done, 903 attach the heatsink to the adapter using thermal epoxy. To simplify light wiring, we use one 904 1000 mA BuckBlock to dirve 3 IR lights in series for 3 boxes on the same level of the shelf. To 905 do this, one needs 2x 7" wires to connect adjacent IR cables and 1x 22" wire connecting the 906 further IR to the BuckBlock. Use another 8" wire to connect the closest IR to the BuckBlock. 907 We recommend using XT60H connectos to link these wires to the IR light cables and connect 908 wires to the BuckBlock for the ease of troubleshooting and replacement. Finally, connect the 909 BuckBlock to 12 V 2 A power supply through pigtail adaptors. Network setup 964 We use a Synology data server as a repository to store behavior data. Hard drives are setup 965 as RAID 10. Each SAMPL rack has its own ethernet switch, which can be connected to other 966 switches as necessary.
968
In this appendix, we discuss algorithms for the data analysis and plotting software. We assume 969 that the user is working with data from larval zebrafish here. If not, the specific parameters 970 identified here are unlikely to translate as other organisms move differently but can nonethe-971 less be used as a starting point. Refer to Appendix 5 for instruction for use. Refer to the Key Re-972 sources
996
All the processes above can be found in the script src/SAMPL_analysis/preprocessing/analyze_dlm_v4.py. 998 Epochs that pass the quality control are used to extract swim bouts using function 999 grab_fish_angle() under src/SAMPL_analysis/bout_ 1000 analysis/grab_fish_angle_v4.py. 1001 We use a swim speed threshold of 5 mm/s to determine swim windows. Adjacent swim win-1002 dows with intervals smaller than 100 ms are combined. Next, we find the time of the peak 1003 speed for each swim window and extract frames in a range of 500 ms before to 300 ms af-1004 ter that. Inter-bout intervals (IBI) are determined as time between adjacent swim bouts with 1005 a 100 ms buffer window deducted from both the beginning and the end and IBI data is ex-1006 tracted accordingly. Baseline is considered the time during which larvae swim slower than 2 1007 mm/s and baseline parameters are extracted accordingly.
1008
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made To calculate fin-body coordination, users need to determine how the rotation is calculated. 1057 One way is to use rotation to time of peak angular velocity which requires estimation of 1058 time of peak angular velocity. To do this, we first calculate angular velocity using smoothed 1059 pitch angles and adjust the signs so that values are positive before time of the peak speed. 1060 Median of angular velocity time series from the same experimental repeat (see Appendix 5 1061 for data organization) is used to find time of peak angular velocity. Lastly, we average results 1062 across experimental repeats to determine the peak angular velocity time. However, this calcu-1063 lation requires a large amount of bout data. Alternatively, one may use a fixed value for time of 1064 peak angular velocity. Generally, we found -50 ms (50 ms before time of peak speed) to be a 1065 good value to use. Once the time of peak angular velocity is determined, rotation is calculated 1066 by pitch change from 250 ms before peak speed to time of peak angular velocity. Some scripts 1067 have the option to sample data from each experimental repeats. See Appendix 5 for instruc-1068 tion.
1069
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 27, 2023. ; https://doi.org/10.1101/2023.01.07.523102 doi: bioRxiv preprint ber of bouts, which will specify the number of boxes and larvae per box required. Outlier boxes 1130 with too few or too many bouts (e.g. more/less than 2SD) can then be excluded from further 1131 analysis according to pre-determined criteria. Finally, we recommend running the full "exper-1132 iment" multiple times to ensure that the findings are reproducible, and to report the variance 1133 across estimated parameters. Certain circumstances may be ill-suited to this approach: for ex-1134 ample, if particular genotypes of larvae are especially rare, such as in the case of doubly biallelic 1135 mutants, or genotypes that simply swim drastically less. In such cases one can combine swim 1136 bouts across experimental repeats, and report the estimated error in pararmeter estimates us-1137 ing statistical resampling techniques such as the jackknife. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made rectory of the root folder) and the frame rate as instructed. This function aligns bouts in .dlm 1200 files within a directory so that peak speed is at time 0 ms, with 500 ms of activity before and 1201 300ms of activity after. It is important to note that all files in the same subfolders under the in-1202 put directory will be combined to extract bout parameters. The analysis script will take the sub-1203 mitted directory and analyze all data files within it, including all subfolders in its search, regard-1204 less of depth. Subfolders can be used to separate analyses, experimental conditions, or repeats. 1205 Data with different frame rates should be analyzed separately to ensure proper parameter cal-1206 culation, as only one can be used at a time. 1207 The program will skip the current .dlm file if it fails to detect a bout in it. However, errors are ex-1208 pected if files contain too little recorded data to extract a bout. Therefore, we suggest removing 1209 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made If the data size from a single repeat is not adequate for parameter calculation, we suggest com-1219 bining data from multiple repeats and use sampling techniques such as Jackknifing for error 1220 estimation.
1221
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 27, 2023. ; https://doi.org/10.1101/2023.01.07.523102 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 27, 2023. ; https://doi.org/10.1101/2023.01.07.523102 doi: bioRxiv preprint (A) An inter-bout interval (IBI, brown area) is defined as the duration when swim speed is below the 5 mm/s homeostasis threshold (dashed line) between two consecutive bouts with a 100 ms buffer window (grey area) deducted from each end. (B) Distribution of IBI duration (left) and mean pitch angle during IBI (right). (C) Bivariate histogram of bout frequency and IBI pitch. Bout frequency is the reciprocal of IBI duration. (D) Bout frequency plotted as a function of IBI pitch and modeled with a parabola (black line, R 2 = 0.14). Brown dots indicate binned average of IBI pitch and bout frequencies calculated by sorting IBI pitch into 3°-wide bins. For all panels, n = 109593 IBIs from 537 fish over 27 repeats.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 27, 2023. ; https://doi.org/10.1101/2023.01.07.523102 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 27, 2023. ; https://doi.org/10.1101/2023.01.07.523102 doi: bioRxiv preprint (C) Illustration of components that contribute to trajectory deviation. Larvae rotate their bodies starting from bout initial (blue) and reach peak angular velocity (asterisk) before peak speed. Any rotation generated during decrease of angular velocity is considered residual (grey). At time of peak speed, there is an offset between the pitch angle (dashed line) and bout trajectory (arrow) which is termed attack angle (orange). Body rotations, residual, and attack angle add up to trajectory deviation. (D) Distribution of attack angles in control fish (left) and fish after fin amputation (right). Dashed lines indicate 0 attack angle. (E) Attack angles plotted as a function of body rotations (left, blue) or residual rotations (right). Rotations and residuals are sorted into 0.5°-wide bins for calculation of binned average attack angles. Swim bouts with negative attack angles while having steering rotations greater the 50th percentile (hollow squares) were excluded for binned-average calculation. (F) Attack angles plotted as a function of body rotations (blue line) and fitted with a logistic model (black line, R 2 = 0.31). Fin-body ratio is determined by the slope of the maximal slope of the fitted sigmoid (magenta). Rotations are sorted into 0.8°-wide bins for calculation of binned average rotations and attack angles (blue line). Swim bouts with negative attack angles while having steering rotations greater the 50th percentile were excluded for sigmoid modeling. (G) Schematic illustration of how fin-body ratio reflect climbing mechanics. For all panels, n = 121979 bouts from 537 fish over 27 repeats.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 27, 2023. ; https://doi.org/10.1101/2023.01.07.523102 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 27, 2023. ; https://doi.org/10.1101/2023.01.07.523102 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 27, 2023. ; https://doi.org/10.1101/2023.01.07.523102 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 27, 2023. ; https://doi.org/10.1101/2023.01.07.523102 doi: bioRxiv preprint Figure S1: Custom breadboard for SAMPL base (A) Custom aluminum breadboard, not anodized, 0.5" thick. All holes (8 total) counterbored for 1/4"-20 cap screw. Grooves to be cut on the side of the breadboard OPPOSITE to the counterbore.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 27, 2023. ; https://doi.org/10.1101/2023.01.07.523102 doi: bioRxiv preprint | 2023-01-10T14:09:35.341Z | 2023-01-07T00:00:00.000 | {
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232135213 | pes2o/s2orc | v3-fos-license | Optical Frequency Comb Fourier Transform Spectroscopy of $^{14}$N$_2$$^{16}$O at 7.8 {\mu}m
We use a Fourier transform spectrometer based on a compact mid-infrared difference frequency generation comb source to perform broadband high-resolution measurements of nitrous oxide, $^{14}$N$_2$$^{16}$O, and retrieve line center frequencies of the $\nu$$_1$ fundamental band and the $\nu$$_1$ + $\nu$$_2$ - $\nu$$_2$ hot band. The spectrum spans 90 cm$^{-1}$ around 1285 cm$^{-1}$ with a sample point spacing of 3 ${\times}$ 10$^{-4}$ cm$^{-1}$ (9 MHz). We report line positions of 72 lines in the $\nu$$_1$ fundamental band between P(37) and R(38), and 112 lines in the $\nu$$_1$ + $\nu$$_2$ - $\nu$$_2$ hot band (split into two components with e/f rotationless parity) between P(34) and R(33), with uncertainties in the range of 90-600 kHz. We derive upper state constants of both bands from a fit of the effective ro-vibrational Hamiltonian to the line center positions. For the fundamental band, we observe excellent agreement in the retrieved line positions and upper state constants with those reported in a recent study by AlSaif et al. using a comb-referenced quantum cascade laser [J Quant Spectrosc Radiat Transf, 2018;211:172-178]. We determine the origin of the hot band with precision one order of magnitude better than previous work based on FTIR measurements by Toth [http://mark4sun.jpl.nasa.gov/n2o.html], which is the source of the HITRAN2016 data for these bands.
Introduction
Nitrous oxide (N 2 O) is of significant importance for atmospheric physics and chemistry. Despite being only a minor constituent of the Earth's atmosphere, it is a potent greenhouse gas [1], and it contributes strongly to stratospheric ozone depletion [2]. Moreover, global N 2 O emissions are increasing due to human activity [3] and will likely be enhanced by global warming [4]. This calls for the ability to monitor atmospheric N 2 O by spectroscopic means, which requires laboratory studies to provide precise and accurate line parameters. One wavelength range of interest for environmental monitoring is the atmospheric window around 8 µm, where N 2 O absorbs due to its strong ν 1 fundamental band as well as several weaker hot and overtone bands. The 8 µm spectral range has been extensively studied using conventional Fourier transform infrared spectroscopy (FTIR). Guelachvili [5] determined line positions of the ν 1 band, the 2ν 2 band, and several hot bands with uncertainties of one to a few MHz, as well as relative line intensities and linewidths. Levy et al. [6] reported absolute line intensities of the ν 1 and the 2ν 2 bands, while Lacome et al. [7] studied self-and N 2 -broadening of lines belonging to these two bands. Toth [8][9][10][11] measured various N 2 O absorption bands over a wide range of the infrared spectrum and compiled an extensive line list [12], including line positions, intensities, and broadening parameters, which to this day forms the basis for a large part of the entries on N 2 O in the HITRAN database [13]. More recently, FTIR was employed by Wang et al. [14] to measure and assign the spectra of the less abundant N 2 O isotopologues around 8 μm. In an early laser-based spectroscopic study of N 2 O, Varanasi and Chudamani [15] used a tunable diode laser to measure the line intensities of the ν 1 band. A few years later, Maki and Wells [16] determined frequencies of the N 2 O absorption lines in several bands spread over almost the entire infrared spectrum (among them the ν 1 band, 2ν 2 band and hot bands at ~8 μm) with uncertainties in the single MHz range using heterodyne frequency measurements, for use in infrared frequency calibration tables. As quantum cascade lasers (QCLs) became available, Grossel et al. [17] used a continuous-wave (CW) QCL to measure line intensities and air broadening parameters of the ν 1 band, while Tonokura et al. [18] measured CO 2 broadening parameters of the ν 1 band. Moreover, Tasinato et al. [19] employed a pulsed QCL to study collisional processes, and hence pressure broadening, of the ν 1 band in He, Ar, N 2, and CO 2 .
The precision and accuracy of line position determination can be significantly improved by using optical frequency combs that directly link the optical frequencies to radio frequency standards. To this date, there exists only one set of studies of line positions of N 2 O in the 8 µm range involving a frequency comb. Lamperti et al. [20] referenced a CW QCL tunable around ~8 µm to a Tm:fiber comb at 1.9 µm through a sum frequency generation scheme, and reported the positions of three lines in the ν 1 band with ~63 kHz uncertainty. AlSaif et al. [21] used this comb-referenced QCL to retrieve positions of the P(40) to R(31) lines of the ν 1 band in the Doppler limit with uncertainties below 200 kHz. More recently, they measured the center frequency of the R(16) line with precision below 50 kHz using sub-Doppler spectroscopy [22]. We show that similar performance (in the Doppler limit) can be obtained by employing an 8 µm comb to directly measure the spectrum, removing the need for complex frequency referencing. Moreover, direct frequency comb spectroscopy allows measuring the entire vibrational bands with tens of thousands of comb lines simultaneously (rather than sequentially line by line, as is done with CW lasers), which reduces the influence of drifts. The most promising comb sources for precision spectroscopy in the 8 µm range are based on difference frequency generation (DFG) from a single femtosecond pump source, deriving the signal comb from the pump laser using a nonlinear fiber [23][24][25], or through intrapulse DFG (IDFG) [26,27]. These DFG combs cover wide bandwidths (up to superoctaves for IDFG sources), and are inherently free from carrier-envelope-offset frequency, f ceo , since f ceo is identical for the pump and signal combs and cancels in the DFG process. The lack of f ceo significantly simplifies the absolute stabilization of the DFG-based comb sources. Timmers et al. [26] employed IDFG sources for dual-comb spectroscopy (DCS) with sample point spacing equal to the comb repetition rate, f rep , of 100 MHz. Much denser sampling point spacing was demonstrated by Changala et al. [28], who used a DFG source [24] and a Fourier transform spectrometer (FTS) with comb-mode-width limited resolution to measure the ro-vibrational spectrum of C 60 . The 8 µm range can also be reached through nonlinear conversion in optical parametric oscillators (OPOs), which, however, are not f ceo -free. 8 µm OPOs have been combined with an FTS [29], the DCS approach [30], and an immersion grating spectrometer [31], all with resolution limited by the instrumental broadening rather than the comb mode linewidth. The latter work investigated the pressure broadening of one hot band N 2 O line, but does not report the line positions. It should be noted that frequency combs based on QCLs [32] operate in the 8 µm range. However, their inherently large comb mode spacing is not ideal for precision spectroscopy of molecule problem; the form We u comb em the ν 1 + ν and 1315 we obtain centrifuga 112 absor band orig N 2 O.
Experi
The main a multi-p Behind the OP-GaP crystal, an optical long-pass filter blocks the near-IR radiation emitted by the dualwavelength source while transmitting the MIR idler produced in the DFG process. The latter is collimated and coupled into a multi-pass absorption cell with an absorption path length of 10 m (Thorlabs, HC10L/M-M02). The cell is connected to a vacuum pump, a pressure transducer (Leybold, CERAVAC CTR 101 N), and the sample gas supply (2.98 % N 2 O diluted in N 2 , supplier: Air Liquide AB), as shown in the right corner of Fig. 1.
After the multi-pass cell, the MIR beam is re-collimated using two lenses and guided to a fast-scanning FTS. The FTS is the same as in Ref. [35], except that the beamsplitter and the detectors have been replaced to extend the operating range to 8 µm. The two out-of-phase interferometer outputs are detected by a pair of thermoelectrically-cooled HgCdTe detectors (VIGO Systems, PVI-4TE-10.6-1x1) in a balanced configuration. The comb beam radius inside the FTS varies between 2 mm at the waist (positioned ~0.5 m after the beamsplitter) and 4 mm at the detectors. Due to the relatively large divergence of the 8 µm beam, the off-axis components give rise to interference fringes that are phase shifted with respect to the interference fringes at the beam center, thus reducing the interferogram contrast. To mitigate this effect, we placed an optical aperture in front of each detector, which yielded an interferogram contrast of ~50% (compared to ~30% without the aperture). The aperture radius (~2 mm) was found empirically as a trade-off between the interferogram contrast and the power level on the detectors (~14 µW). The optical path difference in the FTS is calibrated using a 1556 nm narrow-linewidth CW laser (RIO, PLANEX), whose beam propagates on a path parallel to the MIR comb beam. The CW laser interferogram is registered using an InGaAs detector. The comb and CW laser interferograms are recorded synchronously with an analog-digital converter (National Instruments, PCI-5922) and a LabVIEW program running on a personal computer.
To acquire a spectrum, we first evacuated the cell, purged it with the sample gas, filled it to the desired total pressure, and closed the valve at the input to the cell to isolate it from the rest of the gas system. We stabilized the comb f rep and acquired a set of 50 interferograms. To reduce the sampling point spacing in the spectrum, we stepped f rep in increments of 29 Hz via tuning of the RF generator, and acquired 50 interferograms at each step. In total, we made 14 steps of f rep to scan each comb mode over one mode spacing (f rep ) in the optical domain. We made two measurements, at total pressures of 0.02 mbar and 0.32 mbar, with signal to noise ratio optimized for the ν 1 fundamental band and the ν 1 + ν 2 -ν 2 hot band, respectively. At 0.02 mbar, we made six f rep scans in alternating directions, resulting in a total of 300 interferograms for each f rep setting. One interferogram was acquired in 3.6 s, which yielded a total acquisition time of 4.2 h. At 0.32 mbar, we made two f rep scans with 50 interferograms per f rep step, as well as a final scan with 25 interferograms per step, yielding 125 interferograms per f rep setting and an acquisition time of 1.8 h. To obtain a reference spectrum for normalization, we recorded interferograms with the absorption cell evacuated and the comb locked to the first f rep step. For the measurement at 0.02 mbar, we recorded 150 reference interferograms before and after measuring the N 2 O absorption spectrum, while for the measurement at 0.32 mbar, we recorded 125 reference interferograms before the N 2 O measurement.
We processed the acquired data using a MATLAB code. We first resampled the comb interferograms at the zero-crossings and extrema of the CW laser interferogram and averaged the absolute values of the Fourier transforms of each set of interferograms belonging to one f rep step. We used the method described in Refs [36,37] in order to match the frequency domain sampling points to the comb mode positions at each f rep step and thus minimize the influence of the instrumental line shape. We normalized the averaged spectrum at each f rep step to the averaged reference spectrum, which had been smoothened and interpolated linearly to the sampling points of the pertinent f rep step. Smoothening and linear interpolation was possible since the background features were broad enough to be fully resolved at the comb mode spacing. Using the Lambert-Beer law, we converted each transmission spectrum to an absorption spectrum. To remove the remaining baseline, we fit a model consisting of a sum of a simulated absorption spectrum based on the parameters from the HITRAN 2016 database [13] and a baseline, represented by the sum of a 5 th order polynomial and slowly varying sine terms (periods >6 GHz) to the absorption spectra, and then subtracted the baseline. Finally, we interleaved the averaged and baselinecorrected absorption spectra recorded at different f rep steps to obtain the final spectrum with a sample point spacing of 9 MHz in the optical domain.
High-resolution spectra
Figure 2(a) shows the absorption spectrum of N 2 O measured at 0.02 mbar, dominated by the P and R branches of the ν 1 fundamental band centered at 1285 cm -1 . A second progression of lines, one order of magnitude weaker, belongs to the ν 1 + ν 2 -ν 2 hot band that is split into two components designated by their rotationless parity (e/f). To improve the signal-to-noise ratio (SNR) of these lines, we made a measurement at a higher pressure of 0.32 mbar. Figure 2(b) shows a vertical zoom of the hot band in this second measurement, where the lines belonging to the ν 1 band are plotted in gray for clarity. In addition to the strong P and R branches of the hot band, there is a weaker Q-branch around 1290 cm -1 . Due to minor water impurities in the cell, a few water lines are visible [e.g., around 1260 cm -1 in Fig. 2(a)], some of them accompanied by slight baseline distortions caused by water absorption in the ambient air. The maximum SNR of the lines in the ν 1 band in Fig. 2(a) is ~420, while for the hot band in Fig. 2(b) it is ~200. The SNR is limited by the detector noise. In (b), the spectrum is vertically zoomed in to show the ν 1 + ν 2 -ν 2 hot band, while the lines belonging to the fundamental ν 1 band are plotted in gray.
Line by line fitting
To retrieve the line center positions of the individual absorption lines, we fit Voigt line shapes to them. We applied the fit to lines with line strengths exceeding a threshold of 2 × 10 -20 cm -1 /(molecule/cm 2 ) for the ν 1 band and 2 × 10 -21 cm -1 /(molecule/cm 2 ) for the hot band (based on the values from the HITRAN database [13]). These criteria translate to a minimum SNR of ~30. In the Voigt model, we fit the intensities, the Lorentzian widths, and the center frequencies, together with a linear baseline. We fixed the Doppler widths to the theoretical values calculated at 23 ºC, which are around 71 MHz (FWHM). We used a nonlinear fitting routine based on the Levenberg-Marquardt algorithm, and we chose a fitting range of ±426 MHz for each line, equal to roughly ±6 times the FWHM. Lines separated by less than 9 times the Doppler FWHM were fitted simultaneously in one window. Such cases occurred only for the hot band since the line pairs with different parity overlap in parts of the spectrum. Lines separated by less than twice the Doppler FWHM were excluded from fitting. Weak interfering lines from other N 2 O bands and isotopologues within the fit window were included in the fit with their parameters fixed to the HITRAN values if they exceeded 2% of the line strength threshold. Weaker interfering lines were ignored since they were below the noise floor. We kept the line fits with a quality factor (i.e., the ratio of the peak of the line and the standard deviation of the residuum) larger than 30, thereby eliminating some lines that were affected by baseline distortions, e.g., due to interfering water absorption. Figure 1240 3 shows fits to the R(14) line of the ν 1 band [ Fig. 3(a)], and to a pair of lines belonging to the R(4) transition of the hot band with e-and f-parity [ Fig. 3(b)]. The residuals displayed in the lower panels show no significant structure. The linewidth at the pressures used is dominated by the Doppler broadening, and the pressure broadening is expected to be ~90 kHz for the ν 1 band (at 0.02 mbar) and ~1.5 MHz for the hot band (at 0.32 mbar), as calculated using the pressure broadening parameters from the HITRAN database [13]. The fitted Lorentzian FWHMs are larger by on average 4.2 MHz than the expected values, with no clear J-dependence. We assume that this discrepancy is due to an instrumental broadening, and we made a number of tests to investigate its origin. The characterization of the f rep locking stability described in the experimental section excludes it as a possible cause. To rule out that the broadening stemmed from the drift of the CW reference laser wavelength during the few-hour long measurement, we replaced the RIO diode laser with a CW Er:fiber laser (Koheras Adjustik E15) stabilized to an Er:fiber frequency comb, but this yielded no reduction of the broadening. We also found no dependence of the broadening on the size of the apertures in front of the detectors. We therefore suspect that the broadening is caused by the linewidth of the modes of the 8 µm idler comb, but we have no means of measuring it at this time. Since the line broadening appears symmetric, as indicated by the flat residuals in Fig. 3, we assume it does not skew the retrieved line center positions.
We identify three sources of uncertainty of the fitted line positions. The dominating component is the fit precision, which is in the range of 50-600 kHz. The second contribution stems from the uncertainty of the effective CW reference laser wavelength, which depends on the alignment and divergence of the two beams in the FTS, as well as variations of the refractive index of air that fills the FTS. Using the method described in Refs [35,37] for determination of the reference wavelength and its effect on the retrieved line positions, we estimate its contribution to the uncertainty to be 60 kHz. The third source of uncertainty are the pressure shifts. The pressure shifts of the lines in the hot band at 0.32 mbar are on average 10 kHz (according to HITRAN) and we subtracted them from the fitted line center frequencies. We conservatively assumed the uncertainty of the pressure shift to be as large as the shift itself. The pressure shifts of the lines in the fundamental band at 0.02 mbar are lower than 3 kHz, i.e., more than one order of magnitude below the next largest contribution to the uncertainty budget (originating from the effective reference laser wavelength). We thus neglected pressure shifts in the analysis of the fundamental band.
Line positions of the ν 1 band
The line centers retrieved by fitting to 72 lines in the ν 1 fundamental band are presented in Table 1. The uncertainties (160 kHz on average) are calculated by summing in quadrature the fit precision and the contribution from the reference laser wavelength, described in the previous section. The black dots in Fig. 4(a) show a comparison of the fitted line centers to those from the HITRAN database. A clear systematic discrepancy is visible, though remaining within the HITRAN frequency uncertainty of 3 MHz. Furthermore, this systematic tendency closely follows that reported in a recent work by AlSaif et al. [21], using a comb-referenced externalcavity QCL, shown by the red circles. The root-mean-square discrepancy between the line centers retrieved in that study and in this work is 240 kHz.
Similarly to what was done by AlSaif et al., we fit a model based on the effective ro-vibrational Hamiltonian (see Eq. (1) in [21]) to the retrieved line positions using a Levenberg-Marquardt algorithm implemented in MATLAB. We weighted the lines by the inverse squares of their uncertainties. We fit the vibrational term value G v , the rotational constant B v , and the quartic and sextic centrifugal distortion constants D v , H v of the vibrationally excited state (fixing L v to 0) and fixed the ground-state rotational and centrifugal distortion constants to those determined by Ting et al. [38]. The fitted upper-state constants of the ν 1 band are presented in Table 2, and compared to those obtained by AlSaif et al. [21]. For further comparison, we include the constants reported by Tachikawa et al. [39] from sub-Doppler measurements of the ν 3 -ν 1 and ν 3 -ν 2 bands performed by heterodyning two fluorescence-stabilized lasers. Except for G v , the retrieved upper-state constants from our work and Ref. [21] agree within their combined 1σ uncertainties; the G v constants agree within the combined 3σ uncertainty. The agreement with Tachikawa et al. is noticeably worse for all constants except B v . Note however that their values resulted from fits to two other bands and might be influenced by factors not present in the two other studies, in which the ν 1 band was measured directly. Figure 4(b) shows a comparison of the line centers measured in this work to those calculated from the fitted spectroscopic constants (black dots). The weighted standard deviation (observed -calculated) of the 72 fitted lines is 3.2 × 10 -6 cm -1 (96 kHz). The red circles and blue triangles show a comparison to the line positions calculated using the constants from Refs [21] and [39] respectively. Considerable disagreement with the latter is apparent mainly as a 690 kHz offset. On the other hand, the discrepancy between our model and the one from AlSaif et al. remains within the uncertainties of the measured line positions in the studied wavenumber range. We thus provide an independent support of the accuracy of the line positions and constants reported by AlSaif et al. [21], using a different measurement technique. Fig. 4 (a) The line positions of the ν 1 fundamental band from this work (black dots) and from Ref. [21] (red circles) relative to those from the HITRAN database [13]. (b) The line positions obtained by line-by-line fitting (black dots) relative to the simulation based on the upper state constants from this work. The red circles and blue triangles indicate line positions simulated using constants from Ref. [21] and Ref. [39] relative to the simulation from our work.
Line positions of the ν 1 + ν 2 -ν 2 hot band
The positions of the 112 fitted lines of the P and R branches of the hot band are given in Table 3. The uncertainties include all three contributions described in Section 3.2 summed in quadrature. Their mean is 230 kHz. Figure 5(a) shows the center frequencies compared to those listed in the HITRAN database (black dots) and reported by Maki and Wells [16] (red cicles), where the plotted uncertainty is from our work only. The missing lines between 1277 cm -1 and 1283 cm -1 were excluded from the fitting procedure due to the overlap of the e-and f-parity lines. A second gap between 1288 cm -1 and 1294 cm -1 is due to too low SNR close to the band center.
The discrepancies with respect to HITRAN have a slight J-dependence; the agreement is excellent at the band center, and worse at higher J numbers, but all deviations are within the HITRAN uncertainty of 3 MHz. The discrepancy with respect to Maki and Wells is larger and has a clearer J-dependence, but it is within the 3σ uncertainty (2.3 MHz) reported in their work.
We modeled the hot band with the same effective Hamiltonian as the fundamental band, using a common vibrational term value G v for the e-and f-component, and independent B v , D v and H v constants. We restricted the fit to the G v , B v and D v constants of the upper states, while fixing the upper state H v constants as well as all the lower state constants. (Floating the H v constants resulted in uncertainties comparable to their absolute values.) We took the values for the fixed constants as well as the initial values for the fitted ones from Ref. [12], which is the source of the line positions listed in HITRAN. We applied the fit to all 112 lines weighted by the inverse squares of their uncertainties. Figure 5(b) shows the measured line positions relative to the fit (red solid circles and blue solid squares), where the weighted standard deviation (observed -calculated) is 7.0 × 10 -6 cm -1 (210 kHz). Also indicated are the line positions calculated using the constants from Toth [12] for the e-and f-parity lines (red open circles and blue open squares, respectively). Table 4 shows the band origins, calculated as ν 0 = G v ' -G v ", where G v ' and G v " are the vibrational term values of the upper and lower states, and the B v and D v constants obtained in this work compared to those from Ref. [12]. The uncertainty in the band origin, ν 0 , from Toth was calculated as the in-quadrature sum of the uncertainties of G v ' and G v " reported in Ref. [10] (both equal to 1 × 10 -5 cm -1 ). We note the agreement with Toth in the band origin and the improvement of its precision by one order of magnitude. The significance of the discrepancy in the remaining constants is difficult to evaluate, since no uncertainties are reported on these constants in Ref. [12]. The accuracy of the model also depends on the fixed lower state constants, which are presumably known with lower certainty than their ground state counterparts.
Conclusions
We demonstrated a Fourier transform spectrometer based on a compact fiber-based DFG optical frequency comb source and a multi-pass cell capable of measuring low-pressure spectra with center frequency precision of the order of 100 kHz in the atmospheric window around 8 µm. The DFG comb source is offset-frequency-free, which implies that an RF lock of the repetition rate is sufficient to achieve absolute stability of the comb mode frequencies. This, in combination with the FTS with sub-nominal resolution sampling-interleaving scheme, provides spectra with a calibrated frequency axis. We verified the accuracy of the retrieved line positions of the ν 1 fundamental band of N 2 O by demonstrating excellent agreement with the results of an independent highprecision measurement by AlSaif et al. [21] using an external-cavity QCL referenced to a frequency comb. We reached a comparable precision in the retrieved line positions using a system with reduced experimental complexity. We also retrieved line positions of the ν 1 + ν 2 -ν 2 hot band with up to one order of magnitude improved precision compared to previous studies [5,12,16]. This allowed us to determine the band origin with an uncertainty reduced by one order of magnitude compared to the FTIR-based work of Toth [12]. Longer averaging times will further increase the SNR and hence the precision of the retrieved line positions. Furthermore, the wavelength coverage of the DFG comb can be expanded within the 7-9 µm range by changing the poling period of the crystal and tuning the soliton signal [25]. Our work opens up precision measurements of entire bands of various molecules of interest in atmospheric science and astrophysics, such as methane, ammonia, sulphur dioxide or methanol, in this fingerprint spectral region. | 2021-03-08T02:16:03.698Z | 2021-03-05T00:00:00.000 | {
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3856493 | pes2o/s2orc | v3-fos-license | Astrocytes expressing ALS‐linked mutant FUS induce motor neuron death through release of tumor necrosis factor‐alpha
Mutations in fused in sarcoma (FUS) are linked to amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting both upper and lower motor neurons. While it is established that astrocytes contribute to the death of motor neurons in ALS, the specific contribution of mutant FUS (mutFUS) through astrocytes has not yet been studied. Here, we used primary astrocytes expressing a N‐terminally GFP tagged R521G mutant or wild‐type FUS (WTFUS) and show that mutFUS‐expressing astrocytes undergo astrogliosis, damage co‐cultured motor neurons via activation of an inflammatory response and produce conditioned medium (ACM) that is toxic to motor neurons in isolation. Time lapse imaging shows that motor neuron cultures exposed to mutFUS ACM, but not WTFUS ACM, undergo significant cell loss, which is preceded by progressive degeneration of neurites. We found that Tumor Necrosis Factor‐Alpha (TNFα) is secreted into ACM of mutFUS‐expressing astrocytes. Accordingly, mutFUS astrocyte‐mediated motor neuron toxicity is blocked by targeting soluble TNFα with neutralizing antibodies. We also found that mutant astrocytes trigger changes to motor neuron AMPA receptors (AMPAR) that render them susceptible to excitotoxicity and AMPAR‐mediated cell death. Our data provide the first evidence of astrocytic involvement in FUS‐ALS, identify TNFα as a mediator of this toxicity, and provide several potential therapeutic targets to protect motor neurons in FUS‐linked ALS.
Mutations in fused in sarcoma (FUS) are linked to amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting both upper and lower motor neurons. While it is established that astrocytes contribute to the death of motor neurons in ALS, the specific contribution of mutant FUS (mutFUS) through astrocytes has not yet been studied. Here, we used primary astrocytes expressing a N-terminally GFP tagged R521G mutant or wild-type FUS (WTFUS) and show that mutFUS-expressing astrocytes undergo astrogliosis, damage co-cultured motor neurons via activation of an inflammatory response and produce conditioned medium (ACM) that is toxic to motor neurons in isolation. Time lapse imaging shows that motor neuron cultures exposed to mutFUS ACM, but not WTFUS ACM, undergo significant cell loss, which is preceded by progressive degeneration of neurites. We found that Tumor Necrosis Factor-Alpha (TNFa) is secreted into ACM of mutFUS-expressing astrocytes. Accordingly, mutFUS astrocyte-mediated motor neuron toxicity is blocked by targeting soluble TNFa with neutralizing antibodies. We also found that mutant astrocytes trigger changes to motor neuron AMPA receptors (AMPAR) that render them susceptible to excitotoxicity and AMPAR-mediated cell death. Our data provide the first evidence of astrocytic involvement in FUS-ALS, identify TNFa as a mediator of this toxicity, and provide several potential therapeutic targets to protect motor neurons in FUS-linked ALS. others. Over 35 mutations have been described in FUS, which has been identified as the primary cause of ALS type 6 (Vance et al., 2009).
In healthy controls this multifunctional protein, with roles ranging from DNA repair, transcriptional regulation and mRNA transport (Lagier-Tourenne, Polymenidou, & Cleveland, 2010), is located primarily in the nucleus (Dormann et al., 2010); however, post mortem tissues of FUS-ALS patients reveal cytoplasmic inclusions in affected neurons and glia (Kwiatkowski et al., 2009). Specifically, carriers of the FUS R521C, R521G, R521H, R524W, or G507N mutations show wide-spread FUS pathology (Blair et al., 2009;Hewitt et al., 2010;Rademakers et al., 2010), including glial and neuronal cell loss, with increasing distribution of FUS-immunoreactive inclusions in patients with longer disease durations (Suzuki et al., 2012). Carriers of FUS R521C mutation also show numerous oligodendroglial cytoplasmic inclusions (Mackenzie et al., 2011). Abundant FUS-immunoreactive pathology is also observed in some sALS cases (Oketa, Higashida, Fukasawa, Tsukie, & Ono, 2013). Several lines of evidence suggest that non-neuronal cells play an active role in driving neuronal dysfunction and neurodegeneration in ALS [reviewed (Lasiene and Yamanaka, 2011)]. Among these nonneuronal cells, astrocytes are considered key mediators of disease progression in vivo and of motor neuron death in vitro. Chimeric mice in which mutant SOD1 was selectively deleted in astrocytes showed substantially slowed disease progression (Yamanaka et al., 2008) while both rodent and human patient-derived astrocytes have been shown to play a toxic role through the release of, yet identified, soluble factors [reviewed (Haidet-Phillips et al., 2011;Lasiene and Yamanaka, 2011).
Furthermore, we recently showed that ALS astrocytes, including mut-FUS expressing patient astrocytes, drive up-regulation of the multidrug resistance transporter P-Glycoprotein (P-gp) in endothelial cells of the blood-brain barrier (Qosa et al., 2016). Astrocytes participate in disease also through a "loss of function" [reviewed (Maragakis and Rothstein, 2006)] as demonstrated by several human and rodent studies that have observed defects and/or loss of astrocytic glutamate transporter EAAT2/GLT1 for several ALS subtypes including SOD1-ALS (Benkler, Ben-Zur, Barhum, & Offen, 2013;Howland et al., 2002) sALS (Lin et al., 1998), and C9orf72-ALS (Kwon et al., 2014). In contrast, studies of astrocytic contributions in TDP-43-mediated ALS have shown conflicting results for both gain and loss-of-function toxicity (Haidet-Phillips et al., 2013;Serio et al., 2013;Tong et al., 2013). Important questions still remain about astrocytic contributions to disease across different ALS-subtypes, and in this work, we focused on the role played by astrocytes in FUS-linked ALS.
One of the striking hallmarks of ALS is neuroinflammation. This is characterized by the appearance of reactive astrocytes and microglia, as well as macrophage and T-lymphocyte infiltration, further strengthening the evidence that ALS is a noncell autonomous disease where non-neuronal cells actively participate in neurodegeneration (Keller, Gravel, & Kriz, 2009;Levine, Kong, Nadler, & Xu, 1999;Rizzo et al., 2013). Proinflammatory cytokines such as tumor necrosis factor alpha (TNFa) are upregulated in the spinal cord of ALS patients, and presymptomatically in SOD1 G93A mice (Philips and Robberecht, 2011).
Inflammation may also have a causal link to ALS as haploinsufficiency of the TBK1 gene, a regulator of inflammatory signaling, has been recently shown to cause ALS and frontotemporal lobe dementia (FTD) (Freischmidt et al., 2015). Though inflammation and astrocytemediated toxicity have been identified as part of the pathogenic process of ALS [reviewed (Philips and Robberecht, 2011)], the role of TNFa has been widely debated, at least in the SOD1 mouse model.
Approaches to globally eliminate single inflammatory genes in SOD1-ALS mouse models have largely failed to extend survival (Gowing, Dequen, Soucy, & Julien, 2006) and treatment with the TNFa inhibitor, thalidomide, showed some promising results in rodents but did not appear to effectively modulate disease progression in patients and was additionally accompanied by significant side-effects which prevented patients from reaching the target dosage (Stommel et al., 2009). However, whether inflammation in general and TNFa in particular play different pathogenic roles in other forms (non-SOD1) of ALS remains to be solved.
Nuclear factor kappa B (NF-jB), an inducible cellular transcription factor found in almost all cell types, plays a key role in regulating the inflammatory response and cellular stress response. Dysregulation of NF-jB has been linked to cancer and neurodegenerative diseases such as Huntington's (Ghose, Sinha, Das, Jana, & Bhattacharyya, 2011) and ALS (Frakes et al., 2014;Prell et al., 2014). Interestingly, FUS augments NF-OEB-dependent gene expression induced by physiological stimuli such as TNFa (Uranishi, 2001). TNFa and NF-OEB have been shown to participate in oscillatory positive feedback loops wherein NF-OEB can regulate TNFa transcription while autocrine and paracrine TNFa signals can in turn trigger patterns of NF-OEB activation (Pe R kalski et al., 2013). This process plays a key role in regulating inflammatory responses across a variety of cell types.
TNFa is a pleiotropic molecule and well-studied trigger of apoptosis, however, there has recently been an increased interest in its role in CNS physiology and synaptic functions [reviewed (Santello and Volterra, 2012)]. In addition to classical caspase-8 activation, one mechanism proposed for TNFa-induced neuron death is through the rapid TNFa-induced surface expression changes of AMPA-type glutamate receptors (AMPARs) (Ferguson et al., 2008). Dysregulation of the precise trafficking can alter neuronal calcium permeability and contribute to excitotoxic vulnerability (Ferguson et al., 2008;Leonoudakis, Zhao, & Beattie, 2008;Olmos and Llad o, 2014).
Here, using primary astrocytes expressing mutFUS cocultured with primary motor neurons, we studied whether mutFUS-expressing astrocytes mediate motor neuron dysfunction and began identification of the mechanisms by which mutFUS astrocytes damage the motor neurons. We show that mutFUS-expressing astrocytes damage motor neurons through release of toxic factor(s) in the condition medium (ACM).
Using microarray analysis of mutFUS astrocytes and ELISA and Western Blot analysis of mutFUS ACM and cell lysates, we identified secreted TNFa as the predominant factor, whose toxicity is blocked by anti-TNFa neutralizing antibodies. Preventing astrocytic NF-kB activation inhibits TNFa accumulation in the ACM and prevents mutFUS ACM induced motor neuron degeneration. Further, mutFUS ACM alters expression levels of AMPA receptor subunits GluA1 and GluA2 in motor neurons. We show that these changes sensitize motor neurons to AMPA receptor stimulation resulting in increased calcium influx leading to excitotoxic damage and cell death. We also show that these AMPAR changes are a result of TNFa acting on motor neurons. Our data provide the first evidence of astrocytic involvement in FUS-ALS and identify TNFa and components of the AMPA-mediated excitotoxic pathway as potential therapeutic targets for mutFUS-ALS.
| Primary astrocytic culture
Primary astrocytes were prepared from spinal cord of nontransgenic mice (P2-P4). Briefly, flushed spinal cords were cut into small pieces followed by trypsin-DNase treatment and mechanical resuspension.
| Primary rat motor neuron culture
Motor neurons were prepared from e14.5 rat spinal cords. Briefly, 10-20 spinal cords were dissected and mechanically broken into small fragments before being incubated in 0.025% trypsin, followed by DNase.
After a 4% w/v BSA cushion, cells were centrifuged for 55 min without brake through a 10.4% (v/v) Optiprep (Nycomed Pharma) cushion. Collected bands were then spun through a 4% w/v BSA cushion and treated with rat anti-mouse p75NTR antibody for 30 min. Cells were then washed, spun through a 4% w/v BSA cushion and resuspended in goat anti-mouse IgG microbeads for 15 min at 148C. After 4% w/v BSA cushion magnetically labeled cells were loaded into a column attached to a magnetic base and allowed to pass through, collecting the effluent as a negative fraction on a centrifuge tube. After three rinses, the column was removed from the magnet and a flow resister was used to collect the positive fraction. A final 4% w/v BSA cushion was used to collect motor neurons in complete neurobasal medium (Nb-C) (Neurobasal [Gibco, 21103], B27 supplement (2%), glutamine (0.25%), 2-mercaptoethanol (0.1%), horse serum (2%). Cells were plated on polyornithine-laminin coated coverslips at a density of 5000/18 mm coverslip. Motor neurons were maintained in Nb-C for up to 2.5 weeks.
| Constructs
N-terminally eGFP tagged human FUS in a pcDNA3.1/nV5-DEST backbone was used to create R521G (CGC to GGC) mutant using the Quik-Change Lightning Site-Directed Mutagenesis Kit (Agilent 210518).
Samples were sequenced using BGH pA forward and reverse primers.
To allow for Gateway recombination into adenoviral expression plasmids, pENTR4 (Invitrogen) was opened at EcoR1 and BamH1 sites and the expression cassettes for wild-type and mutant human FUS were released from pcDNA3.1/nV5-DEST plasmids at BglII and NaeI sites and ligated to pENTR4 with EcoRV and BamHI. Finally, both expression cassettes were transferred by Gateway recombinase into adenoviral expression constructs, which were produced and amplified in HEK 293A cells (ViraPower Adenoviral Expression System; Invitrogen).
| Viral induction
After astrocytes were allowed to adhere following trypsinization, an adenoviral N-terminal GFP tagged FUS or C-terminally eGFP tagged SOD1 construct was incubated with cells for 24 hr before being washed with HBSS and cell media. Multiplicity of infection (MOI) ranged from 3 to 5. Cells were then incubated for 3-4 days to allow maximal protein expression and used for experiments. Protein expression stabilized after 4 days and remained constant for 2 weeks or more.
| Protein analysis
To access protein levels, western blotting was done per standard protocols and probed against GFP (Clontech, 632459) and Actin (Abcam,
| Assessment of neuronal length
Each slide was scanned systematically to cover every field of view and each motor neuron was imaged and saved. Motor neurons were identified by their lack of GFP-virus expression, clear morphology, and positive staining of markers. Images were then loaded into Image J and subsequently analyzed using the Neuron J plug-in. Each neurite was individually traced and the sum of neurites for each individual neuron was then used for analysis.
| Enzyme-linked immunosorbent assay
Levels of TNFa in astrocyte culture medium were determined by ELISA (DuoSet ELISA Development System, R&D Systems, DY410). Briefly, 96-well microplates were incubated with the capture antibody overnight, at room temperature. Following coating, plates were blocked with 1% BSA in PBS and then washed with 0.05% Tween 20 in PBS.
Samples and standards were then added and allowed to incubate for 2 hr while shaking at room temperature. Plates were then washed and allowed to incubate with the biotinylated goat anti-mouse TNFa detection antibody for 2 hr while shaking at room temperature, then washed again. Finally, we incubated the plates with Streptavidin-HRP and a substrate solution for 30-min each. A stop solution of 1N H2SO4 was added just prior to plate reading. The optical-density of each well was determined using a microplate reader, set to 450 nm. For inflammatory array, Mouse Inflammatory Cytokines Multi-Analyte ELISArray was run per manufacture instructions (Qiagen, MEM-004A).
| Astrocyte-conditioned medium experiments
Medium was collected from fully confluent cultures of virally transduced astrocytes beginning 48 hr after transduction and at two subsequent time points of 72 and 96 hr. At these three time points, a mixture of 25% astrocyte-conditioned medium and 75% neuronal medium was added onto cultures of primary motor neurons at DIV 4-5. For TNFa neutralization, the medium was incubated with a TNFa neutralizing antibody (abcam ab6671) at a concentration of 5 ng lL 21 for 30 min prior to addition onto motor neurons. SN50 experiments were carried out by pretreating the astrocyte cultures from which the conditioned medium was collected with 18 lM SN50 (Calbiochem 481480). For the CNQX experiments, CNQX (Tocris Bioscience 0190) was added to the conditioned medium just prior to motor neuron treatment, at a final concentration of 10 lM. Individual motor neurons were tracked an imaged each day at 378C and 5% CO 2 using a semiautomated inverted Nikon Eclipse Ti microscope equipped with a Tokai Hit stage top incubator with gas and temperature controller. Neurons were marked as dead by their disappearance or obvious morphological indicators such as fragmentation of the soma and neurites.
| Quantitative real-time PCR analysis
Measurement of motor neuron GluA1 and GluA2 mRNA levels was made by qPCR. Briefly, DIV 4-5 primary motor neuron cultures were treated with astrocyte conditioned medium and left for 72 hr. Total RNA was isolated by lysing the motor neuron cultures with TRIZOL.
Next, phase separation of the sample was done using chloroform, and the upper aqueous phase was retrieved. RNA was then precipitated by isopropanol and washed with ethanol. The subsequent pellet was KIA & MCAVOY ET AL. | 1019 dissolved in DEPC-treated water and those samples were used in a standard reverse transcription protocol using the reverse transcriptase SuperScript IV (Invitrogen). The cDNA was added with TaqMan qPCR primers for rat GAPDH (Rn01775763_g1), and Gria1 (Rn00709588_m1) or Gria2 (Rn00568514_m1). The multi-plex qPCR assay was then performed and analyzed using the QuantStudio 5 Real-Time PCR system (ThermoScientific).
| Statistical analysis
Relative fluorescence levels, neurite lengths, % cells remaining, and time to peak data are presented as mean 6 SEM. Statistical significance was evaluated using Student's t test or one-way analysis of variance with post-hoc Tukey's HSD. p < 0.05 or 0.01 was used to determine significance where indicated. A data transformation was used prior to statistical testing in one case for determining differences between neurite lengths as indicated. A logarithmic transformation was required to address a skew in the distribution.
| MutFUS mislocalizes to the cytoplasm and increases GFAP expression in primary astrocytes
Carriers of FUS mutations, including R521G, show widespread FUS pathology, including neuronal and glial cytoplasmic inclusions in the absence of basophilic inclusions (Blair et al., 2009;Hewitt et al., 2010).
Because the redistribution of nuclear FUS has been observed in a variety of cell culture models (Bosco et al., 2010;Dormann et al., 2010;Farg et al., 2012), we examined it in our in vitro system of cultured astrocytes transduced with mutFUS. Isolated primary astrocytes were firstly tested for purity using the glutamate transporter GLAST as a marker of mature astrocytes (Supporting Information Figure S1a,c).
Immunofluorescence analysis showed negligible staining for cellular markers of other forms of glia or neurons (Supporting Information Figure S1B). Viral transduction was used to introduce N-terminally GFPtagged wild-type (WTFUS) and R521G-FUS mutFUS into primary astrocytes with efficiency around 80% (Supporting Information Figure S1e). Expression levels of both WT FUS and mutFUS in astrocytes were comparable as shown in western blot analysis (Figure 1c). Expression of mutFUS led to cytoplasmic mislocalization in 90% of the transduced astrocytes (Figure 1a,b) thus recapitulating the mislocalization pattern seen in ALS patients. We next asked if mutFUS expression would be sufficient to cause toxicity in astrocytes. Toxicity was measured using CellTiter-Glo Luminescent Cell Viability Assay (CTG), which detects changes in metabolic activity as a measure of overall cell viability (Pedrini et al., 2010). This assay showed that, while 0.5M thapsigargin used as positive control significantly reduced viability, null virus treatment was not significantly different than either WTFUS or mut-FUS (Supporting Information Figure S1f). These data suggest that expression of mutFUS alone does not result in astrocytic cell death.
However, immunofluorescence for GFAP, an indicator of astrogliosis was enhanced in mutFUS astrocytes (Figure 1d). Interestingly the levels of GFAP expression in mutFUS astrocytes were significantly higher compared to WTFUS and null virus (NT) astrocytes (Figure 1e,f).
WTFUS-expressing astrocytes, on the other hand, only showed a modest increase in GFAP expression compared to NT astrocytes. These results suggest that although mutFUS expression is non-toxic to astrocytes, mutFUS induces enhanced astrogliosis.
3.2 | MutFUS-expressing astrocytes are toxic to cocultured motor neurons We next tested whether mutFUS-expressing astrocytes are toxic to co-cultured motor neurons. Immunopanned E14.5 primary rat motor neurons were plated on top of a bed of astrocytes previously transduced with FUS (WT or R521G) and allowed to grow in co-culture for 3 days. Cultures were then fixed and stained with antibodies for motor neuron markers choline acetyltransferase (ChAT), and the 75-kDa low-affinity neurotrophin receptor (p75-NTR). We then assessed neurite lengths in both groups to gauge toxic phenotype as previously done by others (Han et al., 2013;Lasiene and Yamanaka, 2011).
Motor neurons were selected based on ChAT-positive immunostaining (Supporting Information Figure S1d), and neurite length measured using p75-NTR (Anderson et al., 2004). Contrary to motor neurons co-cultured with WTFUS-astrocytes, motor neurons plated on mut-FUS containing astrocytes had less extensive branching and shorter neurites (Figure 2a). To quantify reductions in neurite length, tracings of individual neurons were used (Figure 2b) to create cumulative frequency plots (Figure 2c). Motor neurons cocultured with mutFUS astrocytes, exhibited a 34% 6 6% reduction in neurite length ( Figure 2d), compared to those cocultured with WTFUS-expressing astrocytes. As additional controls, we demonstrated that damage to neurite morphology is also evident in motor neurons co-cultured with mutant, but not WT, SOD1 astrocytes ( Figure 2c). These data suggest that mutFUS expression in astrocytes can damage neurite morphology of wild-type neurons.
| MutFUS astrocytic conditioned medium is toxic to motor neurons
We next asked if the mutFUS-mediated defect in neurite morphology was due to a contact-dependent mechanism or the release of soluble factor(s). Motor neurons were cultured alone and treated with ACM from transduced astrocytes (Figure 3a). Motor neurons were then analyzed for cumulative neurite lengths as they were in Figure 2. We
| MutFUS astrocytes secrete TNFa
Because mutFUS ACM is sufficient to damage the motor neurons, we used a combination of gene expression profiling and protein analysis to begin to identify astrocytic changes that might contribute to toxic gene networks (as ranked by the total number of significantly changed genes found in each pathway), we focused on the secretome pathway, as we have seen ACM toxicity. The top pathway hit that we predicted would influence broad secretomic changes was the TNF-alpha and NF-KB signaling pathway (Table 1), which has been studied extensively for its role in mediating pro-inflammatory cell-to-cell signaling. On the basis of these results, we then measured common pro-inflammatory cytokine levels in the WTFUS and mutFUS ACM by ELISA (Figure 4b).
Among those, we identified TNFa as the most highly altered secreted factor in mutFUS ACM (3.8-fold increase over WTFUS ACM). Further, we validated this result using a second ELISA containing a new set of sandwich antibodies, and found mutFUS ACM contains significantly more TNFa compared to WTFUS or Non-transduced (NT) ACM ( Figure 4c). Additionally, we probed astrocyte cell-lysates for total TNFa via western blot, and found that astrocytes expressing mutFUS have increased expression levels compared to WTFUS and NT astrocytes ( Figure 4d). Together, these data clearly show that TNFa is aberrantly upregulated and secreted by mutFUS astrocytes.
| TNFa neutralization in mutFUS ACM rescues motor neuron degeneration and death
To test the role of TNFa in mutFUS-mediated toxicity, we plated motor neurons in isolation and imaged individual cells before and after ACM treatment 6 5 ng mL 21 anti-TNFa neutralizing antibody (Abcam, ab6671) and tracked cell morphological changes and viability over 96 hr (Figure 5a and Supporting Information Figure S2a). Remarkably, when anti-TNFa antibody is added to mutFUS ACM, motor neurons display a 2.3-fold greater average neurite length compared to motor neurons treated with mutFUS ACM alone (Figure 5b,c). Further, addition of anti-TNFa antibody to mutFUS ACM, significantly prevented mutFUS induced death of motor neurons [cell loss of 12.2% in mutFUS ACM1 anti-TNFa antibody compared to 45.6% with mutFUS ACM alone (Figure 5d)]. While TNFa neutralization inhibits mutFUSmediated toxicity, it does not block mutSOD1 astrocytic toxicity, suggesting that release of TNFa is specific to mutFUS astroctyes (data not shown). These data demonstrate that in mutFUS, astrocytic TNFa is a mediator of motor neuron viability. In our previous work, we have also demonstrated that TNFa is specifically released from mutFUS iPSCastrocytes and drives pathological blood-brain-barrier changes that | 1023 lead to pharmacoresistance (Qosa et al., 2016). Thus, reducing astrocytic TNFa production in patients may have therapeutic potential for multiple features of disease.
| Pharmacological inhibition of NF-OEB in astrocytes rescues motor neuron neurite loss and cell death
Astrocytes can respond to inflammatory cytokines such as TNFa by inducing classical NF-kappaB (NF-OEB) signaling to regulate inflammation and drive the expression of many chemokines and molecules upregulated in ALS. NF-OEB is activated in glia in fALS and sALS patients, and astrocytes derived from human postmortem ALS patients showed NF-OEB as the highest-ranked regulator of inflammation in gene array data (Lasiene and Yamanaka, 2011;Swarup et al., 2011). NF-OEB can also drive the transcription of TNFa and many other inflammatory molecules (Pe R kalski et al., 2013). Further, FUS has been shown to act as a co-activator of NF-OEB by enhancing the NF-OEB-mediated transactivation induced by TNFa (Uranishi, 2001). We next asked if selectively inhibiting NF-OEB in mutFUS astrocytes could block TNFa-induced motor neuron loss. To test this, we used SN50, a cell-permeable inhibitor peptide that inhibits translocation of the NF-OEB active complex into the nucleus, and subsequent NF-kB activation. Treating astrocytes with this peptide reduced TNFa secretion by 67% (data not shown) and, consequently, alleviated mutFUS-induced, reduction of neurite length and reduced motor neuron cell loss by 23% (Figure 6 and Supporting Information Figure S2b).
| MutFUS ACM triggers AMPA receptor changes and AMPA receptor-mediated cell death in motor neurons
Because TNFa has been shown to induce surface expression changes of AMPA-type glutamate receptors (AMPARs), leading to excitotoxicity (Ferguson et al., 2008;Leonoudakis et al., 2008;Santello and Volterra, 2012), we hypothesized that the aberrant release of TNFa by mutFUS astrocytes may also lead to AMPA receptor dysregulation in motor neurons. To test this, we treated motor neurons with ACM (Figure 7a-c). We then determined whether AMPAR changes occur also at the mRNA level. We therefore probed GluA1 and GluA2 mRNA in ACM-treated motor neurons and found GluA1 mRNA to be significantly increased at a time point of 72 hr in the mutFUS ACM condition (Figure 7d). Although GluA2 mRNA remain unchanged at this time point, with the significant increase in GluA1 mRNA levels, the GluA1/GluA2 ratio significantly shifts, similar to the immunofluorescence data shown in Figure 7a-c. GluA2 subunits confer calcium impermeability on AMPARs and their decrease relative to GluA1 is associated with the addition of more calcium-permeable AMPARs (Mansour, Nagarajan, Nehring, Clements, & Rosenmund, 2001). Given that AMPA receptor subunit composition was altered in motor neurons exposed to mutFUS ACM, we hypothesized that AMPA receptor stimulation in these neurons may result in a higher calcium entry. To test this idea, we measured intracellular calcium levels following AMPA stimulation in motor neurons treated with ACM, using the genetically encoded calcium sensor GCaMP6m (Figure 7e). Motor neurons treated with mutFUS ACM for 48 hr showed significantly larger intracellular calcium increases following stimulation with 10 lm AMPA 1 10 lm cyclothiazide compared to WT ACM. When comparing peak GCaMP6m DF/F 0 we found that mutFUS ACM treated motor neurons displayed a 1.8-fold increase over WTFUS ACM-treated neurons on average (Figure 7f). To test the idea that excitotoxic damage might be contributing to neuronal death in our conditioned medium experiments we sought determine whether pharmacological inhibition of AMPA receptors could offer neuroprotection from mutFUS ACM. We treated motor neurons with the AMPA/Kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) alongside treatments of mutFUS astrocytic media. Addition of CNQX significantly increased survival (79% 6 1.3% compared to 59% 6 2.5%) in neurons treated with mutFUS ACM (Figure 7g). Taken together, these results suggest that mutFUS astrocytes sensitize motor neurons to excitotoxic damage. Shown are the top 20 most altered pathways as determined by the combined number of significantly altered genes within each pathway along with the number of changes and the significance score ranking. In bold is the TNF-alpha and NF-KB signaling pathway which was chosen as a candidate pathway for further analysis based on previous evidence of role of the TNF-alpha and NF-KB pathways in regulating cytokines and inflammatory cellto-cell signals. Survival data are presented as mean 6 SEM and statistical significance was evaluated by one-way ANOVA, >50 neurons per condition, n 5 3, *p < 0.01
| TNFa neutralization in mutFUS-ACM prevents AMPA receptor alterations
Next, we sought to determine whether TNFa released by mutFUS astrocytes contributes to motor neuron AMPAR changes as hypothesized. To test this, we measured dendritic GluA1 levels in ACM-treated motor neurons by immunofluorescence (as done in Figure 7a), under conditions in which ACM-TNFa has been ablated. ACM collected from WT or mutFUS expressing astrocytes was incubated with or without a TNFa neutralizing antibody prior treatment of motor neurons. Here, we found that the increase in GluA1 levels observed after treatment with mutFUS ACM was blocked by preincubating the ACM with a TNFa antibody (Figure 8a,b). The results indicate that TNFa signaling is necessary for motor neuron AMPAR changes driven by mutFUS astrocytes. . Peak values are presented as mean 6 SEM and statistical significance was evaluated using Student's t test. n 50, *p < 0.01. (g) Quantification of motor neurons remaining after 96 hr treatment with ACM 6 10 lM CNQX (6-cyano-7-nitroquinoxaline-2,3-dione). Survival data are presented as mean 6 SEM and statistical significance was evaluated using Student's t test, > 60 neurons counted per condition, n 3, *p < 0.01
| D I SCUSSION
Here, we show for the first time that ALS-causative mutations in FUS can cause noncell autonomous motor neuron degeneration. While expression of mutFUS-R521G is not toxic to astrocytes, these cells adopt a reactive phenotype as evidenced by an increase in GFAP expression and a selective increase in some pro-inflammatory cytokines. Importantly, these astrocytes become toxic to motor neurons via the release of harmful factor(s). Interestingly, mutFUS astrocyte toxicity is driven by a cascade of events that include astrocytic NF-kB activation, release of TNFa and AMPA receptor alterations in motor neurons that allow for dysregulated calcium influx and cell death.
Perhaps with the exclusion of TDP-43 astrocytes, it is widely recognized that ALS astrocytes actively kill motor neurons through the release of toxic factor(s) (Haidet-Phillips et al., 2013). While the identification of factors responsible for transmitting toxicity of mutant SOD1 and sporadic ALS astrocytes is still under intense investigation, our work identifies a key toxic factor in mutFUS astrocytes. We show that mutFUS expressing astrocytes have increased expression of TNFa and increased production of soluble TNFa. This is unique to mutFUS astrocytes as mutant SOD1 astrocytes do not release TNFa, as we have shown using patients-derived iPSc-astrocytes cocultured with iPSc-endothelial cells (Qosa et al., 2016). Elevated TNFa production has been observed in a number of neurodegenerative diseases including ALS [reviewed (Philips and Robberecht, 2011)]. TNFa is a pleiotropic molecule that can be produced by a number of different celltypes, including astrocytes (Chung and Benveniste, 1990;Santello and Volterra, 2012). In the CNS in particular, TNFa signaling is complex, and its role in regulating synaptic transmission both physiologically and pathologically is increasingly being appreciated (Ferguson et al., 2008;Leonoudakis et al., 2008;Olmos and Llad o, 2014;Santello and Volterra, 2012 yer, & Antel, 1995;He, Wen, & Strong, 2002;Raivich et al., 2002).
Accordingly, in our model, the TNFa produced by mutFUS astrocytes is toxic to motor neurons and motor neuron death can be rescued by neutralizing anti-TNFa antibodies. In contrast to TNFa, we did not see FIG URE 8 TNFa neutralization in mutFUS-ACM prevents AMPA receptor alterations: (a) 9 DIV motor neurons were treated with ACM for 72 hr following which they were probed for GluA1 and MAP-2 by immunocytochemistry. A representative image is shown along with cropped images of the highlighted region of a single neuron for MAP-2 (green) and GluA1 (red). For TNFa neutralization, ACM was incubated with a TNFa neutralizing antibody (nAb) prior to treatment of motor neurons. (b) Quantification of dendritic GluA1 signal. ROIs along the dendrites of motor neurons were selected on the basis of MAP-2 labeling and fluorescence intensity was measured. A total of >50 dendrites from >15 cells were analyzed per condition. Fluorescence levels are presented as mean 6 SEM. Statistical significance was evaluated using one-way ANOVA, n 5 5, *p < 0.05 In addition to TNFa, our study also points to astrocytic NF-kB as a driver of neurodegeneration. It is well-known that NF-kB plays a role in both driving the expression of TNFa and mediating TNFa signaling pathways (Pe R kalski et al., 2013). In our mutFUS astrocytes, inhibiting the activation of NF-kB reduces the production of TNFa and protects motor neurons, strongly suggesting that NF-kB and TNFa pathways are inter-related. In ALS, NF-kB has been shown to be activated in glial cells in vitro and in postmortem tissues of fALS and sALS and its dysregulation has been shown to have a significant impact on neuroinflammation (Frakes et al., 2014;Haidet-Phillips et al., 2011;Prell et al., 2014;Swarup et al., 2011). Interestingly, studies by Frakes et al. (2014) have found that microglia containing mutant SOD1 also increase their production of TNFa and acquire a neurotoxic phenotype that can be prevented through NF-kB inhibition. Although the sources of TNFa (Swarup et al., 2011;Uranishi, 2001) and augment its function.
The study of FUS and NF-kB interactions however is limited to just a single study that was carried out prior to discovery of FUS as an ALS gene. Future studies will be required to uncover more about interactions between NF-kB and mutFUS variants and the role of these interactions in ALS.
In line with a number of other studies we also found AMPARs as (Udagawa et al., 2015). Motor neurons are known to be especially vulnerable to AMPA-mediated excitotoxicity due to an unusually high density of Ca 21 -permeable AMPA receptors and low calcium buffering ability (Palecek, Lips, & Keller, 1999;Vandenberghe et al. 2000). Furthermore, neighboring astrocytes are known to be key regulators of these AMPA receptors (Beattie, 2002;Van Damme et al., 2007). Here, we show that the AMPAR GluA2 subunits are decreased, while GluA1 subunits are increased in the dendrites of motor neurons exposed to mutFUS ACM but not WTFUS ACM. Edited GluA2 subunits are known to confer calcium impermeability to AMPARs and, therefore, we hypothesized that motor neuron cell death following mutFUS ACM treatment might be attributable to an excitotoxic mechanism involving this AMPAR change. Corroborating with this hypothesis, we found intracellular calcium levels following AMPA stimulation are elevated in motor neurons exposed to mutFUS ACM. Using an AMPA receptor antagonist we then rescued the motor neuron death resulting from mutFUS ACM, indicating the involvement of excitoxicity in our model. Our results extend current evidence for AMPAR involvement in ALS and could prompt further studies of noncell autonomous AMPA regulation in ALS and FUS-ALS.
Interestingly, several groups have reported a role for TNFa as a regulator of surface AMPAR expression in spinal cord motor neurons. These studies have shown that TNFa triggers a change in AMPARs which can promote a shift towards surface expression of GluA2-lacking receptors (Ferguson et al., 2008;Ogoshi et al., 2005;Stellwagen, 2005). Although these studies focused on the rapid effects of short-term TNFa exposure, we have demonstrated here that longer incubations with TNFacontaining mutFUS-ACM, not only alter GluA1 & GluA2 protein levels but also trigger an increase mRNA levels for GluA1. Furthermore, our results indicate that AMPAR changes driven by mutFUS-ACM can be attributed to TNFa signaling, as ablation of soluble TNFa in the ACM prevents an increase in dendritic GluA1 protein levels.
In addition to paracrine effects on motor neurons, astrocytes also express both TNFa receptors (TNFR1 and TNFR2) and TNFa can act on astrocytes to cause a diverse set of changes (Fischer, Wajant, Kontermann, Pfizenmaier, & Maier, 2014;Tezel, Li, Patil, & Wax, 2001) Thus, TNFa released from mutFUS astrocytes may contribute to astro- Despite numerous studies showing both fALS and sALS astrocytes being toxic to motor neurons, the identification of a toxic factor remained elusive. Here we demonstrate that mutant FUS astrocytes contribute to motor neuron death and the principal factor of this toxicity is TNFa secreted from these astrocytes. Although our studies clearly show mutFUS astrocytes are primed to secrete toxic levels of TNFa, the underlying mechanism for such an aberrant secretion remains to be studied. In future studies, it will also be interesting to understand whether changes to the RNA/DNA binding properties of mutant FUS or other cellular stress induced by the mislocalization of mutant FUS mediates the aberrant NF-kB activation and production of TNFa.
In summary, we have extended a significant body of work pointing to a key role for astrocytes in ALS by demonstrating for the first time the neurotoxic properties of astrocytes expressing mutFUS, an ALS causative gene. In our model, we have found key proinflammatory changes to astrocytes, which trigger excitotoxic vulnerability in motor neurons. Although the underlying connection between different genetic forms of ALS is complex and at this time unclear, this work indicates that strategies targeting astrocyte contributions to disease may be useful for a variety of ALS cases. Although future work must be done to confirm these findings in vivo and, eventually in patients, we recently confirmed the clinical relevance of our findings showing that in a patient-derived in vitro model of the blood-brain-barrier, iPSCderived astrocytes from a mutFUS patient, drive drug-efflux transporter mediated pharmacoresistance through TNFa (Qosa et al., 2016). | 2018-04-03T03:36:24.874Z | 2018-01-30T00:00:00.000 | {
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244930830 | pes2o/s2orc | v3-fos-license | Prediction of Cow Calving in Extensive Livestock Using a New Neck-Mounted Sensorized Wearable Device: A Pilot Study
In this study, new low-cost neck-mounted sensorized wearable device is presented to help farmers detect the onset of calving in extensive livestock farming by continuously monitoring cow data. The device incorporates three sensors: an inertial measurement unit (IMU), a global navigation satellite system (GNSS) receiver, and a thermometer. The hypothesis of this study was that onset calving is detectable through the analyses of the number of transitions between lying and standing of the animal (lying bouts). A new algorithm was developed to detect calving, analysing the frequency and duration of lying and standing postures. An important novelty is that the proposed algorithm has been designed with the aim of being executed in the embedded microcontroller housed in the cow’s collar and, therefore, it requires minimal computational resources while allowing for real time data processing. In this preliminary study, six cows were monitored during different stages of gestation (before, during, and after calving), both with the sensorized wearable device and by human observers. It was carried out on an extensive livestock farm in Salamanca (Spain), during the period from August 2020 to July 2021. The preliminary results obtained indicate that lying-standing animal states and transitions may be useful to predict calving. Further research, with data obtained in future calving of cows, is required to refine the algorithm.
Introduction
The study and monitoring of livestock has always been a subject of great interest. Indeed, quantitative measurement of animal behaviour is an important tool for understanding their reproduction, survival, welfare, and interaction with other animals [1]. Animal activity is one of the most important indicators associated with animal health and welfare [2], and animal behaviour is an indicator of the well-being and health of cows [3]. Detecting changes in the behaviour and activity of cows is a good preventive tool to determine the animal's health status [4].
Every year an average of 8.5% of perinatal calves are lost due to natural abortions, stillbirths, and complications during parturition (calving) [5], which translates into higher economical costs and reduced animal wellbeing. Ideally the calving process should be carefully overviewed by experts to avoid or correct any problems that may arise (e.g., dystocia). Even today, the analysis of cow behaviour and calving detection is mainly carried out by experienced workers through unaided monitoring. These approaches are however expensive and time consuming, and not effective for extensive livestock farming, where many animals are kept under grazing in the open air on large areas of surface.
An automated solution based on cow data collection from sensors could provide better calving predictions. This will allow the farmer to better identify those cows that require intensive supervision and to focus on caring for cows with upcoming calving, reducing possible risks and improving the health and wellbeing of the animals.
observation. This will allow the farmer to have a more accurate estimation of the expected calving date of the cow and to identify those cows that require intensive supervision due to the proximity of calving. It will make possible both reducing the workload of farmers, who can focus on caring for cows with upcoming calving and improving the health of the cow.
In this paper a low-cost neck-mounted sensorized wearable device was designed and an algorithm was developed to detect the onset of calving of cows in extensive livestock farming. This work was divided in three main phases: (1) development of a wearable solution for data collection based on different sensors; (2) data collection in extensive livestock farming by using the aforementioned solution and human-based observations; and (3) development of algorithms to detect the onset of calving and creation of a decision function based on the frequency and duration of lying and standing behaviour (lying bouts).
This paper is organized as follows. Section 2 presents the sensorized wearable device, the software developed to collect data from different cows by human observers, gives an overview of the collected data, as well as the methodology followed during this study and introduces the proposed algorithm for parturition detection. Results and discussion of this algorithm are presented in Section 3. Finally, Section 4 shows the conclusions and future work.
Materials and Methods
The goal of the first phase of the study is the development of a solution for recording large quantities of behavioural information, which will later be combined with human observation of the animals. The captured information will be used for the development, validation, and quantification of algorithms for calving prediction.
A new low-cost sensorized wearable device was developed and integrated into a collar, which can be placed around the cow's neck. The developed device incorporates three sensors: an Inertial Measurement Unit (IMU), a GNSS and a thermometer. Human observers helped with monitoring, labelling, and recording the animals' state using our own development PC software. The designed collars were tested on cows from an extensive livestock farm in Salamanca (Spain), during the period August 2020-July 2021.
Sensorised Wearable Device
The sensorized wearable device is a collar which is placed on the animal's neck. The collar houses a nRF52840-dongle (Nordic Semiconductor, Trondheim, Norway) microcontroller, three sensors (thermometer, 9-axis IMU-3-axis accelerometer, 3-axis gyroscope and 3-axis magnetometer-and GNSS), a microSD card breakout board (AdaFruit, NY, EEUU) for data storage and lithium batteries. Figure 1 shows the overall architecture of the collar. The microcontroller nRF52840 is a small, low-cost device built around the 32-b ARM ® Cortex™-M4 CPU which supports short-range wireless standards including BLE For measuring the cow's body temperature, a DS18B20 digital thermometer with a tem perature range of −55 to 125 °C (accuracy ± 0.5 °C) is placed in such a way that contac between the skin of the cow and the sensor occurs. The microcontroller acquires the 9-1 bits configurable Celsius temperature measurements using a unique 1-wire interface The microcontroller nRF52840 is a small, low-cost device built around the 32-bit ARM ® Cortex™-M4 CPU which supports short-range wireless standards including BLE. For measuring the cow's body temperature, a DS18B20 digital thermometer with a temperature range of −55 to 125 • C (accuracy ± 0.5 • C) is placed in such a way that contact between the skin of the cow and the sensor occurs. The microcontroller acquires the 9-12 bits configurable Celsius temperature measurements using a unique 1-wire interface, which only requires one port pin for the communication. The IMU integrated in the collar is the ICM-20948 (InvenSense, Berkeley, CA, USA, EEUU), which is a low-power 9-axis motion tracking device embedding a 3-axis gyroscope, a 3-axis accelerometer, and a 3axis compass. A SAM-M8Q (U-blox, Thalwil, Switzerland) receiver is used for precise geographic positioning of the animal and is configured to work in PSM (Power Save Management) mode to minimize power consumption. Both the IMU and the GNSS communicate with the nRF52840 microcontroller using I 2 C at 400 KHz. The microSD card, which allows the storage of data from the sensors, communicates with the microcontroller using SPI. Four lithium ion NCR18650GA (Sanyo, Osaka, Japan) batteries of 3.7 V and 3350 mA were used to power the device. Figure 2 shows the developed collar. To avoid damage to the electronic components and the batteries, a protective box was designed and produced using additive manufacturing in a 3D printer (Ultimaker ® 2+). The polymer selected was ABS, a low-cost plastic material with good mechanical properties. The cover box has two areas (Figure 2a,b). The lower part contains the electronics, whereas the upper area houses the batteries. This design facilitates the replacement of the batteries in the collar without affecting the electronic components. To waterproof the container, a 2 mm diameter nitrile rubber O-ring was fitted in the junction between the two covers. The box was attached to the neck of the cows using an adjustable leather belt (Figure 2c), which allows for a good fit on animals of different sizes. The material of the belt is soft, which maximises the animal's comfort. The microcontroller nRF52840 is a small, low-cost device built around the 32-bit ARM ® Cortex™-M4 CPU which supports short-range wireless standards including BLE. For measuring the cow's body temperature, a DS18B20 digital thermometer with a temperature range of −55 to 125 °C (accuracy ± 0.5 °C) is placed in such a way that contact between the skin of the cow and the sensor occurs. The microcontroller acquires the 9-12 bits configurable Celsius temperature measurements using a unique 1-wire interface, which only requires one port pin for the communication. The IMU integrated in the collar is the ICM-20948 (InvenSense, Berkeley, CA, USA, EEUU), which is a low-power 9-axis motion tracking device embedding a 3-axis gyroscope, a 3-axis accelerometer, and a 3-axis compass. A SAM-M8Q (U-blox, Thalwil, Switzerland) receiver is used for precise geographic positioning of the animal and is configured to work in PSM (Power Save Management) mode to minimize power consumption. Both the IMU and the GNSS communicate with the nRF52840 microcontroller using I 2 C at 400 KHz. The microSD card, which allows the storage of data from the sensors, communicates with the microcontroller using SPI. Four lithium ion NCR18650GA (Sanyo, Osaka, Japan) batteries of 3.7 V and 3350 mA were used to power the device. Figure 2 shows the developed collar. To avoid damage to the electronic components and the batteries, a protective box was designed and produced using additive manufacturing in a 3D printer (Ultimaker ® 2+). The polymer selected was ABS, a low-cost plastic material with good mechanical properties. The cover box has two areas (Figure 2a,b). The lower part contains the electronics, whereas the upper area houses the batteries. This design facilitates the replacement of the batteries in the collar without affecting the electronic components. To waterproof the container, a 2 mm diameter nitrile rubber O-ring was fitted in the junction between the two covers. The box was attached to the neck of the cows using an adjustable leather belt (Figure 2c), which allows for a good fit on animals of different sizes. The material of the belt is soft, which maximises the animal's comfort. All electronic components of the collar were configured to work in low power mode, to minimize power consumption, and to extend battery life. The data from the accelerometer and the gyroscope embedded in the IMU were sampled at a frequency of 17.6 Hz. The temperature and geolocation information (longitude, latitude, altitude, and speed) were collected at 1 Hz. The orientation of the IMU axis is shown in Figure 2d.
Initial tests were performed using BLE 4.2 for the communication between the board and a computer where data was registered. Due to high power consumption, however, it was decided to store the information onboard instead, using a microSD card. This ap- All electronic components of the collar were configured to work in low power mode, to minimize power consumption, and to extend battery life. The data from the accelerometer and the gyroscope embedded in the IMU were sampled at a frequency of 17.6 Hz. The temperature and geolocation information (longitude, latitude, altitude, and speed) were collected at 1 Hz. The orientation of the IMU axis is shown in Figure 2d.
Initial tests were performed using BLE 4.2 for the communication between the board and a computer where data was registered. Due to high power consumption, however, it was decided to store the information onboard instead, using a microSD card. This approach increased the average collar battery life to up to 15 days.
Each datapoint includes the information from the sensors and a timestamp, using the format described in Table 1. The data are then saved to the microSD card using binary format. To minimize data loss if the collar runs out of batteries or fails, a new file is created every hour. Table 1. Raw data collected by the sensorized collar and saved in the microSD card.
Variable Datatype Units
Timestamp int32 ms since Unix Epoch Temperature IMU float32 degrees C Temperature DB18B20 float32 Although the data transmission is no longer performed wirelessly, the collar still makes use of BLE: during collar initialization a timestamp will be exchanged between the collar and the laptop, which allows for later data synchronization. A keepalive message is also sent periodically (every minute), from collar to laptop, using BLE, to allow the human observer monitoring the cow to detect if the collar is still operative or another action is required (e.g., replacement of batteries).
Data Annotation with Computer Software
Direct visual observations were used to collect states and actions of cows in their natural environment at the cattle farm. A PC-based software (Figure 3a) was designed and developed to help the user register the observed state of multiple cows. The program includes the following functionalities:
Collar initialization
During collar initialization, a timestamp is sent to the collar using BLE. This timestamp will be used for the synchronization of the sensor data collected by the collar and the states and actions of the cows recorded by the human observer.
Collar management (Figure 3b)
According to the European Commission, individual identification and registration of bovine livestock is mandatory to ensure full traceability and, consequently, enhance food safety and better safeguard animal health. The application uses an alpha-numeric code as the cowl's ID. Similarly, each device is identified by a unique 64-bit collar ID, which corresponds to the serial number of the microcontroller housed in the collar. Sensors 2021, 21, x FOR PEER REVIEW 7 of 16
Animals, Facility and Data Collection
For this study, ten collars were developed. However, cow monitoring was limited to a maximum of three animals simultaneously in order to improve the quality of the annotated data. The rest of the produced collars were saved as replacements, to minimize dead times and data loss in case a collar stopped working. The monitored beef cattle belong to an extensive livestock farm located in the municipality of Carrascal de Barregas, in the province of Salamanca, Spain.
During the study, collars were fitted to six cows (see Table 2). For eleven months (August 2020-July 2021) two experienced observers (one working in the morning shift and the other during the afternoon shift) annotated the actions of the animals for a total of 6 h every day. It is to be noted that although the observers were following the animals continuously, some situations introduced uncertainty in the data, for example, when cows stampede from one location to another. To mitigate the data deterioration, observers left the annotations blank when detecting these situations. To record the different states a laptop running the application previously presented was used while maintaining a clear line of sight with the animals. Every week, the data stored in the microSD card of the collar was downloaded, and the batteries were replaced. A detailed overview of the cow-wise distribution of the data is presented in Table 2. As datapoints were dumped in the SD card on an hourly basis, it is straightforward to know the number of hours we got data from. The total quantities shown in Table 2 accumulate the number of hours the collar was recording data (raw data) and the number of hours the observer labelled behaviours for every studied cow. During operation, it was noted that human observers sometimes had difficulty identifying cows when recording actions, especially over long distances or when animals were close to each other. To solve this, each manufactured collar was printed with a different colour (Figure 2c shows a red collar). Collar management allows registering and assigning a new collar to an animal, unregistering, or reassigning an existing one and updating the colour of the collar.
Collar scanner (Figure 3c)
This feature allows to check active collars within the BLE's range, by listening to a keepalive message containing the ID that the devices send every minute. The discovered collars are listed on an overview and their status is changed to active (Figure 3c). The application prevents the annotation of data from not active collars.
Data annotation (Figure 3d)
It allows the annotation, by a human observer, of the status and the action the cow is performing. Additional observations can also be recorded. All annotated data and observations are stored on the PC. Once the annotation activity is finished, the software automatically generates a file containing the recorded information. Two predetermined sets of states have been considered: • General behaviour. The tags considered are: "Grazing/Eating", "Ruminating", "Neutral" and "Walking". • Standing behaviour. The tags considered are: "Standing" and "Lying" position.
This reduction was necessary to identify transitions between lying and standing positions (lying bouts) which would not be registered in the general behaviour tag set, where the neutral state can happen both standing and lying (if the cow is doing nothing else). Import data: Once the operator recovers the collar and obtains the sensor data files stored in the microSD, the application allows the generation of a final file, using the timestamp in both files to combine and synchronize the data from the collar with the data from the file containing the visual observation data (stored in the PC). This synchronized data set is used to develop our own parturition prediction algorithm described in Section 2.3.2.
. Animals, Facility and Data Collection
For this study, ten collars were developed. However, cow monitoring was limited to a maximum of three animals simultaneously in order to improve the quality of the annotated data. The rest of the produced collars were saved as replacements, to minimize dead times and data loss in case a collar stopped working. The monitored beef cattle belong to an extensive livestock farm located in the municipality of Carrascal de Barregas, in the province of Salamanca, Spain.
During the study, collars were fitted to six cows (see Table 2). For eleven months (August 2020-July 2021) two experienced observers (one working in the morning shift and the other during the afternoon shift) annotated the actions of the animals for a total of 6 h every day. It is to be noted that although the observers were following the animals continuously, some situations introduced uncertainty in the data, for example, when cows stampede from one location to another. To mitigate the data deterioration, observers left the annotations blank when detecting these situations. To record the different states a laptop running the application previously presented was used while maintaining a clear line of sight with the animals. Every week, the data stored in the microSD card of the collar was downloaded, and the batteries were replaced. A detailed overview of the cow-wise distribution of the data is presented in Table 2. As datapoints were dumped in the SD card on an hourly basis, it is straightforward to know the number of hours we got data from. The total quantities shown in Table 2 accumulate the number of hours the collar was recording data (raw data) and the number of hours the observer labelled behaviours for every studied cow. The schema of data acquired with the collars is shown in Table 3. In total, more than 855 million (855,319,572) raw datapoints have been recorded, of which approximately 114 million (114,167,178) are labelled. Previous research work related to monitoring of pregnant cows around calving, (Jensen, 2012) and (Titler et al., 2015), has been focused on the period immediately around the time of calving (one and four days, respectively). This approach however is not practical for application where monitoring is less frequent, such as extensive livestock farms where large herds are held. Therefore, our research was focused on the long-term monitoring of the pregnant animals, which extended up to two months after calving. Using this approach, we could analyse the individual behavioural change during different stages of pregnancy, which previous research has proved can differ greatly between individuals [21]. The labels used for data annotation are presented in Tables 4 and 5. These two sets of labels distinguish the two datasets introduced before, namely general behaviour and standing/lying behaviour. Considering both label sets, approximately 1.777 h of cow behaviour have been annotated by two experienced observers (working part-time in morning and afternoon shifts). Table 4. General behaviour annotations.
B1 Standing B2 Lying
The distribution of the annotated actions is presented in Figure 4 for general behaviour actions and in Figure 5 for standing/lying behaviour.
around the time of calving (one and four days, respectively). This approach however is not practical for application where monitoring is less frequent, such as extensive livestock farms where large herds are held. Therefore, our research was focused on the long-term monitoring of the pregnant animals, which extended up to two months after calving. Using this approach, we could analyse the individual behavioural change during different stages of pregnancy, which previous research has proved can differ greatly between individuals [21].
The labels used for data annotation are presented in Tables 4 and 5. These two sets of labels distinguish the two datasets introduced before, namely general behaviour and standing/lying behaviour. Considering both label sets, approximately 1.777 h of cow behaviour have been annotated by two experienced observers (working part-time in morning and afternoon shifts) The distribution of the annotated actions is presented in Figure 4 for general behaviour actions and in Figure 5 for standing/lying behaviour.
ID Action B1
Standing B2 Lying General behaviour (Table 4) action prediction [33] has been explored during the study as additional input for calving prediction. Furthermore, data already gathered allows for further investigation in this field without the need of additional human-labelling. However, the general behaviour label set can lead to an imperfect classification of standing/lying behaviour (lying bouts) of the cow when transformed, as some actions can only be carried out in one posture (e.g., walking-standing), but other may occur both standing and lying.
For this reason, a second set of labels (Table 5) only accounting for lying and stand- General behaviour (Table 4) action prediction [33] has been explored during the study as additional input for calving prediction. Furthermore, data already gathered allows for further investigation in this field without the need of additional human-labelling. However, the general behaviour label set can lead to an imperfect classification of standing/lying Sensors 2021, 21, 8060 9 of 15 behaviour (lying bouts) of the cow when transformed, as some actions can only be carried out in one posture (e.g., walking-standing), but other may occur both standing and lying.
For this reason, a second set of labels (Table 5) only accounting for lying and standing postures has been used to ensure a correct classification of these two postures when needed due to the importance of lying bouts detection for calving detection (as discussed in the introduction).
The annotated data from human observations show a small delay between the change of action of the cow and the annotation of this new action due to the response time of the observer and their need to operate the annotation software. For this reason, the datapoints recorded two minutes before an action change were ignored. This time period was chosen as a balance between loss of data and minimizing mislabelled data in the dataset. As cows did not change actions with high frequency in the recorded labels, two minutes resulted in enough certainty without losing a significative volume of data for each action.
As discussed previously, the sensor readings from the collars and the annotations from the observers can be joined using the timestamp of each data point to form the final dataset.
Three datasets were generated using the recorded data: • Non-annotated data from the devices. These data have been proved to be useful for unsupervised and semi supervised learning tasks. • General behaviour annotated data (Table 4). This dataset could be used to classify and predict the animal actions based on new reading from the devices. • Standing/lying behaviour annotated data (Table 5). This dataset, although smaller compared to b, serves for statistical learning tasks, as well as semi-supervised learning techniques.
Parturition Prediction Algorithm
Calving prediction is the main objective of this pilot study. To usefully notify parturition, it is necessary to detect it with enough anticipation using a low-memory algorithm suited for the microcontroller.
The number of transitions of the animal between lying and standing has been empirically proved to be a good indicator of parturition in different studies [27,40,41]. This measure serves as an indicator of the proximity of calving due to the relative increase of its value in the 8 to 2 h before the parturition event. Furthermore, a notable decrease in the number of lying bouts in the hours after calving is also observed. However, these works study intensive dairy cattle, while our work studies extensive beef cattle, with the according significantly less restricted environment since calving barns are not used. A new low-memory algorithm based on classification of two cow postures (lying and standing), from the collar sensors readings has been developed. To classify these behaviours, accelerometer readings, commonly used to distinguish between lying and standing, as well as GPS altitude readings were initially considered. However, GPS readings were discarded due to insufficient sensor resolution.
In [21] is indicated that is more difficult to distinguish between a lying and standing position with a neck-based accelerometer since the two positions show similar accelerometer readings. Leg-based accelerometers show a distinct crossover of two axes and can easily be utilized to determine a standing or lying position. However, we have analyzed accelerometer signals read by our neck-mounted collars on the Y and Z axes ( Figure 6). This figure shows that the analysis of the accelerometer signals provided by the cow's collar, allow us to clearly distinguish cow lying position (green colour), and cow standing position (blue colour). These results are similar to those presented in [29] to classify the cow posture as standing or lying. erometer readings. Leg-based accelerometers show a distinct crossover of two axes and can easily be utilized to determine a standing or lying position. However, we have analyzed accelerometer signals read by our neck-mounted collars on the Y and Z axes (Figure 6). This figure shows that the analysis of the accelerometer signals provided by the cow's collar, allow us to clearly distinguish cow lying position (green colour), and cow standing position (blue colour). These results are similar to those presented in [29] to classify the cow posture as standing or lying. Our algorithm for cow posture classification (lying/standing) based on accelerometer readings has been implemented based on an heuristic threshold [27]. This approach is focused on simplicity and is based on the different distributions of acceleration recordings along the Y axis depending on the posture of the animal. Figure 7 shows the distribution of all available data labelled with the standing/lying action (11 months of data collection). Accelerometer Y-axis readings (left) indicates that standing behaviour is characterized by a larger mean (denoted by a grey triangle) than those recorded with the animal lying down. Accelerometer Z-axis readings (right), indicates that standing position have a larger interquartile range than the lying ones. Our algorithm for cow posture classification (lying/standing) based on accelerometer readings has been implemented based on an heuristic threshold [27]. This approach is focused on simplicity and is based on the different distributions of acceleration recordings along the Y axis depending on the posture of the animal. Figure 7 shows the distribution of all available data labelled with the standing/lying action (11 months of data collection). Accelerometer Y-axis readings (left) indicates that standing behaviour is characterized by a larger mean (denoted by a grey triangle) than those recorded with the animal lying down. Accelerometer Z-axis readings (right), indicates that standing position have a larger interquartile range than the lying ones. To achieve "a low-memory algorithm", the accelerometer data is resampled from 17.6 Hz to 0.27 Hz as our experiments have proven that this data rate is enough for the algorithm. This way, memory requirements during the sliding window operations decreases and battery life of the device increases. Based on these appreciations and given the series of readings from the Y-axis of the accelerometer at discrete timestamps t , denoted by ( ), the following algorithm has been developed: 1. First, a rectangular sliding window of the last 15 min is used to count the number of readings in the Y-axis between a given superior and inferior threshold, ℎ and ℎ , respectively. The values of these thresholds are obtained from the distribution shown in Figure 7 to maximize the difference in the resulting count while To achieve "a low-memory algorithm", the accelerometer data is resampled from 17.6 Hz to 0.27 Hz as our experiments have proven that this data rate is enough for the algorithm. This way, memory requirements during the sliding window operations decreases and battery life of the device increases. Based on these appreciations and given the series of readings from the Y-axis of the accelerometer at discrete timestamps t i , denoted by f ay (t i ), the following algorithm has been developed: First, a rectangular sliding window of the last 15 min is used to count the number of readings in the Y-axis between a given superior and inferior threshold, thr sup and thr in f , respectively. The values of these thresholds are obtained from the distribution shown in Figure 7 to maximize the difference in the resulting count while standing and lying. This operation results in a function f count (t) (1) given by: 2.
Next, f count (t) is thresholded to obtain a binary signal f standing (t) (2) depending on the value of the function in each instant relative to a threshold thr standing . This way, any value greater than thr standing will be denoted as 1 (standing), while values smaller than the threshold will be converted to 0 (lying). 3.
This binary function f standing (t) is converted to a discrete transition signal f lb (t) (3) taking the absolute value of the difference between f standing (t) at any given time and its immediately previous value with each transition from standing to lying down and vice-versa represented by a 1.
4. Finally, with this discrete transition signal f lb (t) computed, another rectangular sliding window is used to count the number of transitions that took place in the previous 5 h of each reading. This function f parturition (t), acts as a proxy to predict parturition based on the lying bouts occurrence increasement before calving.
Results and Discussion
As indicated in Table 2, cow number 03 calved on 5 May 2021 at 4:45 PM. Figure 8 shows the values of function f parturition (t) in the last five hours calculated with a rolling window for cow 03, for a week (from 4 May to 11 May), using the proposed algorithm for parturition prediction. A notable increase of this function is observed near the parturition instant, signalled with a vertical red dotted line.
Results and Discussion
As indicated in Table 2, cow number 03 calved on 5 May 2021 at 4:45 PM. Figure 8 shows the values of function ( ) in the last five hours calculated with a rolling window for cow 03, for a week (from 4 May to 11 May), using the proposed algorithm for parturition prediction. A notable increase of this function is observed near the parturition instant, signalled with a vertical red dotted line. Figure 8 represents the function ( ) during a week. This figure shows that it is during the hours before calving (2~3 h) when this function takes the highest values, reaching the maximum at the instant of calving. Furthermore, a horizontal dotted green line in Figure 8 denotes a value that is only surpassed during the calving event ( ( ) = 10). This value could be used as a trigger of the parturition detection for this cow 03. It is noted that this signalled value differs between individuals since there is variance between the activity and energy expenditure of each animal, and therefore this value has to be dynamically calculated (for each cow) on the collar based on previous readings. As indicated in Table 2, during the data collected over a period of eleven months (August 2020-July 2021), five cows calved. Figure 9 shows the mean of function ( ) in the last five hours calculated with a rolling window, and generated from the algorithm showed before (from the five calving events that took place). To calculate this mean, the values of this function have been aligned on the moment of parturition (0 h relative to calving). It is observed in Figure 9 that the mean value of the function ( ) increases two hours before the calving of the cows. This increase allows us to determine that the cow is close to parturition. As previously mentioned, the parturi- Figure 8 represents the function f parturition (t) during a week. This figure shows that it is during the hours before calving (2~3 h) when this function takes the highest values, reaching the maximum at the instant of calving. Furthermore, a horizontal dotted green line in Figure 8 denotes a value that is only surpassed during the calving event ( f parturition (t) = 10). This value could be used as a trigger of the parturition detection for this cow 03. It is noted that this signalled value differs between individuals since there is variance between the activity and energy expenditure of each animal, and therefore this value has to be dynamically calculated (for each cow) on the collar based on previous readings.
As indicated in Table 2, during the data collected over a period of eleven months (August 2020-July 2021), five cows calved. Figure 9 shows the mean of function f parturition (t) in the last five hours calculated with a rolling window, and generated from the algorithm showed before (from the five calving events that took place). To calculate this mean, the values of this function have been aligned on the moment of parturition (0 h relative to calving). It is observed in Figure 9 that the mean value of the function f parturition (t) increases two hours before the calving of the cows. This increase allows us to determine that the cow is close to parturition. As previously mentioned, the parturition trigger value of 10 signalled in Figure 8 is only applicable to cow 03. This can be shown in Figure 9, where the f parturition (t) signals from the rest of the cows have brought the signal mean value slightly down. Table 2.
The heuristic-based algorithm we have developed traces lying bouts with enough resolution to detect their increase in a time frame, which can be used to detect cow calving. This is accomplished without the need to calculate a calving indicator index that requires tracking the step count and the time spent lying down by the animal as in [27], mainly due to this variables stability when compared with the number of lying bouts near calving.
A very important aspect of the developed calving detection algorithm is its simplicity. This algorithm must be programmed in the microcontroller housed in the collar placed on the cow's neck, which represents a significant limitation in the microcontroller's computing and memory fields. Furthermore, a coarse estimate of the calving date in the receiving end of the calving prediction is also useful to use in conjunction with the algorithm, as it ensures the rejection of any naturally inviable false positive from the algorithm (i.e., calving detection one month before and after mount).
The dataset used in previous studies [27,40], usually includes data in a small temporal window around calving (1-5 days). The data collected in our study enable the back test of algorithms in a much more extensive temporal window, something essential to validate any algorithm that would run in a real-world environment, such as the proposed sensorized wearable device.
Although the proposed algorithms of this study are focused on calving prediction, the developed sensorized wearable device and the collected data enable the development of different algorithms that could be of great help to farmers both in extensive and intensive livestock. Additionally, the generalist design of the presented wearable device could be equally helpful to develop hardware solutions oriented to different animals, benefiting their caretakers with the localization data from the collar, the suite of sensors that it incorporates, and other algorithms that could be implemented within the device.
Conclusions and Future Work
In this paper, a low-cost neck-mounted sensorized wearable device to continuous long-term monitoring cow data has been presented. To incorporate the ability to detect Table 2.
The heuristic-based algorithm we have developed traces lying bouts with enough resolution to detect their increase in a time frame, which can be used to detect cow calving. This is accomplished without the need to calculate a calving indicator index that requires tracking the step count and the time spent lying down by the animal as in [27], mainly due to this variables stability when compared with the number of lying bouts near calving.
A very important aspect of the developed calving detection algorithm is its simplicity. This algorithm must be programmed in the microcontroller housed in the collar placed on the cow's neck, which represents a significant limitation in the microcontroller's computing and memory fields. Furthermore, a coarse estimate of the calving date in the receiving end of the calving prediction is also useful to use in conjunction with the algorithm, as it ensures the rejection of any naturally inviable false positive from the algorithm (i.e., calving detection one month before and after mount).
The dataset used in previous studies [27,40], usually includes data in a small temporal window around calving (1-5 days). The data collected in our study enable the back test of algorithms in a much more extensive temporal window, something essential to validate any algorithm that would run in a real-world environment, such as the proposed sensorized wearable device.
Although the proposed algorithms of this study are focused on calving prediction, the developed sensorized wearable device and the collected data enable the development of different algorithms that could be of great help to farmers both in extensive and intensive livestock. Additionally, the generalist design of the presented wearable device could be equally helpful to develop hardware solutions oriented to different animals, benefiting their caretakers with the localization data from the collar, the suite of sensors that it incorporates, and other algorithms that could be implemented within the device.
Conclusions and Future Work
In this paper, a low-cost neck-mounted sensorized wearable device to continuous long-term monitoring cow data has been presented. To incorporate the ability to detect cow parturitions with enough anticipation using only this device an algorithm has been developed. This algorithm can detect an increment in the number of times the cow stands up and lies down (lying bouts) using a signal calculated from the accelerometer's readings. To test the proposed algorithm, data collection using the cow neck-mounted devices and annotations from in-situ human observers has been carried out for eleven months (August 2020-July 2021). The data gathered by the neck-mounted collars correspond to six different cows monitoring in extensive livestock farming.
This preliminary study (of six cows, with five calving events), provides evidence that cow approaching parturition shows an increase in lying bouts behavioural pattern that can predict calving on average two hours before calving. To confirm these results, however, more pregnant cows need to be monitored and further research is required to refine this algorithm. The long-term character of the data acquired will allow for individualization of the thresholds for calving detection by calculating the baseline "restlessness level" of a particular animal. This could be used by the collar to generate an alert system to warn the farmer of the onset of calving.
The low-cost of this device (≈100 € for small-scale production) would benefit most large livestock holdings by greatly reducing the number of hours the human experts must manually monitor individual animals. Economies of scale would allow the unitary price of the collar to be lowered even more, which would allow massive adoption even for the monitoring of large herds. However, smaller farms with less resources would also benefit from the reduced cost of the device, which lowers the entry barrier into a technology such as this, thus allowing for its adoption.
Future work will focus in two separate points. The first one, data related, requires the acquisition of calving data from a larger number of cows to validate the developed algorithm and to develop a new data driven algorithm that learns from the available data from different cows (both from the same breed and from different breeds, to deal with the variance between different species). This new algorithm could be dedicated to the classification of standing and lying behaviour or to the detection of the birth event as a time series task. Furthermore, the data already gathered from the animals long before calving and therefore related mostly to the normal activity of the animal would allow for the development of algorithms that analyse cow behaviour (grazing, ruminating, etc). A deviation of the normal behaviour of a particular animal due to sickness, heat, or an abortion could be signalised to the farmer.
The second point englobes the development of a new neck-mounted collar with IMU, GNSS and wireless low energy, and long-range communication using the LoRa protocol. This wireless communication would allow farmers to physically localize cows in extensive livestock farming, as well as receiving notifications of the detected parturition event, reducing the workload associates with parturition, and preventing dystocia in unattended calving. The energy autonomy of the device is an aspect of great importance in terms of its practical utility. In the tests carried out, it has been identified as an improvement parameter. For this, solar-powered technology is being incorporated in order to increase overall autonomy. | 2021-12-08T16:09:48.890Z | 2021-12-01T00:00:00.000 | {
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254915558 | pes2o/s2orc | v3-fos-license | Density Functional Theory Study of the Point Defects on KDP (100) and (101) Surfaces
Surface defects are usually associated with the formation of other forms of expansion defects in crystals, which have an impact on the crystals’ growth quality and optical properties. Thereby, the structure, stability, and electronic structure of the hydrogen and oxygen vacancy defects (VH and VO) on the (100) and (101) growth surfaces of KDP crystals were studied by using density functional theory. The effects of acidic and alkaline environments on the structure and properties of surface defects were also discussed. It has been found that the considered vacancy defects have different properties on the (100) and (101) surfaces, especially those that have been reported in the bulk KDP crystals. The (100) surface has a strong tolerance for surface VH and VO defects, while the VO defect causes a large lattice relaxation on the (101) surface and introduces a deep defect level in the band gap, which damages the optical properties of KDP crystals. In addition, the results show that the acidic environment is conducive to the repair of the VH defects on the surface and can eliminate the defect states introduced by the surface VO defects, which is conducive to improving the quality of the crystal surface and reducing the defect density. Our study opens up a new way to understand the structure and properties of surface defects in KDP crystals, which are different from the bulk phase, and also provides a theoretical basis for experimentally regulating the surface defects in KDP crystals through an acidic environment.
Introduction
Potassium dihydrogen phosphate (KH 2 PO 4 , KDP) crystal is the only nonlinear optical crystal material that can be used in inertial confinement nuclear fusion devices [1][2][3][4][5][6][7] because of its excellent nonlinear optical properties, high laser-damage threshold, wide light-transmission range, and ability to grow a large-sized single crystal. However, there is a certain gap between the actual nonlinear optical properties and the laser-damage threshold of KDP crystals in practical applications and for theoretical values. Numerous studies have shown that intrinsic point defects significantly influence the optical properties of KDP crystals. For example, Guillet et al. pointed out that the stress field caused by dislocation or the growth-sector boundary increases the concentration of intrinsic defects, which leads to a reduction in the laser-damage threshold [8]. The first-principles calculation results pointed out that hydrogen and oxygen defects may cause additional optical absorption and reduce the laser-damage threshold of KDP [9][10][11][12][13]. Sui et al. found that point defects such as hydrogen vacancy (V H ), oxygen vacancy (V O ), etc., can not only introduce defect states in the band gap but also cause large structural deformation and local lattice stress near the defect sites, which can reduce the optical performance of KDP crystals [14,15].
It should be noticed that apart from point defects in the crystals, the defects of KDP surfaces also affect the optical properties of KDP crystals. For example, by combining spectral detection and theoretical calculation, Ding et al. found that the intrinsic defects convergence criteria of structural relaxation were set to be 0.02 eV/Å and 10 −5 eV/atom, respectively. After the convergence tests, a Monkhorst-Pack k-point grid [42] of 3 × 3 × 1 was used to sample the Brillouin zone for structural optimization, and a denser grid of 7 × 7 × 1 was used for the electronic structure calculations.
According to the experimentally reported growth faces of KDP crystals [27,28], we constructed the (100) and (101) surface models by cutting four KDP molecule layers from a bulk 2 × 2 × 2 tetragonal supercell. A 15 Å vacuum layer was added perpendicular to both the (100) and (101) surfaces, to reduce the interaction between the neighboring periodic images. The surface V H and V O models were constructed by moving a hydrogen or oxygen atom from its original site. All the possible defect sites on the surface and sub-surface were considered, and only the defect with the lowest formation energy was chosen for the structural and property analysis. We simulated the charged defects by setting the total number of electrons and adding electrons or holes to the background of the whole system. Herein, we considered 0 and −1 charge states for the V H defect and 0, +1, and +2 charge states for the V O defect. The acid and alkali growth environments were simulated by placing H + and OH − ions at about 1.5 Å above the optimized surfaces. In order to better investigate the effect of the acid and alkali growth environments on the intrinsic point defects on the surfaces, we mainly considered the adsorption of H + and OH − near the defect sites of the surfaces. During the structural optimization, the bottom-most KDP molecule layer was fixed, and the top three KDP molecule layers were allowed to fully relax.
The relative stability of these defects can be determined by comparing their defect formation energies. The calculation formula is as follows [43][44][45][46]: where E total (X q ) and E total (surface) are the total energies of the surface with and without the defect X, respectively. q represents the charge of the defect. n i represents the number of atoms of species i that are added or removed when the defect is created, and µ i are the corresponding chemical potentials. E f is the Fermi level of the bulk valence-band maximum E v , and ∆V is a correction term that aligns the reference potential in the surface with the defect, with respect to the surface without the defect. We calculated the chemical potential of the required elements by referring to the work of Sui [25], namely that the chemical potential of H and O were taken as half of the energies of H 2 and O 2 , and the chemical potentials of P and K were calculated according to the formation enthalpies of their stable compounds P 2 O 5 and K 2 O. The obtained chemical potential of H, O, P, and K are −3.39, −4.47, −7.39, and −3.63 eV, respectively.
Stability and Structures of the Point Defects on the KDP Surfaces
Firstly, we constructed the most stable structures of the (100) and (101) surfaces, which correspond to the cylindrical and pyramidal surfaces of the KDP crystals, respectively, according to the exposed surface atoms reported by previous experiments [27,28], as can be seen in Figure 1. The calculated surface energies of the (100) and (101) surfaces are 0.0321 eV/Å 2 and 0.0327 eV/Å 2 , respectively. The slightly higher surface energy of the (101) surface indicates a faster growth rate and a higher activity than the (100) surface. This is consistent with the experimental result: the growth rate of the pyramidal surface is higher than that of the cylindrical surface [27,47]. We can also infer from the calculated surface energies that the (101) surface has a stronger adsorption capacity for ions and small molecules.
is higher than that of the cylindrical surface [27,47]. We can also infer from the calculat surface energies that the (101) surface has a stronger adsorption capacity for ions a small molecules. Since VH and VO defects have been reported as the main defects in KDP crystals a also have a significant influence on the optical properties, we then focused on the stru tures and electronic structures of these two point defects on the surfaces of KDP crysta The selected sites to construct the VH and VO defect models are shown in Figure 1, in whi they reflect the different locations and coordination environments at the surface, subsu face, and interior of the surface models. We compared the difficulty of forming VH and defects at different sites on the (100) and (101) surfaces and in the bulk KDP crystal calculating their defect formation energies, as seen in Table 1. It can be seen that the f mation energies of both the VH and VO defects on both the (100) and (101) surfaces a more than 1 eV lower than those in the bulk KDP. This indicates that these vacancy defe are preferable to be formed on the surfaces rather than in the bulk. Meanwhile, their f mation energy on the (101) surface is about 0.3 eV lower than that on the (100) surfa which indicates that the structure and properties of the (101) surface are more sensiti and susceptible to intrinsic defects. It should be noted that the VH defect prefers to form the outermost surface site on both the (100) and (101) surfaces, while the VO defect sho different stable sites on the different surfaces of KDP crystals. For the (100) surface, the defect prefers to form at the surface site connected to the other PO4 group by the hydrog bond. For the (101) surface, the formation energy of the VO defect at the inner site is 1 eV lower than that at the surface sites, indicating that the VO defects formed inside t crystal are extremely stable. In this case, the structure and properties of the (101) surfa are mainly influenced by the VH defect instead of the VO defect. By comparing the f mation energies, we selected Hsurf and Osub on the (100) surface and Hsurf and Oin on t (101) surface as the VH and VO defect sites for the following studies. Since V H and V O defects have been reported as the main defects in KDP crystals and also have a significant influence on the optical properties, we then focused on the structures and electronic structures of these two point defects on the surfaces of KDP crystals. The selected sites to construct the V H and V O defect models are shown in Figure 1, in which they reflect the different locations and coordination environments at the surface, subsurface, and interior of the surface models. We compared the difficulty of forming V H and V O defects at different sites on the (100) and (101) surfaces and in the bulk KDP crystal by calculating their defect formation energies, as seen in Table 1. It can be seen that the formation energies of both the V H and V O defects on both the (100) and (101) surfaces are more than 1 eV lower than those in the bulk KDP. This indicates that these vacancy defects are preferable to be formed on the surfaces rather than in the bulk. Meanwhile, their formation energy on the (101) surface is about 0.3 eV lower than that on the (100) surface, which indicates that the structure and properties of the (101) surface are more sensitive and susceptible to intrinsic defects. It should be noted that the V H defect prefers to form at the outermost surface site on both the (100) and (101) In order to further understand the impact of surface vacancy defects on the surface structures, we compared the local lattice distortion caused by the neutral and charged V H and V O defects on the (100) and (101) surfaces of KDP crystals in Figure 2. The change in the related bond lengths and bond angles are listed in Table 2. For the (100) surface, the removal of a surface hydrogen atom leads to two oxygen atoms that were initially In order to further understand the impact of surface vacancy defects on the surface structures, we compared the local lattice distortion caused by the neutral and charged VH and VO defects on the (100) and (101) surfaces of KDP crystals in Figure 2. The change in the related bond lengths and bond angles are listed in Table 2. For the (100) surface, the removal of a surface hydrogen atom leads to two oxygen atoms that were initially connected through this hydrogen atom (O-H…O hydrogen bond) moving away from each other, and, thus, the distance between O1 and O2 increases by 1.82 Å (Figure 2b). At the same time, the two PO4 tetrahedra connected by this hydrogen atom distort with the bond angle ∠O2P2O3 increasing by about 8°. The influence of an electron captured by the neutral VH defect on the structure can be neglected ( Figure 2c). For the VO defect, the adjacent hydrogen atom falls into the original vacancy defect, which leads to the movement of the hydrogen atom into the adjacent hydrogen bonds toward the PO4 tetrahedron. The bond length of H-O2 is, thus, shortened by 0.12 Å (Figure 2d). The effect of a one and two electron loss from the neutral VO defect on the structure can also be neglected (Figure 2e,f). 4 tetrahedron, instead of occupying the vacancy site like in the case of the (100) surface, due to the electrostatic repulsive force. A similar phenomenon has also been observed in the case of V O 2+ . The increase in electrostatic repulsion after the loss of two electrons moves the hydrogen atom farther away from the P atom in PO 3 . As a result, the H-P distance increases by 0.43 Å compared with the structure of V O + . However, the loss of two electrons causes a serious distortion in the structure of PO 3 . For instance, the O 1 -O 2 distance increases by 0.05 Å, the P-O 2 distance decreases by 0.05 Å, and the bond angle ∠O 2 PO 3 increases by~12 • , compared with the structure of V O 0 . Through the above analysis, it can be concluded that both the V H and V O defects have relatively local influences on the structure of the (100) surface and do not cause significant surface reconstruction. However, the V O defect on the (101) surface is mainly formed in the deep layers of the surface and, thus, causes a large degree and range of lattice distortion on the surface. Especially as the V O charge states increase from 0 to +2, the V O defect shows an increasing effect on the lattice distortion of the surface structure. We, thus, speculate that the V O defect will also have a great impact on the electronic structure of the (101) surface.
Electronic Structures of KDP Surfaces with Vacancy Defects
To confirm our suspicions, we calculated the partial density of states (PDOS) of the (100) and (101) surfaces with different charged V H and V O defects, as shown in Figure 3. For both the (100) and (101) surfaces, the valence band maximum (VBM) is mainly contributed by the O 2p states, while the conduction band minimum (CBM) is mainly contributed by the K 4s states. However, the contribution of the K 4s states to the CBM of the (101) surface is much stronger than that of the (100) surface because the (101) surface is exposed by the K + ions. When a V H 0 is formed on the (100) surface, a defect state is introduced near the VBM, and the charge disperses in the nearby O atoms. When V H 0 gains one electron, the Fermi energy level shifts slightly upward, without a significant change in the contribution from the electron states. The V H on the (101) surface shows a similar influence on the VBM, but changes the contribution of the K 4s states to the CBM. Since the absence of a H atom leads to the transfer of the surrounding electrons to the defect site and a weakening of the charge transfer between the surface group and the K + ions, the V H 0 on the (101) surface reduces the contribution of the K electronic states to the CBM. When V H 0 captures an electron to form V H − , the charge balance is restored, and the contribution of K + ions at the CBM is significantly enhanced. This indicates that when the concentration of V H is high enough, the electrostatic force of the K + ion layer on the (101) surface is weakened. When the surface adsorbs the growth groups, reaching the equilibrium state is not favorable and, thus, affects the microscopic growth process. the absence of a H atom leads to the transfer of the surrounding electrons to the defect site and a weakening of the charge transfer between the surface group and the K + ions, the VH 0 on the (101) surface reduces the contribution of the K electronic states to the CBM. When VH 0 captures an electron to form VH -, the charge balance is restored, and the contribution of K + ions at the CBM is significantly enhanced. This indicates that when the concentration of VH is high enough, the electrostatic force of the K + ion layer on the (101) surface is weakened. When the surface adsorbs the growth groups, reaching the equilibrium state is not favorable and, thus, affects the microscopic growth process. The generation of VO usually creates a donor level below the CBM in the bulk [13], but no donor level can be observed in the band structure of VO on the (100) surface ( Figure 3a). This is because after forming a VO on the (100) surface, the neighboring H atom moves to the VO site to form a stable H-PO3 configuration, which causes two additional electrons to be transferred to the H atom. When VO + and VO 2+ defects are formed, the lost electrons are distributed over the surface instead of being localized around the vacancy site. Therefore, the charged VO defects do not introduce any defect levels in the band gap. In general, surface VO defects can be immediately filled with H atoms, thereby reducing the effect of defects on the structure and electronic structure of the (100) surface. Therefore, the acidic environment may be more conducive to eliminate the defect state of VO defects and maintain the good optical characteristics of the (100) surface.
The structure and electronic structure of the VO 0 on the (101) surface are similar to those on the (100) surface due to the filling of the vacancy site by the neighboring H atom. However, when the VO 0 loses one electron, the H atom leaves the VO site and forms a H-O chemical bond with its nearest neighboring O atom. Therefore, a normal VO defect can be observed (Figure 2k), which can introduce a deep and half-filled defect level in the The generation of V O usually creates a donor level below the CBM in the bulk [13], but no donor level can be observed in the band structure of V O on the (100) surface (Figure 3a). This is because after forming a V O on the (100) surface, the neighboring H atom moves to the V O site to form a stable H-PO 3 configuration, which causes two additional electrons to be transferred to the H atom. When V O + and V O 2+ defects are formed, the lost electrons are distributed over the surface instead of being localized around the vacancy site. Therefore, the charged V O defects do not introduce any defect levels in the band gap. In general, surface V O defects can be immediately filled with H atoms, thereby reducing the effect of defects on the structure and electronic structure of the (100) surface. Therefore, the acidic environment may be more conducive to eliminate the defect state of V O defects and maintain the good optical characteristics of the (100) surface.
The structure and electronic structure of the V O 0 on the (101) surface are similar to those on the (100) surface due to the filling of the vacancy site by the neighboring H atom. However, when the V O 0 loses one electron, the H atom leaves the V O site and forms a H-O chemical bond with its nearest neighboring O atom. Therefore, a normal V O defect can be observed (Figure 2k), which can introduce a deep and half-filled defect level in the band gap (Figure 3b). This defect level is composed by the 3p states of the adjacent P atom and the 2p states of the adjacent O atom. When the V O + loses one more electron, this defect level moves below the CBM. In addition, another defect level at 2.2 eV can also be observed from the PDOS figure for the V O 2+ on the (101) surface, which mainly comes from the 2p states of the three O atoms of the surface PO 3 group. The deep defect levels introduced by V O + and V O 2+ defects can introduce additional optical absorption that damage the optical performance of KDP crystals. On the other hand, the V O defect forms in the inner layer of the (101) surface, so it is difficult to repair it in the process of crystal growth, and it is easy to enter the interior of a crystal with the stability of the growth layer and the process of the crystal's growth. Therefore, the V O defect becomes the initial point of defect extension in the crystal and the prone point of laser damage, which has an adverse effect on the optical absorption and laser-damage properties of KDP crystals.
Effects of Acidic Environment on the Defective Surface of KDP Crystals
We simulated the effect of an acidic environment on the crystal surface by adsorbing H + and compared the structural change of the perfect surfaces and defective surfaces, as shown in Figure 4. It has been found that the adsorption energies of H + on the (100) and (101) surfaces are −0.43 eV and −1.08 eV, respectively. This indicates that the H + is adsorbed on the (100) surface by very weak physisorption. This can be confirmed by the isolated charge distribution around the H + (Figure 4a) and the fact that H + is as far as 3.011 Å from the (100) surface. In order to further analyze the interaction between the adsorbed H + and the surface, we plotted the PDOS of the adsorbed ions and their adjacent surface atoms in Figure 5a. Previous studies have found that if the adsorbed particles and the surface are bonded together, the PDOS of the bonded atoms will have the same resonance peaks [48,49]. As shown in Figure 5a, there is no resonance peak between H + and the nearest atoms on the (100) surface, indicating that the H + is not bonded to the surface. On the other hand, the much lower adsorption energy of the H + on the (101) surfaces indicates that the adsorption of H + on the (101) surface is quite stable. This is because H + is connected to the outermost O atom on the (101) surface by a weak H-O bond. This can be judged by the charge distribution along the H-O bond (Figure 4d), by the bond length (1.674 Å) between the typical H-O bond length (0.96 Å), and by the hydrogen bond length (2.55 Å). The interaction between H and O has been proven to have a significant impact on the molecular arrangement and characteristics of materials [50]. This phenomenon can also be verified by the overlapped resonance peaks between the H ls states and O 2p states over a wide range, as shown in Figure 5a. When the (100) surface contains a VH, the adsorbed H + falls into the VH site, and the defect is repaired. According to Figure 5a, there are resonance peaks between the H + adsorbed to the surface and the O atoms on both adjacent sides, indicating that H + and O atoms form chemical bonds connecting the two PO4 tetrahedrons. This is consistent with the significant decrease in the adsorption energy of H + on the defective surface (−4.87 eV). Therefore, the PDOSs of the H + adsorbed (100) surface with and without VH are almost exactly the same (Figure 5b). For the surface containing the VO, the reduced H + adsorption energy, with respect to that on the perfect surface and the overlapped resonance peaks shown in Figure 5a, all indicate that the surface VO defect promotes the interaction between the adsorbed H + and the surface defect. It is interesting to note that the H that fell into the VO site combines with the adsorbed H + and forms H2. Then, a Vo defect is formed at the (100) surface, and the H2 molecule is adsorbed at 1.23 Å above the defective surface. Therefore, a defect state of VO can be clearly observed near the VBM from the PDOS in Figure 5b. It should be noted that the formation of the H2 molecule represents an extreme situation, because we are examining the surface under vacuum conditions. In real experimental conditions, a KDP crystal's growth surface is exposed to the solvent environment, and the water molecules, K + ions, and growth groups in the solvent weaken the interaction between the surface H atom and the adsorbed H + ions, making it difficult to form a real H2 molecule. However, our study shows that because the surface H atom and adsorbed When the (100) surface contains a V H , the adsorbed H + falls into the V H site, and the defect is repaired. According to Figure 5a, there are resonance peaks between the H + adsorbed to the surface and the O atoms on both adjacent sides, indicating that H + and O atoms form chemical bonds connecting the two PO 4 tetrahedrons. This is consistent with the significant decrease in the adsorption energy of H + on the defective surface (−4.87 eV). Therefore, the PDOSs of the H + adsorbed (100) surface with and without V H are almost exactly the same (Figure 5b). For the surface containing the V O , the reduced H + adsorption energy, with respect to that on the perfect surface and the overlapped resonance peaks shown in Figure 5a, all indicate that the surface V O defect promotes the interaction between the adsorbed H + and the surface defect. It is interesting to note that the H that fell into the V O site combines with the adsorbed H + and forms H 2 . Then, a Vo defect is formed at the (100) surface, and the H 2 molecule is adsorbed at 1.23 Å above the defective surface. Therefore, a defect state of V O can be clearly observed near the VBM from the PDOS in Figure 5b. It should be noted that the formation of the H 2 molecule represents an extreme situation, because we are examining the surface under vacuum conditions. In real experimental conditions, a KDP crystal's growth surface is exposed to the solvent environment, and the water molecules, K + ions, and growth groups in the solvent weaken the interaction between the surface H atom and the adsorbed H + ions, making it difficult to form a real H 2 molecule. However, our study shows that because the surface H atom and adsorbed H + ion tend to attract each other and form a stable chemical bond, the acidic environment makes the V O defect on the (100) surface more stable, thereby damaging the optical properties of KDP crystals.
For the adsorption of H + on the defective (101) surface, it can be seen from Figure 4e that the adsorbed H + also restores the V H on the (101) surface, resulting in a serious reduction in the H + adsorption energy, with respect to that on the perfect surface. This phenomenon is similar to that on the (100) surface, which can be verified by the resonance peaks at the same position and with similar intensity from the PDOSs of the H + adsorption on the (100)
Effects of Alkaline Environment on the Defective Surface of KDP Crystals
By adsorbing OH − on the (100) and (101) surfaces, we investigated the influence of an alkaline environment on the structures and electronic structures of the V H and V O defects on KDP crystals' surfaces. As shown in Figure 6, the adsorption energies of OH − on the (100) and (101) surfaces are −1.08 eV and −1.37 eV, respectively. From the moderate adsorption energy values for H + on the (100) and (101) surfaces, we can make a preliminary inference that OH − is chemically adsorbed on the (101) surface. From Figure 6a, we can see that the OH − binds with the exposed H atom on the surface and forms a water molecule. The generated H 2 O molecule is adsorbed on the (100) surface and connects to the surface through a hydrogen bond. This phenomenon can be verified by the calculated PDOS in Figure 7a, where the resonance peaks of O on the OH − and the surface H atom overlap at about −7.5 eV, indicating the chemical interaction between the OH − and the H atom on the (100) surface. This phenomenon is consistent with the result of Zhang [51]. It can be seen from Figure 6a that a serious charge transfer occurs between the H 2 O molecule and the (100) surface, which greatly reduces the adsorption energy to −3.02 eV [52]. Therefore, the alkaline environment causes the (100) surface to lose H atoms and then destroy the surface structure to form V H defects. For the adsorption of OH − on the (101) surface, since there is no exposed H atom in the outermost layer, the OH − adsorbs near the surface O atoms with a H-O bond length of 1.941 Å. The adsorption of OH − has a slight influence on the structure and energy of the (101) surface. By comparison, it can be seen that OH − has a greater impact on the (100) surface than the (101) surface. This is consistent with experiments that grew high-quality KDP crystals in a weakly acidic environment. When OH − adsorbs on the (100) surface with a VH defect, OHalso combines wi outmost H atom to form a H2O molecule and then adsorbs on the crystal surface (F 6b). Due to the attractive force from the VH, the distance between the H2O molecu the surface slightly increases by 0.13 Å. Since the adsorbed H2O molecule has little When OH − adsorbs on the (100) surface with a V H defect, OH − also combines with the outmost H atom to form a H 2 O molecule and then adsorbs on the crystal surface ( Figure 6b). Due to the attractive force from the V H , the distance between the H 2 O molecule and the surface slightly increases by 0.13 Å. Since the adsorbed H 2 O molecule has little influence on the structure and electron distribution of V H , the OH − adsorption has little influence on the electronic structure of the (100) surface with a V H defect (Figure 7b). In contrast, when OH − is adsorbed on the (100) surface with a V O defect, OH − is far away from the surface with a distance of 3.689 Å, indicating that there is no interaction between the OH − and the surface. This can also be verified by the PDOS in Figure 7a, which shows that the O atom in OH − and the H atom on the surface have no overlapped resonance peaks. Therefore, the OH − adsorption has little influence on the electronic structure of the (100) surface with a V O defect.
For the (101) surface with a V H defect, the adsorbed OH − could insert into the V H site, leading to an obvious charge transfer between the OH − and the adjacent O atom. This can be seen from the calculated charge distribution in Figure 6e and can be verified by the many overlapped resonance peaks over a wide energy range in Figure 7a. Due to the charge transfer, the surface K + contributes more electrons, and the contribution of their electronic states at the CBM is, thus, enhanced. The effect of OH − adsorption on the structure and electronic structure of the (101) surface with a V O defect is similar to that of the (100) surface. Both the calculated charge distribution in Figure 6f and the PDOS in Figure 7a verify that there is almost no charge transfer between OH − and the surface. Therefore, the adsorption energy (−0.78 eV) is as high as that on the (100) surface. On the other hand, the introduced defect states of the charged V O defects in the band gap of the (101) surface cannot be eliminated because of the very weak interaction between OH − and the surface. Therefore, the alkaline environment cannot improve the electronic structure of the defective surfaces, but it leads to more V H defects on the surface, which is not conducive to the improvement of the surface quality and properties.
Conclusions
In summary, we investigated the structure, stability, and electronic structure of hydrogen and oxygen vacancy defects (V H and V O ) on the (100) and (101) surfaces of KDP crystals using density functional theory. The effects of acidic and alkaline environments on the structure and properties of these defects were also studied. The results show that both V H and V O defects are more likely to be formed on the crystals' surface than in the bulk phase. The effect of a V H defect on the electronic structures of the (100) and (101) surfaces is almost negligible, while a V O defect on the different surfaces shows different sites and electronic structure characteristics compared with that in the bulk KDP. Different from the donor energy level introduced by a V O defect in the bulk KDP, a V O defect on the (100) surface does not introduce any defect level and can maintain the good optical properties of the crystal. However, a V O defect on the (101) surface tends to be formed in the deep layer and introduce deep defect states, resulting in extra optical absorption and a reduction in the optical properties of KDP crystals. In addition, an acidic environment is conducive to the repair of surface V H and can eliminate the defect states introduced by the surface V O defects, which is conducive for improving the quality of the crystals' surface and reducing the defect density. An alkaline environment leads to the formation of more V H defects, which should be avoided. Our study can provide theoretical guidance for the experimental optimization of the crystal-growth parameters needed to improve the quality of KDP crystals.
Author Contributions: Investigation, data curation, visualization, writing-original draft preparation, X.Z. (Xiaoji Zhao); investigation, writing-review and editing, funding acquisition, Y.L.; writing-review and editing, supervision, X.Z. (Xian Zhao). All authors have read and agreed to the published version of the manuscript. | 2022-12-21T16:29:17.254Z | 2022-12-01T00:00:00.000 | {
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14776809 | pes2o/s2orc | v3-fos-license | Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron–sulfur deficiency and pulmonary hypertension
Iron–sulfur (Fe-S) clusters are essential for mitochondrial metabolism, but their regulation in pulmonary hypertension (PH) remains enigmatic. We demonstrate that alterations of the miR-210-ISCU1/2 axis cause Fe-S deficiencies in vivo and promote PH. In pulmonary vascular cells and particularly endothelium, hypoxic induction of miR-210 and repression of the miR-210 targets ISCU1/2 down-regulated Fe-S levels. In mouse and human vascular and endothelial tissue affected by PH, miR-210 was elevated accompanied by decreased ISCU1/2 and Fe-S integrity. In mice, miR-210 repressed ISCU1/2 and promoted PH. Mice deficient in miR-210, via genetic/pharmacologic means or via an endothelial-specific manner, displayed increased ISCU1/2 and were resistant to Fe-S-dependent pathophenotypes and PH. Similar to hypoxia or miR-210 overexpression, ISCU1/2 knockdown also promoted PH. Finally, cardiopulmonary exercise testing of a woman with homozygous ISCU mutations revealed exercise-induced pulmonary vascular dysfunction. Thus, driven by acquired (hypoxia) or genetic causes, the miR-210-ISCU1/2 regulatory axis is a pathogenic lynchpin causing Fe-S deficiency and PH. These findings carry broad translational implications for defining the metabolic origins of PH and potentially other metabolic diseases sharing similar underpinnings.
Reviewers' concerns must be addressed including with additional experimental data where appropriate and that acceptance of the manuscript will entail a second round of review.
Please note that it is EMBO Molecular Medicine policy to allow a single round of revision only and that, therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript.
As you know, EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection. However, I do ask you to get in touch with us after three months if you have not completed your revision, to update us on the status. Please also contact us as soon as possible if similar work is published elsewhere.
I look forward to seeing a revised form of your manuscript as soon as possible.
***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System): The authors discovered that deficiency of ISCU1/2, a core protein involved in Fe-S cluster assembly, promoted pulmonary hypertension in mice. They use a myriad of elegant genetic and biochemical approaches as well as a study in a human subject with ISCU deficiency to validate their conclusions. I anticipate that this study will have high impact in the pulmonary hypertension, Fe-S cluster and mitochondrial metabolism field. It is also possible that this work could lead to potential therapeutics for pulmonary hypertension.
Referee #1 (Remarks):
This interesting manuscript from the Chen laboratory (White et al.) shows that chronic activation of miR-210-ISCU1/2 axis in mice promotes pulmonary hypertension (PH). The data presented are an extension of a previous study (Chen, et al.) showing that miR-210 repression of ISCU1/2 leads to Fe-S cluster deficiency, reduced mitochondrial respiration and increased glycolysis. In this current manuscript, their conclusion that alterations in miR-210-ISCU1/2 promote Fe-S deficiency and PH is validated by several elegant approaches using cultured cells, mice and humans. They show that overexpression of miR-210 in lungs of mice leads to increased endothelin 1, right ventricular systolic pressure (RVSP), vessel muscularization and vessel wall thickness, all features of PH, while depletion of miR-210 either genetically (miR-210 KO mice) or pharmacologically (anti-sense oligonucleotides) protected against PH or improved existing PH. They further showed that ISCU1/2 depletion alone in the pulmonary vasculature of mice promoted PH and that a patient with an ISCU mutation displayed exercise-induced pulmonary dysfunction, which was not previously recognized. Elegant biochemical approaches using a fluorescent-based detection assay and EPR spectroscopy was used to confirm a reduction in Fe-S clusters in endothelial cells and diseased pulmonary vasculature in mice and humans. The work is of high quality and the use of multiple approaches convincingly demonstrates the importance of miR-210-ISCU1/2 axis in promoting PD. I believe that these studies will be of particular interest to researchers in the PH, Fe-S cluster and mitochondrial metabolism fields and also to researchers interested in therapeutic interventions for PH.
The work has high potential impact and is suitable for publication in EMBO Molecular Medicine. There are a few issues noted below that could improve the manuscript. Some are minor editorial issues. One issue is clarification of the Discussion. Some sentences are not interpretable and should be rewritten (indicated below). Also, a more in depth, but concise discussion of mechanistic issues related to specific regulation of ISCU1/2 by miR-210 and metabolic dysfunction in diseases linked to mitochondrial Fe-S cluster biogenesis would be useful.
1. What is the significance of extracellular miR-210?
2. ISCU1 and ISCU2 -provide a brief description of the relevance of the two forms.
9. Discuss potential mechanisms through which dysregulated mitochondrial respiratory activity and the metabolic shift to glycolysis as a consequence of altered miR-210-ISCU1/2 activity affect vascular cell proliferation and remodeling.
10. Page 17 (Discussion), briefly state how altered iron transport causes PH in mice (reference 32). The statement "Such dysregulation of iron homeostasis is also consistent with our finding of increased oxidative stress during vascular Fe-S deficiency in vivo." However, Fig. S9 shows that total iron pulmonary iron content is not significantly changed in mice treated with hypoxia + SU54167 for 7 or 21 days vs day 0. Therefore, there are no studies in this manuscript showing dysregulated iron homeostasis. Clarify this statement. Diseases linked to mutations in Fe-S cluster assembly core proteins (e.g. frataxin, ISD11) are characterized by increased mitochondrial iron accumulation and alterations in iron homeostasis. While total iron content is not altered in diseased mice, it is possible that subcellular iron content is changed. Is mitochondrial iron increased under conditions where ISCU1/2 expression decreased? Also, Fig. S9 indicates that iron was measured by an "ELISA assay", but this assay is described in the Methods section as QuantiChromeTM Iron Assay Kit, which is not an ELISA.
11. The data convincingly show that ISUC1/2 deficiency promotes PH. A question arises whether deficiencies in other core Fe-S cluster biogenesis proteins (e.g. frataxin, ISD11, NFS1 or ferrodoxin 2) promote PH. Another related question is whether reduced mitochondrial respiration and increased glycolysis due to causes unrelated to Fe-S cluster biogenesis also promote PH. Briefly comment on these questions in the Discussion.
12. Last sentence of the discussion should be rewritten.
Referee #2 (Comments on Novelty/Model System):
This is a very interesting study by an active group of investigators and physician scientists who have made significant contributions to the field. The study provides some conceptually novel observations that will befit all the investigators in the field. Overall, this is an excellent manuscript; I only have some minor concerns and questions.
Referee #2 (Remarks):
This is a well conducted study demonstrating that miR-210-ISCU1/2 regulates the development of pulmonary hypertension. The authors previously reported that hypoxia up-regulated miR-210 leading to specific mitochondrial and metabolic alterations. In current study, they demonstrated that miR-210-ISCU1/2 axis was activated in both mouse models and human examples of PH. Over-expression of miR-210 repressed ISCU1/2 and promoted PH. Furthermore, deletions of the miR-210 and antisense inhibition of miR-210 protect against the development of PH. Overall, the experiments are well designed and executed, and the study bears conceptual novelty and is of great interest. The data are clearly presented in the manuscripts. I only have some minor concerns that may need the authors' attention to improve the manuscript.
Minor comments: 1. The study may need more and better discussion/description on the interaction of PA endothelial and smooth muscle cells according to the activated miR-210-ISCU1/2 axis. Induction of miR-210 was also found in whole lungs of mice with chronic hypoxia-induced PH by other investigators and up-regulation of miR-210 in PASMC may inhibit cell apoptosis during hypoxia by repressing E2F3 levels. Does ISCU1/2 expression change in PASMC with increased miR-210 levels in PH mice? Does activated miR-210-ISCU1/2 in endothelial cells contribute to pulmonary vascular remodeling or concentric vascular wall thickening (in addition to the enhanced PA smooth muscle cell proliferation)?
2. Several PH animal models have been used to study the pathogenesis of PH. Hyp-mouse and MCT-rat are most commonly used and well characterized. In this study, the authors found that miR-210-ISCU1/2 was activated in VHL-/-, Hyp+Su5416, IL-6 TG, and S. mansoni infection PH models. Are miR-210-ISCU1/2 activated in Hyp-mouse or MCT rat models?
3. Did you see significantly changes in RV/LV hypertrophy in the miR-210 knockout or antisense inhibited mice treated with Hyp+SU5416?
Response to Reviewers (White et al.)
We thank the Reviewers and the Editors for their positive remarks regarding this manuscript. We have taken the comments seriously, and we believe that all concerns have now been addressed comprehensively with new data and revised discussion. Consequently, we believe this manuscript is much improved, and we hope that it is now acceptable for publication.
What is the significance of extracellular miR-210?
We appreciate the opportunity to clarify this point. There is a growing literature showing that diseased tissues, which express increased levels of specific microRNAs, can release those microRNAs into the extracellular space and into the circulating plasma. These circulating microRNAs have been proposed as unique biomarkers for specific diseases. In addition, we and others have shown that extracellular miR-210 can be taken up by recipient tissue to induce true biological effects beyond the source tissue (Hale et al, 2014). Thus, the fact that PH patients carry increased levels of plasma-based miR-210 is significant as these data could be the basis for the future study of miR-210 as a biomarker for PH. These data also may suggest that diseased pulmonary vasculature may even use circulating miR-210 as a molecular messenger to communicate with other recipient tissues during disease progression. These points are now more clearly stated in the Results (p. 6) and Discussion (p. 22-23).
ISCU1 and ISCU2 -provide a brief description of the relevance of the two forms.
The description of these two isoforms of ISCU is now in the Introduction (p. 4).
3. It is intriguing that miR-210 targets, E2F3 and ephrinA3 are not altered in WT mice exposed to Hyp+Su5416. Is the expression of miR-210 targets COX10 and SDHD altered in PH-mice? Briefly comment in the Discussion on a potential mechanism for miR-210 selective regulation of ISCU1/2.
We thank the Reviewer for this suggestion. In addition to our original immunohistochemical data measuring E2F3 and Ephrin A3 expression in remodeled pulmonary vessels, we have quantified the expression of ISCU1/2, COX10, SDHD, E2F3, and Ephrin A3 (all reported direct targets of miR-210) by flow cytometry in PECAM-positive pulmonary vascular endothelial cells derived from mice suffering from hypoxic PH (chronic hypoxia x 3 weeks) versus normoxic control mice. As displayed in Fig. E6-7 and described in p. 6-7, while miR-210 was up-regulated and ISCU1/2 was downregulated, these other targets of miR-210 either remain unchanged or even increased in expression, thus emphasizing the unique importance of ISCU1/2 as a canonical miR-210 target gene in this context.
In the Discussion (p. 19), we now offer a more in-depth explanation for these findings. Similar to our previous findings for miR-21 and PH (Parikh et al, 2012), this is not an uncommon scenario in miRNA biology where, with increased specific miRNA expression (such as miR-210), only a subset of its validated gene targets may display a net down-regulation. We previously have postulated that specific stoichiometry of miRNA to target transcript levels can dictate the overall effectiveness of target gene down-regulation (either translational repression or mRNA degradation). Alternatively, indirect regulatory pathways may certainly exist (such as during hypoxic stress) that ultimately may be more powerful stimuli on a specific target gene than any direct miR-210 engagement of the target transcript itself. Such complex regulation may serve as homeostatic rheostats. Both possibilities may be active in this case, resulting in an apparent context-specific predilection for only certain targets, such as ISCU1/2, to be subject to net down-regulation.
Page 16, tricylic should be tricarboxylic acid cycle.
This term has been corrected (p. 17).
Discuss potential mechanisms through which dysregulated mitochondrial respiratory activity and the metabolic shift to glycolysis as a consequence of altered miR-210-ISCU1/2 activity affect vascular cell proliferation and remodeling.
We thank this Reviewer for this suggestion. We have included a discussion of putative mechanisms in the Discussion with references (p.20-21).
Page 17 (Discussion), briefly state how altered iron transport causes PH in mice (reference 32).
The statement "Such dysregulation of iron homeostasis is also consistent with our finding of increased oxidative stress during vascular Fe-S deficiency in vivo." However, Fig. S9 We thank this Reviewer for allowing us to address this confusing point. We have removed the statement "Such dysregulation of iron homeostasis is also consistent with our finding of increased oxidative stress during vascular Fe-S deficiency in vivo." In its place, we have included new data and discussion to clarify these points p. 23).
As this Reviewer suggests, although total iron levels are not altered, we believe that ISCU1/2 modulation can regulate subcellular iron content and is the subject of our ongoing investigation beyond the scope of this manuscript. First, mutations in ISCU1/2 have previously been linked to mitochondrial iron overload, and we now cite those studies in the text (Haller et al, 1991;Mochel et al, 2008). Second, to determine whether miR-210-specific activity mirrors that finding, we generated whole cell lysates and isolated mitochondria from human PAECs over-expressing this miRNA. While iron content in whole cell lysates were not different, mitochondrial iron levels were greater in miR-210-expressing PAECs as compared with control PAECs (now shown in Fig. E16). Furthermore, our unpublished work indicates that miR-210 likely regulates additional gene targets important in iron homeostasis and transport, consistent with prior reports from other groups (Qiao et al, 2013;Yoshioka et al, 2012). In the Discussion (p. 18-19, p. 23), we now clarify that, although our primary focus in this manuscript is the control of Fe-S biogenesis by miR-210, the complex actions of the miR-210-ISCU1/2 axis in iron handling in general may be relevant to PH pathogenesis and may be of particular significance for the known clinical association of iron deficiency and PH. We hope this Reviewer agrees, however, that any additional studies of complex iron transport here would distract from the focus on Fe-S biology in this already expansive manuscript.
Also, Fig. S9 indicates that iron was measured by an "ELISA assay", but this assay is described in the Methods section as QuantiChromeTM Iron Assay Kit, which is not an ELISA.
This sentence has been corrected (now Fig. E10 legend), and we apologize for the error.
11. The data convincingly show that ISUC1/2 deficiency promotes PH. A question arises whether deficiencies in other core Fe-S cluster biogenesis proteins (e.g. frataxin, ISD11, NFS1 or ferrodoxin 2) promote PH. Another related question is whether reduced mitochondrial respiration and increased glycolysis due to causes unrelated to Fe-S cluster biogenesis also promote PH. Briefly comment on these questions in the Discussion.
We thank this Reviewer for this suggestion. We have included these insightful points in the Discussion (p. 20-21, 23).
Last sentence of the discussion should be rewritten.
This sentence has been revised and shortened (p.24).
The study may need more and better discussion/description on the interaction of PA endothelial and smooth muscle cells according to the activated miR-210-ISCU1/2 axis. Induction of miR-210 was also found in whole lungs of mice with chronic hypoxia-induced PH by other investigators and up-regulation of miR-210 in PASMC may inhibit cell apoptosis during hypoxia by repressing E2F3 levels. Does ISCU1/2 expression change in PASMC with increased miR-210 levels in PH mice? Does activated miR-210-ISCU1/2 in endothelial cells contribute to pulmonary vascular remodeling or concentric vascular wall thickening (in addition to the enhanced PA smooth muscle cell proliferation)?
We thank the Reviewer for giving us a chance to clarify. We now have included data regarding cultured human PASMCs, whereby hypoxia indeed induces miR-210 while decreasing ISCU1/2 expression (Fig. E16, p. 21). Notably, hypoxic induction of miR-210 is more modest in PASMCs than in PAECs, thus reinforcing the idea that miR-210 has particularly pronounced effects on endothelial cells, as we have discussed in a prior report (Chan et al, 2009). In vivo, via serial sections of immunohistochemical staining, we found miR-210 up-regulation (Fig. 1c) and ISCU1/2 down-regulation (Fig. 1f) specifically in the a-smooth muscle actin-positive medial layer of diseased and remodeled pulmonary arterioles. Thus, these data demonstrate the activity of the miR-210-ISUC1/2 axis in PASMCs in PH mice. Notably, however, while miR-210-specific actions in PASMCs may certainly contribute to overall PH manifestations, we reiterate that our findings in this manuscript focused on the endothelial contribution to disease. Notably, we demonstrated a substantial amelioration of histologic pulmonary vascular remodeling with inhibition of miR-210 specifically in endothelial cells (Fig. 6hi), thus proving the notion that, indeed, miR-210 is crucial in endothelial cells for control of vascular wall thickening. These points are made in the Results (p. 13) and Discussion (p. 20-21). We thank this Reviewer for this suggestion. In the revised manuscript, we have added a study of chronically hypoxic mice (normobaric 10% O2 x 3 weeks) that suffer from PH versus normoxic littermate control mice. In this case, we isolated PECAM-positive pulmonary vascular endothelial cells for study. In these cells, miR-210 was up-regulated (as determined by RT-qPCR) while ISCU1/2 expression was down-regulated (as determined by flow cytometry), consistent with the other rodent models of hypoxia-relevant PH. These data are included in Fig. E6a-b and described on p. 6-7.
Did you see significantly changes in RV/LV hypertrophy in the miR-210 knockout or antisense inhibited mice treated with Hyp+SU5416?
We thank the Reviewer for the chance to clarify. As compared with miR-210-replete controls, miR-210-/-mice displayed a blunted increase of RV/LV+S (Fulton index) under disease versus baseline conditions (expressed as a ratio of RV/LV+S under Hyp+SU5416 vs. Norm+SU5416). These data (Fig. 3j, p. 11) indicated at least partial protection from the RV hypertrophic response and were consistent with the hemodynamic improvements of RVSP. Notably, LV ejection fraction, fractional shortening, and interventricular thickness were not altered in miR-210 knockout mice as compared with controls either with Norm+SU5416 or Hyp+SU5416 (as reported in Fig. E13).
2nd Editorial Decision 17 February 2015
Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees that were asked to re-assess it. As you will see the reviewers are now globally supportive and I am pleased to inform you that we will be able to accept your manuscript pending the following final minor amendments: 1) The quality of some images specifically of the blots in Fig.2 is not ideal. Specifically, there is excess contrasting that should be reduced to an acceptable level.
2) Please provide your manuscript in word (.doc) format.
3) We are now encouraging the publication of source data, particularly for electrophoretic gels and blots, with the aim of making primary data more accessible and transparent to the reader. Would you be willing to provide a PDF file per figure that contains the original, uncropped and unprocessed scans of all or at least the key gels used in the manuscript? The PDF files should be labeled with the appropriate figure/panel number, and should have molecular weight markers; further annotation may be useful but is not essential. The PDF files will be published online with the article as supplementary "Source Data" files. If you have any questions regarding this just contact me.
Please submit your revised manuscript as soon as possible so that we can proceed with formal acceptance.
***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System): The authors have responded to the critiques and have made the appropriate changes. The discussion has been revised and provides a more in depth analysis of the data.
Referee #1 (Remarks):
The manuscript is suitable for publication.
Referee #2 (Comments on Novelty/Model System):
This is a very interesting study from an excellent and productive research team. The manuscript provides some novel and important results that may yield more thoughts on developing novel therapeutic approaches for pulmonary vascular disease. The manuscript is well written with high quality of data. I think it is an important paper in the field of pulmonary vascular pathobiology and pulmonary hypertension, and the revised manuscript is now deemed ready for publication in the journal.
Referee #2 (Remarks): The authors have adequately and appropriately addressed all my concerns.
2nd Revision -authors' response 20 February 2015 We thank the Reviewers and the Editors for their positive remarks regarding this manuscript. We have now made corrections based on the final recommendations.
1) The quality of some images specifically of the blots in Fig.2 is not ideal. Specifically, there is excess contrasting that should be reduced to an acceptable level.
We have now revised the immunoblot images utilizing less contrast in Fig. 2. In a similar manner, we have also improved the quality of the Western blot images in Fig. E7.
2) Please provide your manuscript in word (.doc) format.
We have provided the main text in a .doc format.
3)
We are now encouraging the publication of source data, particularly for electrophoretic gels and blots, with the aim of making primary data more accessible and transparent to the reader. Would you be willing to provide a PDF file per figure that contains the original, uncropped and unprocessed scans of all or at least the key gels used in the manuscript? The PDF files should be labeled with the appropriate figure/panel number, and should have molecular weight markers; further annotation may be useful but is not essential. The PDF files will be published online with the article as supplementary "Source Data" files. If you have any questions regarding this just contact me.
We have provided source data for all of our immunoblots in Fig. 2, Fig. E4, Fig. E7, and Fig. E15. Please note that we routinely cut our membranes in order to blot for different sized proteins derived from the same gel. The immunoblots from these original membranes are shown in the source data. | 2016-03-14T22:51:50.573Z | 2015-03-01T00:00:00.000 | {
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187187624 | pes2o/s2orc | v3-fos-license | SOME ETHICAL POINTS IN ORTHOPAEDIC PRACTICE ( Part II )
Research and publications rthopaedic surgeon should always stay on the current by learning what is new and by keeping his standard high. This can only be achieved by reading, writing and by performing research projects, research is the life blood of our wonderful specialty. We are currently witnessing profound and far-reaching progress in the technology and biosciences with breakthroughs in human genetic, organ transplantation and the highly sophisticated means for internal fixation. We should go hand in hand with the recent advances, which should be harnessed and guided for the welfare and happiness of mankind and not to be left to go astray for decremental uses. At this juncture it suffices to state that biomedical area which has intimate relations to human life and well being, should receive careful analysis under the broad teaching of medicine and religion in order to ascertain their legality. The cost of each study should be weighed against the anticipated benefits to be derived from it. It is not only appropriate but also an obligation of clinicians who develop new interventions or diagnostic assessments to perform proper clinical research to objectively demonstrate the efficacy and safety of any new procedure. The World Medical Association developed specific guidelines for performing a research with several basic principles. O Among these are: • Health and well-being of human subject must be the first consideration. • Patients and families must be fully informed of all risks as well as benefits of research and so indicated in a proper informed consent. The protocol must be scientifically based. There must be no other way to obtain the information except through use of human subject. Results must be reported accurately. Statements should not just accept because they are said or written by an authority. We have to be aware of those authors who have a special financial interest in the product under review. Continuous education which is the heart and soul of orthopaedic surgery is very vital for the physician, the patient and the public. It is absolutely essential for our practice and economic well-being. The products of orthopaedic research are truly golden eggs and we should forever nourish and cherish these magnanimous geese. Good reasons for performing a study and for publishing its results are to provide information to improve patient care and to record data from a well known study in an accurate fashion, so that the present and future generations of readers will be able to build up upon solid research. Regrettably many
Introduction
odes of Medical ethics have been recognized for thousands of years as essential requirements to shape up the proper and dignified conduct of medical practitioners. From the Hippocrates Oath to the Geneva and Helsinki declarations, the basic Percepts of Medical Conduct have not changed 1 . Orthopaedic surgeons must understand the fundamental of orthopaedic surgery. Adequate knowledge is assumed to mean that the behavior associated with that knowledge would be carried out properly 2 . Educational need in all aspect of Orthopaedic is the primary concern, so we must commit ourselves to a lifetime of learning. We should stay warm and willing to answer questions, but appropriate responses to questions and scenarios do not necessarily translate into appropriate behaviors 2 . Vie have inherited a troubled profession and specialty confronted with problems that are not of our making 3 . The patients are in need for a surgeon who treat his moral and emotion in addition to treating joints, bones and incision. The career needs physician rather than a technician, healing takes more than a surgical skill. The heart muscle or valve may heal but scars from emotionally difficult recovery unsupported by the surgeon, may never heal 4 .
We have to have a good relationship with our patients who will create an environment in which cure take place for the patients, the family and the physician. We must also take on the responsibility of improving the quality of our own practice and monitoring the quality of care. It appears that organized orthopaedic surgery need to clarify its position not only in the minds of the rest medical profession but in the minds of the people who are, will be, or should be our patients 5 . We should always remind ourselves of the ethical reasoning which is of particular importance in orthopaedic career. The patient's best interest should stay with us as one of our fundamentals and the welfare of our patients will always be our higher consideration. We have to do everything in power to uphold the confidence that we have gained from our patients. A mutual trust is mandatory, without trust the success of the healing 4 process would be seriously diminished. There is and always has been one constant in medicine and orthopaedic, that constant is honest caring for our patients 6 . The young may be intelligent but can not be wise; wisdom is not inborn, it is acquired by experience and experience is often painful 7 . The required personality for orthopaedic career Because of the specification of orthopaedic career, special characters are always needed. Orthopaedic surgeon must be sincere and devoted believer in the honesty of his career, he should try his best to keep it in the best possible shape. He must always perfect himself in his specialty or subspecialty which he practices. He should save no effort to acquire all possible details and aspects of his profession and excels in them to the best of his abilities. His belief in his specialty should be manifested in behavior and attitude at the professional as well as the personal level. He should be truthful when lie speaks, writes or gives testimony, should be humble, modest, free from arrogance and self-glorification. Because of the prolonged time usually required for healing, he needs to be kind, patient, comforting and friendly. His word should be soothing, and his effort towards his patient complaints should be prompt and caring. Extending his knowledge and experience to his colleague and medical students is a duty. He should provide medical care to those who need it regardless of race, ethnicity, color or religion. He should have a specific recognized qualification and good practice under supervision for several years in a recognized center before he takes up the responsibility alone. He should work diligently to avoid emotional involvement with his patients. His piety must restrain him from inappropriate physical or emotional feelings during patient's care. Professional secrecy is mandatory and a seal should be placed on the confidentiality on all information lie acquires; any breach of these would be detrimental to the practice and the proper doctor-patient relationship. Finally we should not forget the notable names in Orthopaedic history, those who made the very rough terrain smooth for us to walk on without frequent falls. Their outstanding contributions to science and humanity should not be forgotten. Thanks would not be complete without acknowledgement to their original work. The public that we serve points an accusing finger and say, in effect you did not give us the kind care and help your fathers and your grandfathers gave their patients 5 . Isaac Newton said: "If I have seen further, it is by standing on the shoulders of giants".
Doctor-patient relationship
The fundamental act of professional medical care is the assumption of responsibilities for patient's welfare, an unwritten contract assured by few words, handshake, and eye contact, devoting mutual understanding or acknowledgement by the physician 8 . So we have to provide service even at a physical loss and despite physical discomfort or inconvenience. Whenever we interact with a patient, we must put aside our personal agenda, and interact with the patient for the benefit of the patient, it is not always easy to do this, but it is always necessary 9 . A strong doctor-patient relationship is essential to successful treatment; the doctor must gain his patients trust and respect, which can be achieved by telling the patient an accurate and truthful picture about his or her condition as well as realistic expectations about the result of management 6 . Unfortunately the study of Wenger and Lieberman showed that there was a poor understanding of proper ethical conduct with regard to physician-patient relationship 2 . Since orthopaedic care, frequently extended over a prolonged time and may involve surgery, casting or bracing, a longstanding bond is mandatory, with the patient and the family to establish the good ground work for years of treatment and follow up that line ahead, we have to be sympathetic with our patients, but it is much better to obtain a proper scientific goal than to allow one's motion to overrule sound medical judgment, so that a happy result can be achieved. The first golden moments when we meet the patient may make the difference between acceptance and total rejection. It is important to emphasize that we are not assuming God's role, but merely managing a medical condition, to the best of our scientific ability. The family and the patient must realize this fact and should also understand that the effort must be a team one, which requires their help and patience, if the outcome is this, and then the result will be much more successful for all concerned. The family and the patient should also realize that the nature does not have a specific time clock and can not be hurried, if they are not patient and cooperative at this time, this certainly indicates that they are totally unable to face the bad results of treatment. There is abundance of evidence that attitude affects healing, anxiety and depression have a negative effect on healing. A patient who feels supported and cared for by the physician and the family will be certainly in the best position for healing. The physician who is able to hold a patient's hand, to touch a shoulder, to hug a grateful or sad patient creates a healing environment 4 . When a patient has operation, the family is wounded, with even the simplest and shortest operation. A caring physician who is able to listen to a family's concern will help that family to heal and the family can help the patient to heal 4 . We have to be friend with our patient and their family, we have to prove for them that we are honest, but our relation should always stay at a professional level, probably the best insurance against malpractice is a good doctor-patient relationship. A good doctor-patient relationship can never be achieved with a pernicious practice. We have to look at the patient as a person and not just as an anatomical problem to be fixed. It was in the twelfth century when the philosopher and physician Maimonides prayed "May I never forget that the patient is a fellow creature in pain. May I never consider him merely a vessel of disease" 6 . Sir Francis Fraser's famous observation that the patient come not in search of treatment but of peace of mind 10 . If the patient chose us for sacred purpose we let that happen too 11 . The easy, fast and cheep ways should be considered in the first line of achieving diagnosis and precise treatment, sometimes orthopaedic surgeon gets exposed to multiple pressure from the insurance company, the patient himself or the creator of the accident in that case he should be frank and strong enough to tell the truth as it is irrespective of the severity of the pressure or the different convincing offers. The patient's psychological acumen should be studied carefully so that compensation neurosis can be discovered easily. Religious belief must be taken into serious consideration as in cadaveric transplant, blood transfusion, or even shaving certain parts of the body, there is always strange or special belief for each particular religion, which we need to learn from the patient himself. So that we do not hurt his beliefs, which may be reflected badly on the doctor-patient relationship. It is always necessary to avoid procrastination and to acquire the power of admitting failure and to tell the patient the truth as it is and then to advice him what to do next. A good doctor-patient relationship is mandatory in medical practice and it is of prime importance in orthopaedic career. So we should weld the links of this golden chain to immobilize it. Mankin 12 believes that we were put on earth to help others to recover from their illness, curing a few, making a better world for some, controlling suffering for others and for a few helping them to a reasonable and dignified end. That is for all of us the best of times and is a great feeling and wonderful way to spend our life.
The economic aspect of medical care
Orthopaedists should remember always to place the patient's interest ahead of their own interest including the financial aspect, unfortunately the financial conflicts are inherited in medicine, some of our previous colleagues weakened medicine by wanting too much and now it is reflected badly on our profession and threatens its future. Greed can certainly destroy one's source of wealth; medicine is certainly a profession rather than a business. One of the worst by product of insecurity is greed; greed like love of comfort is a kind of fear 13 . Economic well being is not the patient's pocket or the insurance company, rather as a physician's underlying source of wealth relates more to 'the professional ethic than the business ethic of medicine 3 . Particularly in orthopaedic practice there is always a good chance of collecting money in a short period of time, by simply changing the line of treatment, e.g. from gypsona cast to an internal fixation, but this is certainly against the professional ethic and surely it is a malpractice. Financial gain is indeed shameful when it comes from compromising the health care that we provide to our patient 13 . So we have to put aside the self concern by helping the sufferers. We should try to do our utmost best work not to disappoint any patient simply because he can not cover the expenses. The patient needs us not only for what we can do, but for what they feel we can do. The patient should be protected from the undue conflict of interest, the physician is obliged to provide or recommend treatment when he believes that the treatment will materially benefit his patient, it is unethical to knowingly provide unnecessary care or to be wasteful in providing the needed care because of a hidden financial or personal benefit. Patient should never be kept at an unfair disadvantage under any condition. The financial incentive should always be based on quality rather than the quantity of service. Sadly medicine has not been immune to the materialization and greed, there is increasing interest to change the art of medicine into business, the hospital and the patient care pass from control by physician to control by giants from insurance and industry, a situation that we should stand against. We have to prove to everybody on this earth by our humanistic and ethical handling of our patient that medicine is an honest profession and can never be considered as a business for making money at the expenses of the miserable sufferers. The fundamental objective of commerce in providing medical care is achieving an excess of revenue over cost 14 . We need to focus together on counteracting the pernicious behavior of the industries and institutions that threaten the golden quality of care which we need to keep on for our dear patients. Medical ethics prohibit solicitation and cheep advertising and any other form of commercialism. It has become painfully obvious that still some Orthopaedic surgeons are still performing operations basically for their own financial or scientific interest and what is more painful that operations sometimes performed to satisfy the producer company.
Professional respects towards our colleagues
It is of vital importance to keep a high standard of relation with our colleagues which is based on respect and harmonious cooperation in all aspects. Because there is various methods used to treat identical pathology, a lot of opinions may appear for solving one identical clinical problem, so respect and caution for our previous colleagues is indicated. Suggestions and advice should be considered. Some surgeons unfortunately are unwilling to face their own complications, but at the same time they are very eager to search for the complications of their colleagues. The author feels that this tendency should be reversed and it is much better to look seriously for our own complications rather than to be proud of our good results or to look with widely opened eyes for complications of our colleagues. We have to remember that alone we can do so little but together we can do so much. Unfortunately sometimes colleagues malignantly attack each other, certainly this is unethical attitude and we have to maintain a degree of professional respect towards one another under any condition. it is important not to criticize prior treatment or even the delay of it, since this only add to the sense of ill feeling, guilt and defeat already present. We have to stand shoulder to shoulder in support of our career at every turn. We should never recommend treatment for condition that needs no treatment just because if we don't do, someone else will, and the real motive behind it is usually financial.
Let's avoid making bitter remarks or demeaning comments about our colleagues in public or in front of patients. We have to criticize constructively when we believe that the best possible care has not been provided. We must emphasize advice and education rather than punishment as a remedy for a defeat in quality care 5 . All human being are bound to make mistakes and lucky is the one whose mistakes can be counted, we have to keep ourselves busy in analyzing and then solving our mistakes, rather than concentrating on the mistakes of our colleagues, but we have in the same time to learn from their mistakes rather than learning from our mistakes because we are not going to live to make all the possible mistakes in our career. Disaster may arise from an incompetent colleague practice, unfortunately this is not rare, and it is reflected badly on the miserable patient and our career reputation alike. It is certainly not an easy problem to solve, probably the best answer is to repair the defect created by the incompetent colleague, to keep the patient totally unaware of the guilt and then to proceed with education and advice to the competent colleague, in a direct or indirect way. We should make him fully convinced that he is not competent, but if he continues doing surgical crime, I think the final answer is to keep him out of our highly respected specialty through the court. It is a duty of the wise an experienced senior colleague to support and help the young and inexperienced by education and advice in a very wise manner. Perkins 7 said that: The Young may be intelligent but can not be wise, wisdom is not inborn, it is acquired by experience and experience is often painful. | 2019-06-13T13:23:57.578Z | 2010-12-28T00:00:00.000 | {
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210165497 | pes2o/s2orc | v3-fos-license | Fingolimod (FTY720) improves postoperative cognitive dysfunction in mice subjected to D-galactose-induced aging
Neurocognitive dysfunction is a common postoperative complication, especially in older adult patients. Fingolimod (FTY720) is a sphingosine-1-phosphate receptor modulator that has been found to be neuroprotective in several animal models of central nervous system disease. However, few reports have examined whether FTY720 could mitigate postoperative cognitive dysfunction. In this study, we investigated whether FTY720 could prevent postoperative neurocognitive impairment in mice subjected to D-galactose-induced aging. We induced an accelerated model of aging by administering an intraperitoneal injection of D-galactose. Subsequently, we performed a partial hepatolobectomy under sevoflurane anesthesia. FTY720 (1 mg/kg) was administered intraperitoneally 3 hours before and 24 hours after anesthesia and surgery. Our results indicated that anesthesia and surgery significantly impaired spatial memory in the Y-maze test 6 hours after surgery. We also found that problem solving ability and long-term memory in the puzzle box test on postoperative days 2-4 were significantly improved by FTY720 treatment. Immunohistochemical staining and western blot assay demonstrated that FTY720 significantly inhibited microglial activation in the hippocampal CA1 region of mice 6 hours and 3 days after anesthesia, and down-regulated the expression of synaptic-related proteins postsynaptic density protein 95 and GluR2 in the hippocampus. These results indicate that FTY720 improved postoperative neurocognitive dysfunction in mice subjected to D-galactose-induced aging. This study was approved by the Experimental Animal Ethics Committee of the Third Xiangya Hospital of Central South University of China (approval No. LLSC (LA) 2016-025) on September 27, 2016.
Introduction
Postoperative neurocognitive dysfunction (POCD) is a complication related to the central nervous system. It is more common in older vs. younger patients following surgery (Li et al., 2019;Song et al., 2019), and includes postoperative delirium, delayed neurocognitive recovery (up to 30 days after the procedure), and postoperative neurocognitive disorder (up to 12 months) (Evered et al., 2018). Although this postoperative complication usually persists for a period of days to weeks after surgery, it can last for decades in severe cases, and even develop into dementia and affect mortality. The incidence rate of POCD can range from 17% to 43%, depending on the patient characteristics (Evered et al., 2011). To date, the prevention and treatment of POCD are not optimal, and are still mainly focused on adjusting the risk factors, enhancing cognitive reserves, and administering symptomatic treatment (Feinkohl et al., 2017;Pappa et al., 2017;Kotekar et al., 2018;Liu et al., 2018). Among the many risk factors for neurocognitive dysfunction, including patient characteristics, surgery type, anesthesia, and environment (Kotekar et al., 2014;Kulason et al., 2017;Kubota et al., 2018), age was identified as an independent risk factor (Kotekar et al., 2014;Kubota et al., 2018). A prospective study by Kotekar et al. (2014) found that the incidence of POCD in individuals aged 60 years, 61-70 years, and 71-80 years was 12.5%, 20.5%, and 40.9%, respectively, suggesting that POCD is strongly associated with age. Therefore, as many societies face aging populations, new and effective methods for preventing POCD in older adult patients are urgently needed.
Fingolimod (FTY720) is a new immunosuppressant that is primarily used to treat relapsing-remitting multiple sclerosis (Kappos et al., 2010;Calabresi et al., 2014). FTY720 has been found to have neuroprotective and anti-inflammatory effects in several pre-clinical animal models of central nervous system diseases, such as Alzheimer's disease (Hemmati et al., 2013;Aytan et al., 2016), ischemic stroke (Kraft et al., 2013;Nazari et al., 2016), cerebral hemorrhage (Lu et al., 2014), hyperoxia (Serdar et al., 2016) and Parkinson's disease (Motyl et al., 2018). FTY720 has a carbon backbone, making it a highly lipophilic compound. Accordingly, it can easily traverse the blood-brain barrier where it becomes localized in the white matter in the central nervous system (Foster et al., 2007). FTY720 also modulates the sphingosine-1-phosphate receptor (S1PR), which is highly present in the central nervous system (Cruz et al., 2014;Martin et al., 2014;Healy et al., 2016). Cannon et al. (2012) found that FTY720 combined with S1PR1 and quickly but reversibly reduced P-glycoprotein activity. As P-glycoprotein activity facilitates the entry of small-molecule drugs into the central nervous system through the blood-brain barrier, it appears that FTY720 can influence the blood-brain barrier. Therefore, FTY720 may enter the central nervous system and exert a neuroprotective effect on S1PR in central nervous system cells, including microglia (Noda et al., 2013;Cipriani et al., 2015), astrocytes (Dusaban et al., 2017;Rothhammer et al., 2017), oligodendrocytes (Segura-Ulate et al., 2017), and neurons (Di et al., 2013). Many animal and clinical studies have confirmed that central nervous system cells and neuroinflammation play an indispensable role in the pathogenesis of POCD (Berger et al., 2019;Safavynia et al., 2019). Therefore, we hypothesized that FTY720 may be useful as a preventive drug that could alleviate postoperative cognitive impairment. Zhou et al. (2013) evaluated the effects of FTY720 on sevoflurane-induced neurotoxicity in rat pups. They found that 1 mg/kg of FTY720 before exposure to sevoflurane significantly inhibited neuronal apoptosis, and that this could be abrogated by VPC23019 (S1P antagonist). Unfortunately, few studies have examined the impact of post-surgical administration of FTY720 in aged animals. Thus, the neuroprotective mechanisms of FTY720 remain unknown.
Because animals injected with D-galactose exhibit a number of aging-related features, this technique has been extensively applied to the study of aging-related diseases (Ali et al., 2015;Sadigh-Eteghad et al., 2017;Shwe et al., 2018). Therefore, in the present study, we induced aging in mice via an intraperitoneal injection of D-galactose (1000 mg/kg). We then evaluated whether FTY720 could improve POCD in mice subjected to D-galactose-induced aging and explored the underlying mechanisms.
Animals
All experiments were performed in accordance with the National Institutes of Health guidelines. The protocol was approved by the Animal Ethics Committee of the Third Xiangya Hospital of Central South University, China on September 27, 2016 (approval No. LLSC (LA) 2016-025). We purchased 2-month-old male C57BL/6J mice that weighed 20-25 g from the Central South University of China [license No. SCXK (Xiang) 2016-0002]. All mice were housed for 7 days before the experiments in a controlled environment (22-25°C, 12-hour light/dark cycle). The mice were allowed free access to water and food.
Experimental groups
The C57BL/6J mice received 1000 mg/kg of D-galactose (Sigma-Aldrich Co., St. Louis, MO, USA) via intraperitoneal injection, once daily for 60 consecutive days. The same person conducted injections at the same time every day. This accelerated aging model was successfully established in a previous study in our laboratory (Duan et al., 2018). All mice subjected to D-galactose-induced aging were randomly divided into four groups (n = 12/group): (1) The C group received no anesthesia, surgery, or FTY720; (2) the C + FTY720 group received FTY720 (1 mg/kg, intraperitoneally), but no anesthesia or surgery; (3) the S + vehicle group received 2% sevoflurane anesthesia for 2 hours, underwent a partial hepatolobectomy, and received injections of vehicle (0.5 mL, intraperitoneally) 3 hours before and 24 hours after surgery, and (4) the S + FTY720 group received 2% sevoflurane anesthesia for 2 hours, underwent a partial hepatolobectomy, and received injections of FTY720 (1 mg/kg, intraperitoneally) 3 hours before and 24 hours after surgery.
Drug administration
FTY720 is sparingly soluble in aqueous buffers. As per the instructions that accompanied FTY720, we first dissolved FTY720 (Cayman Chemical Co., Ann Arbor, MI, USA) in ethanol with a concentration of 20 µg/µL for maximum sol-ubility in aqueous buffers, and then diluted it with saline. FTY720 was freshly prepared for each intervention. The dose of FTY720 (1 mg/kg, administered intraperitoneally) was selected according to previous studies in neonatal rats (Zhou et al., 2013;Serdar et al., 2016). FTY720 was administered 3 hours before and 24 hours after surgery. The vehicle solution was the same as the FTY720 solution except we did not add FTY720. The injection volume and age of the vehicle solution were the same as those for the FTY720 solution.
Anesthesia and partial hepatolobectomy
The anesthesia and surgery were conducted in accordance with previous studies (Tang et al., 2017;Duan et al., 2018). Mice were placed into an anesthesia induction chamber that was prefilled with 5% sevoflurane (Maruishi Pharmaceutical Co., Ltd., Osaka, Japan) mixed with high-flow oxygen (5 L/min). After the mice lost the righting reflex, they were given 3% sevoflurane and oxygen (80-85%) for 2 hours through a mask over the mouth and nose. Sevoflurane and oxygen concentrations were monitored using a multifunctional detector (Datex-Ohmeda, Helsinki, Finland). During anesthesia, the mice were subjected to a partial hepatolobectomy. After skin antisepsis and disinfection, a 2-cm incision was made just below the xiphoid process. Cutting the muscle layer exposed the abdominal cavity. The left lobe of the liver was then visualized and isolated, ligated, and carefully resected. Subsequently, the incision was sutured using 5-0 thread. Finally, to relieve pain caused by the incision, lidocaine cream (2.5% lidocaine and 2.5% prilocaine) was applied to the skin incisions immediately after the surgery and three times per day for the following 2 days. Anesthesia was induced for 2 hours, after which we removed the mask and placed the mice in a warm environment to recover naturally.
Behavioral tests
We used the Y-maze test and puzzle box test to assess whether the anesthesia and surgery impaired cognitive function and whether FTY720 could reverse this change. Spatial memory was evaluated via the Y-maze test 6 hours after surgery, and executive function was assessed via the puzzle box on postoperative days 2-4.
Y-maze test
We used the Y-maze test to evaluate spatial memory ability, as previously described (Peng et al., 2016). The Y-maze consisted of a start arm (always open), second arm (always open), and novel arm (blocked during the first trial, open during the second trial). The angle between each arm was 120 degrees. The Y-maze test consisted of two trials separated by a 2-hour interval. In the first trial (training), which was 10 minutes long, the mouse freely explored the two arms (start arm and second arm) of the maze with the novel arm blocked. After a 2-hour interval, we conducted a second trial (retention) in which the mouse was placed in the maze at the start arm and allowed to explore all three arms for 5 minutes. A logitech video camera was placed directly above the Y-maze such that it captured activity in all three arms. The number of entries and the time spent in each arm were recorded and analyzed. More entries in the novel arm (%) and a longer duration of time spent in the novel arm (%) indicated better spatial recognition memory. At the end of each experiment, a 75% ethanol solution was sprayed on the bottom and inner wall of the maze to remove odors, feces, and urine.
Puzzle box test
In accordance with a previous study (Zurek et al., 2016), we used the puzzle box test to assess executive function, including problem solving and cognitive flexibility. The puzzle box was composed of a light box (58.0 × 28.0 × 27.5 cm 3 ) and dark goal box (14.0 × 28.0 × 27.5 cm 3 ), which were connected by a door and also by a covered tunnel. The mouse was required to travel from the light box to the dark box (the goal box). The puzzle box test consisted of four trials: the 1 st trial (door open, tunnel open), 2 nd trial (door closed, tunnel open), 3 rd trial (door closed, bedding in tunnel), and 4 th trial (door closed, obstacle in tunnel). Thus, the task difficulty was gradually increased from the 1 st trial to the 4 th trial. The mouse was placed in the middle of the bright box and allowed to explore freely until it reached the dark box. If the mouse did not reach the dark box within 5 minutes, it was then gently guided to the dark box. The whole experiment was conducted over 3 days with three steps on each day. On the 1 st day (trials 1-2-2), we conducted trial 1 (door open, tunnel open), in which the mouse could reach the dark box through the open door or the tunnel. After a 2-minute interval, we conducted trial 2 (door closed, tunnel open), in which the mouse could get to the dark box only through the tunnel (problem-solving). After a 2-minute interval, we tested short-term memory for this task by repeating trial 2. On the 2 nd day (trials 2-3-3), we conducted trial 2 to assess long-term memory for the task. After a 2-minute interval, we conducted trial 3 (door closed, bedding in tunnel), in which the mouse was required to burrow into the clean bedding material to find the entrance to the tunnel and enter the dark box (more difficult problem-solving). After another 2-minute interval, we repeated trial 3 to assess short-term memory for this task. On the 3 rd day (trial 3), we repeated trial 3. We found that trial 4 was too difficult for the mouse, so we excluded this task. We recorded and analyzed the time required to solve each task. The mice were given 5 minutes for each trial, which was considered complete if the mouse had all four paws inside the goal box. Shorter task latency indicated better problem-solving, short-term memory, and long-term memory.
Immunohistochemistry
After anesthetizing the mice, they were perfused with 4% paraformaldehyde from the heart to the brain until the body was stiff. The brain tissue was then removed from the skull and fixed in 4% paraformaldehyde. After 2 days, the brain tissue was dehydrated with different concentrations of sucrose (15%, 30%, 30%, and 35%) at 4°C. When the tissue sank to the bottom of the container, the brains were embedded in OCT compound and stored at -80°C.
We used a pre-freezing sliding microtome (Leica CM1950, Wetzlar, Germany) to continuously cut brain tissue containing the hippocampus into 20-µm sections. These were washed three times using phosphate buffered saline and then exposed to 3% hydrogen peroxide for 10 minutes at room temperature.
Activated microglia cells were counted using a microscope (Nikon, Tokyo, Japan). The procedure for counting activated microglia cells in CA1 was consistent with that used in a previous study (Cerbai et al., 2012). First, we established criteria for determining whether microglia cells were "resting" or "reactive". According to the literature, resting microglia cells were defined as small, round, thin, and branched with protuberations around the cell body. Reactive microglia cells were defined as multipolar (bipolar, tripolar, or spindle/rod shaped), short branched, wound, or asymmetrically distributed, with larger cell bodies compared with resting cells.
western blot assay
We used a western blot assay to assess the expression of synaptophysin (SYN), postsynaptic density protein 95 (PSD95), and GluR2 in the hippocampus. After anesthesia and perfusion with 4% paraformaldehyde, the hippocampal tissues were extracted and stored in liquid nitrogen. The hippocampal samples were then treated with histone lysate, which contained NP40 lysate, a 1% phosphatase inhibitor, and a 1% protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA). The tissue samples were homogenized via a probe, centrifuged at 12,000 × g at 4°C, and the supernatant was obtained. Total protein concentration was assessed according to the instructions that accompanied the bicinchoninic acid protein assay kit (CWBio, Beijing, China). We added the marker and protein sample directly to sodium dodecylsulfate-polyacrylamide electrophoresis gel. We then conducted electrophoresis at a constant voltage of 80 V until the marker separated, at which point we increased the voltage to 110 V. After transferring the sample onto a polyvinylidene fluoride membrane (BioRad, Hercules, CA, USA), the membrane was placed in Tris-buffered saline Tween + 5% non-fat milk, and sealed at room temperature for 60 minutes. Subsequently, the membrane was incubated with rabbit anti-SYN (1:500; Proteintech, Chicago, IL, USA; a marker for presynaptic terminals, polyclonal antibody), rabbit anti-PSD95 (1:1000; Abcam, Cambridge, MA, USA; a marker for postsynaptic terminals, polyclonal antibody), rabbit anti-GluR2 (1:1000; Proteintech; a glutamate receptor subunit, polyclonal antibody), and rabbit anti-β-actin (1:2000; Proteintech; polyclonal antibody) at 4°C overnight. After three washes with Tris-buffered saline Tween, the membrane was treated with the diluted secondary antibody (1:8000; anti-rabbit, 926-32211, Li-CORr, polyclonal antibody) and slowly shaken at room temperature for 60 minutes. The immunoblot bands were detected using Odyssey-CLX infrared imaging visualizer (Li-CORr). The relative protein levels of SYN, PSD95, and Glu R2 compared with β-actin were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Statistical analysis
We used GraphPad Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA) for statistical analysis. All data are presented as the mean ± SEM. The results of the Y-maze test, microglia activated ratio, and western blots were analyzed using a one-way analysis of variance followed by Tukey's post hoc test. The results of the puzzle box test were analyzed using a two-way analysis of variance followed by Tukey's post hoc test. P-values < 0.05 were considered statistically significant.
Results
Perioperative FTY720 administration ameliorates postoperative cognitive impairment in mice subjected to D-galactose-induced aging In the Y-maze test, the percentage of entries in the novel arm and the percentage of time spent in the novel arm were statistically different among the groups (entries in novel arm% F (3, 43) = 3.470, P = 0.024; time spent in novel arm% F (3, 43) = 4.485, P = 0.008; Figure 1A and B). Tukey's post hoc analysis confirmed that the percentage of entries in the novel arm was significantly lower in the S + vehicle group compared with the C group, and that the percentage of time spent in the novel arm was significantly lower in the S + vehicle group compared with the C + FTY720 group (entries in novel arm%: S + vehicle vs. C, P < 0.05; time spent in novel arm%: S + vehicle vs. C + FTY720: P < 0.05; Figure 1A and B). There was no statistical difference between the S + FTY720 group and S + vehicle group (entries in novel arm%, P = 0.498; time spent in novel arm%, P = 0.788). These data suggest that anesthesia and surgery impaired spatial memory in mice subjected to D-galactose-induced aging.
Finally, we assessed changes in problem solving and memory, as revealed by performance in the Puzzle Box test. Problem solving ability was assessed according to the time taken to enter the dark box. The difficulty of the task was progressively increased. As shown in Figure 1, in terms of problem solving ability, the mice in all groups took longer to reach the dark box when they were required to burrow into the tunnel, compared with when the tunnel was open and when both the door and tunnel were open (two-way analysis of variance: group, F (3, 99) = 6.117, P < 0.001; interaction, F (6, 99) = 4.611, P < 0.001; Figure 1C). Tukey's multiple comparisons showed that the mice in the S + vehicle group spent significantly more time to solve the burrowing task than those in the other groups (S + vehicle vs. C, P = 0.0001; S + vehicle vs. C + FTY720, P < 0.0001; S + vehicle vs. S + FTY720, P < 0.0001; Figure 1C). We assessed short-term memory and long-term memory by retesting the mice 2 minutes and 24 hours, respectively, after first exposing the mice to the task. In terms of short-term memory, we found no significant difference among the four groups (two-way analysis of variance; group, F (3, 66) = 1.067, P = 0.369; interaction, F (3, 66) = 0.297, P = 0.828; Figure 1D). In terms of longterm memory, the four groups of mice exhibited a similar latency in the open tunnel task. However, the time required to complete the burrowing task was longer in the S + vehicle group compared with the other groups (two-way analysis of variance; group, F (3, 66) = 3.504, P < 0.05; interaction, F (3, 66) = 2.374, P = 0.078. Tukey's multiple comparisons: S + vehicle vs. C, P < 0.01; S + vehicle vs. C + FTY720, P < 0.01; S + vehicle vs. S + FTY720: P < 0.05; Figure 1E).
These data suggest that anesthesia and surgery impaired problem-solving ability and long-term memory and that while mice subjected to D-galactose-induced aging had more difficultly completing the tasks, FTY720 ameliorated these impairments.
Perioperative FTY720 administration inhibits microglial activation after anesthesia and surgery in mice subjected to D-galactose-induced aging
Microglial over-activation has been reported to drive neuroinflammation via positive feedback mechanisms, which is an important pathological mechanism of postoperative cognitive impairment (Hovens et al., 2014). Next, we examined the degree to which microglia were activated in the hippocampal CA1 area.
Our results showed that the percentage of activated microglia was statistically different among the four groups (6 hours: F (3, 12) = 17.24, P = 0.0001; 3 days: F (3, 12) = 12.82, P = 0.0005). Tukey's post hoc analysis showed that microglia in the S + vehicle group were significantly activated in the CA1 area compared with those in the C group and C + FTY720 group at 6 hours and 3 days after surgery (6 hours: S + vehicle vs. C, P < 0.001; S + vehicle vs. C + FTY720, P < 0.001; 3 days: S + vehicle vs. C, P < 0.01; S + vehicle vs. C + FTY720, P < 0.001; Figure 2). Compared with that in the S + vehicle group, the ratio of activated microglia in the S + FTY720 group was clearly lower at 6 hours and 3 days after surgery (6 hours: S + vehicle vs. S + FTY720, P < 0.05; 3 days: S + vehicle vs. S + FTY720, P < 0.05; Figure 2). These results suggest that FTY720 inhibited microglial activation after anesthesia and surgery in mice subjected to D-Gal-induced aging.
Perioperative FTY720 administration increases synaptic protein expression in mice subjected to D-Gal-induced aging mice
Synaptic plasticity-associated proteins including presynaptic protein SYN, PSD95, and AMPAR are involved in postoperative cognition (Jiang et al., 2018;Zhang et al., 2018;Zhou et al., 2018). Here, we measured the expression of SYN, PSD95, and the AMPAR subunit GluR2 in the hippocampus at 6 hours and 3 days after anesthesia and surgery (Figure 3). The expression of PSD95 and GluR2 proteins in the hippocampus was statistically different among the four groups at 6 hours and 3 days after surgery (PSD95: 6 hours: Tukey's post hoc tests showed that, compared with that in the C group, C + FTY720 group, and S + FTY720 group, hippocampal expression of PSD95 and GluR2 proteins in the S + vehicle group was significantly lower at 6 hours and 3 days after surgery (all P < 0.05; Figure 3). There were no significant differences in SYN expression among the four groups at 6 hours and 3 days after surgery (all P > 0.05; Figure 3). These results showed that anesthesia and surgery induced significant decreases in GluR2 and PSD95 expression in the hippocampus, which were then alleviated by perioperative FTY720 treatment.
Discussion
In this study, we sought to determine whether FTY720 could improve POCD in mice subjected to D-Gal-induced aging, and to examine the possible mechanisms underlying this phenomenon. Our results demonstrated that FTY720 treatment alleviated postoperative decreases in problem solving ability and long-term memory in the puzzle box test on postoperative days 2-4. Corresponding with this behavioral improvement, FTY720 also alleviated postoperative microglial activation and the loss of synaptic plasticity-associated proteins (PSD95, GluR2). These results suggest that FTY720 is neuroprotective and thus represents a potential preventive reagent for POCD.
POCD is a common central nervous system complication after surgery. Age, frailty, surgery-induced inflammation, anesthetic toxicity, sleep disturbances, and pain all are closely associated with the occurrence and development of POCD (Callaway et al., 2015;Hovens et al., 2016;Gu et al., 2018). To date, the main preventive strategies have addressed various risk factors involving the patient, surgery methods, and anesthesia. Pharmacological agents such as acetylcholine esterase inhibitors, COX-2 inhibitors, dexmedetomidine, and statins have been studied in terms of their potential to relieve the symptoms of POCD (Safavynia et al., 2019). However, the pathogenesis of POCD is not fully understood, and so an optimal solution for preventing and treating POCD has yet to be established. The high prevalence of POCD remains a clinical challenge. S1P receptors are widely expressed in cells in the brain, heart, liver, stomach, and retina, with the exception of leucocytes and lymphocytes (Subei et al., 2015;Chaudhry et al., 2017). FTY720 is a functional S1P receptor modulator that has been used to treat patients with multiple sclerosis, immune diseases, organ transplants, myasthenia gravis, and some metastatic cancers (Mandal et al., 2017;Huwiler et al., 2018). FTY720 has been reported to exert neuroprotective and anti-inflammatory effects in the central nervous system disease model, while neuroinflammation is one mechanism of POCD Safavynia et al., 2019). However, few studies have examined the use of FTY720 for treating POCD. In this study, we measured the preventive role of FTY720 in mice subjected to POCD-induced aging. We found that FTY720 treatment alleviated postoperative impairment in terms of problem solving ability and long-term memory in the puzzle box test on postoperative days 2-4. Corresponding with this behavioral improvement, FTY720 also alleviated postoperative microglia activation and synaptic plasticity-associated protein loss (PSD95, GluR2). These results are in accordance with previous studies (Zhou et al., 2013;Miguez et al., 2015;Nazari et al., 2016;Serdar et al., 2016;Xu et al., 2017). For example, Zhou et al. (2013) demonstrated that FTY720 attenuated sevoflurane-induced neurotoxicity in rat pups, whereas VPC23019 (S1P antagonist) inhibited the protective action of FTY720. These results suggest that FTY720 has a protective effect against sevoflurane-induced neurotoxicity in developing rats. In a neonatal model of hyperoxia, Serdar et al. (2016) found that FTY720 could reduce hyperoxia-induced cognitive dysfunction, microglial activation, and associated pro-inflammatory cytokine expression. Furthermore, to ascertain Latency to solve task in problem solving (s) Latency to solve task in short-term memory (s) Latency to solve task in long-term memory (s) S + FTY720 S + vehicle C + FTY720 C S + FTY720 S + vehicle C + FTY720 C S + FTY720 S + vehicle C + FTY720 C Underpass Burrowing Underpass Burrowing Baseline Underpass Burrowing C C + FTY720 S + vehicle S + FTY720 C C + FTY720 S + vehicle S + FTY720 Puzzle box test performance on postoperative days 2-4 (mean ± SEM, n = 12; two-way analysis of variance followed by Tukey's post hoc test). *P < 0.05. C group: Received no anesthesia, surgery, or FTY720; C + FTY720 group: received FTY720, but no anesthesia or surgery; S + vehicle group: received vehicle, anesthesia, and surgery; S + FTY720 group: received FTY720, anesthesia, and surgery.
C C + FTY720 S + vehicle S + FTY720 C C + FTY720 S + vehicle S + FTY720 C C + FTY720 S + vehicle S + FTY720 Representative images showing Iba-1 staining (yellow) in CA1 at 6 hours and 3 days after surgery. Typical Iba-1-stained activated microglia cells in CA1 had bigger cell bodies and shortened or twisted branches (indicated by black arrows). The percentage of activated microglia was statistically different among the four groups. Scale bar: 50 μm. Data are expressed as the mean ± SEM (n = 4 per time point per group; one-way analysis of variance followed by Tukey's post hoc tests). *P < 0.05. C group: Received no anesthesia, surgery, or FTY720; C + FTY720 group: received FTY720, but no anesthesia or surgery; S + vehicle group: received vehicle, anesthesia, and surgery; S + FTY720 group: received FTY720, anesthesia, and surgery.
Figure 3 Perioperative fingolimod (FTY720) administration selectively increases postoperative synaptic protein expressions in mice subjected to D-galactose-induced aging.
Representative images and analysis showing the expression levels of SYN, GluR2, and PSD95 in the hippocampus, as revealed by western blot assay, at 6 hours (A) and 3 days (B) after surgery. Data are expressed as the mean ± SEM (n = 3; one-way analysis of variance followed by Tukey's post hoc tests). *P < 0.05. C group: Received no anesthesia, surgery, or FTY720; C + FTY720 group: received FTY720, but no anesthesia or surgery; S + vehicle group: received vehicle, anesthesia, and surgery; S + FTY720 group: received FTY720, anesthesia, and surgery. GluR2: Glutamate receptor 2; PSD95: postsynaptic density protein 95; SYN: synaptophysin.
whether the memory-enhancing effect of FTY720 was correlated with synaptic plasticity in the hyperactivity disorder model, Miguez et al. (2015) examined the expression of PSD-95 in the hippocampus and found that FTY720 treatment prevented the expected decrease in PSD-95 protein levels, indicating a role for FTY720 in modulating structural synaptic plasticity. All of these previous studies demonstrate that FTY720 has a protective effect on the central nervous system and that it can rescue impaired cognitive function. S1P receptors are widely expressed in lymphocytes and neural cells. Furthermore, FTY720 is fat-soluble and can penetrate the blood-brain barrier. In future work, we hope to determine whether FTY720 improves the symptoms of POCD by limiting the infiltration of lymphocytes into the brain or by directly acting on neural cells. Few studies have examined the protective effect of FTY720 on POCD. In this exploratory study, we examined the effect of FTY720 on postoperative cognitive function in a mouse model of rapid aging. We found that FTY720 could improve postoperative cognitive impairment in mice subjected to D-galactose-induced aging, which is associated with inhibitory microglial activation and the loss of synaptic proteins (PSD95, GluR2). However, additional factors might be involved in the neuroprotective mechanisms of FTY720. S1PR is also expressed in astrocytes, oligodendrocytes, and neurons. Thus, further studies are needed to investigate other possible mechanisms of the effect of FTY720 on POCD, and particularly to determine which brain cells are mainly affected.
Acknowledgments:
We are very grateful to Jie Chen, MD, from the Third Xiangya Hospital, Central South University, China for the assistance of laboratory measurement. Author contributions: Study design and supervision, main investigator, behavioral test and immunohistochemistry, western blot assay, and statistical analysis, and paper writing: JZ, BX, CXL and YW. All authors have read and approved the current version of the manuscript.
Conflicts of interest:
The authors declare that there are no conflicts of interest associated with this manuscript. Financial support: This work was financially supported by the National Natural Science Foundation of China, No. 81500932 (to YW). The funding source had no role in study conception and design, data analysis or interpretation, paper writing or deciding to submit this paper for publication. Institutional review board statement: All experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of the Third Xiangya Hospital of Central South University of China (approval No. LLSC (LA) 2016-025) on September 27, 2016. The experimental procedure followed the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996 | 2020-01-14T14:08:06.503Z | 2020-01-09T00:00:00.000 | {
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203847564 | pes2o/s2orc | v3-fos-license | Transmission efficiency of Cotton leaf curl Multan virus by three cryptic species of Bemisia tabaci complex in cotton cultivars
Cotton leaf curl Multan virus (CLCuMuV) is a serious and economically important viral disease agent in cotton and ornamental plants like Hibiscus in many regions of the world, especially in South Asia. CLCuMuV is transmitted exclusively by Bemisia tabaci cryptic species complex. This virus was recently recorded in southern China, presumably an invasion from South Asia. This study was performed to estimate the efficiency of three species of the B. tabaci whitefly complex (tentatively named as MEAM1, MED and Asia II 7, respectively) to transmit CLCuMuV and Cotton leaf curl multan virus betasatelite (CLCuMuB). Transmission assays and real-time quantitative PCR were conducted using three cultivars of cotton, Gossypium hirsutum, including 112-2, Xinhai-21 and Zhongmian-40. The results indicated that Asia II 7 was able to transmit the virus to two of the cotton cultivars, i.e. 112-2 and Xinhai-21, with the highest transmission efficiencies of 40% and 30%, respectively, but was unable to transmit the virus to the cotton cultivar Zhongmian-40. MEAM1 and MED failed to transmit CLCuMuV and CLCuMuB to any of the three cotton cultivars. After the three cryptic species of whiteflies had fed on virus-infected cotton plants for 48 h, the relative quantity of CLCuMuV in Asia II 7 was detected to be significantly higher than that in both MEAM1 and MED (P < 0.05). These results indicate that among the three species of whiteflies Asia II 7 is likely the most efficient vector for CLCuMuV and CLCuMuB in Malvaceae crops in China. Our findings provide valuable information to the control of viral diseases caused by CLCuMuV in the field.
INTRODUCTION
The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), causes significant damage in different agricultural regions, not only by direct feeding as a pest but also by its ability to efficiently transmit several Geminiviruses, and is responsible for a number of epidemics worldwide (Gilbertson et al., 2015). The whitefly taxonomic group is believed to be a complex of over 39 cryptic species (Liu, Colvin & De Barro, 2012;Boykin & De Barro, 2014;Alemandri et al., 2015). Different cryptic whitefly species may transmit specific begomoviruses with various levels of efficiency, and each of the whitefly species may only be able to transmit certain viruses (Bedford et al., 1994;Polston, Barro & Boykin, 2014). Cotton leaf curl disease (CLCuD) is one of the most significant diseases in Pakistan and India (Briddon & Markham, 2000;Briddon et al., 2003;Sattar et al., 2013), the whitefly vectors of begomoviruses associated with the development of this disease. Field survey of whitefly vectors have been conducted in the area of CLCuD-epidemic field. Asia II 1 is the dominant cryptic species of the whiteflies in Pakistan (Ahmed et al., 2011;Ashfaq et al., 2014;Masood et al., 2017;Islam et al., 2018). Asia I, Asia II 5, Asia II 7 and Asia II 8 are identified in the field of cotton in India (Dinsdale et al., 2010;Rajagopalan et al., 2012;Ellango et al., 2015). In field surveys, it was observed that plants exhibiting symptoms of CLCuD in Guangdong Province, China, were also infested with the whitefly populations comprising of the MEAM1 and Asia II 7 cryptic species (Chen et al., 2016a). There are 11 viruses that have been identified to be associated with the leaf curl disease of cotton (Pan et al., 2018). However, whitefly transmission of viruses in cotton and Hibiscus was experimentally determined without stating the species of virus and whitefly used (Singh, 2001;Khan & Ahmad, 2005;Rajeshwari et al., 2005).
Cotton leaf curl Multan virus (CLCuMuV), associated with a betasatellite called Cotton leaf curl Multan betasatellite (CLCuMuB), was the major pathogen to cause CLCuD in Multan, Pakistan, in the 1990s (Briddon & Markham, 2000;Mansoor et al., 2003). The virus rapidly spread to all cotton-growing areas of Pakistan, Northwestern India (Sattar et al., 2013). Thus far, CLCuMuV has been found in Pakistan, India and the Philippines (Briddon et al., 2003;Srivastava et al., 2016;She et al., 2017). In China, CLCuMuV spread rapidly in the last thirteen years and became established in Southern China. It infects at least five malvaceous plant species, H. rosa-sinensis, H. esculentus, Malvaviscus arboreus, Gossypium hirsutum and H. cannabinus (Cai et al., 2010;Du et al., 2015). Experimental inoculation demonstrated that CLCuMuV and CLCuMuB infectious clones can cause typical symptoms of the disease on cotton and several other Malvaceae plants. The symptoms include upward or downward curling of the leaf margins, vein thickening and enation (Tang et al., 2015).
Much more attention has been paid to the whitefly species associated with the CLCuMuV in recent years. Previous studies in the laboratory showed that CLCuMuV and CLCuMuB was transmissible through indigenous Asia II 7 and Asia II 1 species and caused CLCuD symptoms in kenaf and okra plants (Chen et al., 2016b). Lately, tobacco plant was used as a virus source; and the indigenous Asia II 1 species was able to transmit disease-causing CLCuMuV to cotton and tobacco (Pan et al., 2018). Field surveys conducted on Malvaceae hosts showed that Asia II 7 was the dominant indigenous cryptic species of whitefly in South China. Hibiscus serves as an ornamental flower and is transported from southern China to flower markets in other provinces in China. Perhaps via international trade in ornamental plants, this plant serves as a continuous source of begomoviruses. Therefore, an evaluation of the potential of whitefly species to cotton plants will help prevent the spread of the virus.
MEAM1and MED are the most abundant whiteflies across china and in many other countries (De Barro et al., 2010). In this study, we conducted laboratory experiments to
MATERIALS & METHODS
Whitefly insects belonging to three cryptic species (MEAM1, MED and Asia II 7) were collected from three localities in Guangdong Province, China, and were maintained in the Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou (Table 1). Pure populations of three whitefly species were maintained on cotton, G. hirsutum cv. Zhongmian-838, which is a suitable host for all three species and a non host of CLCuMuV (Tang et al., 2017). The whiteflies rearing were conducted in insect proof cages (60 cm × 60 cm × 60 cm) in a climate-controlled room at 26 ± 1 • C, 16/8 h light/dark, and 50-70% relative humidity. The purity of each culture was tested on a monthly basis by a random sampling of 30 adults as described by Chen et al. (2016b) using the mitochondrial cytochrome oxidase I (mtCOI ) PCR technique.
Virus source for inoculation
An infectious clone of CLCuMuV was used to inoculate cotton plants (G. hirsutum cv. 112-2), CLCuMuV (accession number KP762786) and CLCuMuB (KP762787) was isolated from diseased cotton in the Guangdong province of China. The plants were agroinoculated with CLCuMuV and CLCuMuB infectious clone pGreenII049-1.6A and pGreenII049-2.0β at the 2-3-true-leaf stage (Wang et al., 2016). At 20 days post-inoculation, the success of virus infection of source cotton plants was assessed by the appearance of leaf curling and vein thickening. The diseased plants were maintained in an insect-free room at 26 ± 1 • C with a 16 h and 8 h of light and dark period respectively, for transmission tests.
Experimental plants
The cotton plants G. hirsutum cv. 112-2, Xinhai-21, and Zhongmian-40 were planted in plastic pots (15 × 15 cm). The plants were grown and maintained in separate cages following standard procedures for growing cotton crops. All plants were grown in an insect-free greenhouse with a controlled temperature of 24-28 • C and 16 h and 8 h of light and dark period respectively. Cotton plants were raised to the 3-5-true-leaf stage in pots for transmission tests.
Detection of CLCuMuV and CLCuMuB DNA in diseased cotton plants
Nucleic acids from cotton plants were extracted using the method of Kidwell & Osborn (1992) with slight modifications (primer sequences used for PCR analysis are shown in Table 2). PCR was conducted with 25 micro liter volumes containing 12.5 µL PCR Mixer (Takara Bio, Mountain View, CA, USA), two µL template DNA lysate (∼20 ng), one µM of each primer, 0.25 µM of each deoxynucleoside triphosphate, and 8.5 µL sterile water. Amplifications were performed in a DNA Engine PTC-200 Thermal Cycler (Bio-Rad, Hercules, CA, USA). The PCR was set to an amplification cycle consisting of an initial denaturing at 94 • C for 4 min, followed by 35 cycles of 94 • C for 45 s, 52 • C for 45 s and 52 • C for 60 s and a final extension of 72 • C for 10 min. PCR products of a volume of 10 micro liters were electrophoresed on a 1.0% agarosgel in a 1× TAE buffer and visualized by a Universal Hood II system (Bio-Rad, Hercules, CA, USA).
Detection of CLCuMuV and CLCuMuB DNA by qPCR in whitefly adults
Quantification of CLCuMuV and CLCuMuB in whitefly individuals was performed after various feeding durations on CLCuMuV and CLCuMuB -infected cotton plants. Five hundred nonviruliferous 7 to 8-day-old adults of each species were transferred to feed on CLCuMuV and CLCuMuB -infected cotton plants to acquire the virus. Two detection experiments were conducted on the whiteflies. In the first experiment, 10 female and 10 male adults were collected after a 48 h AAP. In the second experiment, 10 adults without the discrimination of sex were randomly collected from each plant at the end of a 6, 12, 24 and 48 h AAP, respectively. Each experiment was replicated three times. The collected whitefly samples were stored at −20 • C. The DNA of the 10 whitefly adults was extracted with an Easy Pure Genomic DNA Kit (Trans Gen Biotech, Beijing, China). The DNA samples were stored at −20 • C. The primer sequences used for real-time PCR analysis are shown in Table 2. The β-actin gene was used in each sample as a normalization gene to verify equal quantity of whitefly genomic DNA. Amplifications were performed using SYBR R Premix Ex Taq TM (Takara, Liaoning, China) and a CFX 96 TM real-time PCR system (Bio-Rad, Hercules, CA, USA) with SYBR green detection (Takara, Liaoning, China). The relative quantity of viral DNA was calculated using the 2− Ct method (Guo et al., 2015).
Statistical analyses
The relative quantity of CLCuMuV and CLCuMuB in ten adult whiteflies were used for the transmission tests and were compared by one-way analysis of variance (ANOVA) at a 0.05 significance level followed by least significant difference (LSD) tests. The virus variance between males and females was compared by Student's t -test. All data analyses were performed using SAS 8.0.
Efficiency of CLCuMuV and CLCuMuB transmission by whiteflies
Three species of whiteflies MEAM1, MED and Asia II 7 were compared for their efficiency of CLCuMuV and CLCuMuB transmission. Adult MEAM1 and MED whiteflies did not transmit CLCuMuV to 112-2, Xinhai-21 and Zhongmian-40, the transmission efficiency were 0 based on symptom inspection and PCR detection of viral DNA as well. Asia II 7 was able to transmit the virus, the average transmission efficiencies were 40.0%, 30.0% and 0 shown by both methods (Table 3). The symptoms appeared approximately 4-8 weeks after virus inoculation, included downward curling of leaf margins and vein swelling (Tang et al., 2015).
The accumulation of CLCuMuV in whitefly female and male adults
The difference in the quantity of CLCuMuV acquired between male and female adults that were fed CLCuMuV and CLCuMuB-infected cotton plants for 48 h was determined by qPCR. The quantity of virus in adult female or male insects of MEAM1, MED and Asia II 7 was not significantly different (t = 0.02, P = 0.9882; t = −1.04, P = 0.4006; t = 0.17, P = 0.8764), but the relative quantity of viral particles in Asia II 7 were significantly higher as compared to the rest of the two tested cryptic species (Fig. 1).This result indicated that the different sex of whitefly adults had no significant difference in ability to acquire the virus from CLCuMuV and CLCuMuB-infected source plants.
Acquisition of CLCuMuV and CLCuMuB by whiteflies
A comparison of the relative quantity of virus in the three species of whitefly fed on CLCuMuV and CLCuMuB-infected cotton plants for a 6, 12, 24 and 48 h AAP was conducted. The results showed that all three species ingested both CLCuMuV and CLCuMuB. The relative quantity of CLCuMuV (Fig. 2) and CLCuMuB (Fig. 3) in the whiteflies increased significantly in each treatment i.e., 6 h, 12 h and 24 h of AAP (F = 1.45, P = 0.3061; F = 4.96, P = 0.0536; F = 0.77, P = 0.5050); however, the virus quantity after 48 h AAP in Asia II 7 was much higher than that in MEAM1 and MED (F = 4.09, P = 0.0212).
DISCUSSION
We have showed that all MEAM1, MED and Asia II 7 of B. tabaci tested in this study were able to acquire CLCuMuV and CLCuMuB, but only Asia II 7 was able to transmit CLCuMuV and CLCuMuB. The lower transmission capacity of MEAM1and MED is consistent with the results of Pan et al. (2018). It has been recognized that there are differences in transmission rates of begomoviruses owing to various whiteflies (Bedford et al., 1994;Jiu, Zhou & Liu, 2006;Wei et al., 2014). Evidence suggests that the distribution of CLCuD in South Asia is governed predominantly by a specific vector population, not by a host plant or geographic origin (Seal, Van Den Bosch & Jeger, 2006). Asia II 1 provided had the higher capacity than other species in the transmission of CLCuMuV among host plants of Malvaceae in China (Chen et al., 2016b;Pan et al., 2018). Even though Asia II 7 is not the predominant species in Pakistan and India (Islam et al., 2018;Ellango et al., 2015), while the cryptic species (Asia II 7) is found in China to be involved in CLCuMuV transmission.
Based on Bayesian analysis of mtCO I haplotypes, Asia II 7 and Asia II 1 belong to the same Asia II genetic group (De Barro et al., 2010). It's reasoned possibly that they have similarity in genomics and then share the capacity to transmit beogomovirus. Begomoviruses are transmitted by whiteflies in a persistent and circulative way. Viruses acquisition, retention and transmission in host plants belonging to different families are associated with the successful spread of the viruses. The whitefly-mediated transmission of cotton leaf curl virus to cotton plants showed that the least amount of time required to acquire the virus by whiteflies was 15 min to 4 h, while the least amount of time required to inoculate the plant was from 5 min to 1 h (Mann & Singh, 2004). Based on qPCR analysis, all three B. tabaci-complex species acquired CLCuMuV and CLCuMuB after a 6 h, 12 h, 24 h and 48 h AAP. Whereas transmission experiments revealed that Asia II 7 could transmit the virus efficiently, but MEAM1 and MED were unable to transmit CLCuMuV and CLCuMuB, which might be due to a transmission barrier present in non-vector whiteflies. According to a previous report that employed immunofluorescence assays, CLCuMuV and CLCuMuB had a poorer capacity to cross the midguts of MEAM1 and MED than the midgut of Asia II 1 (Pan et al., 2018). Similarly, a study by Ohnishi et al. (2009) suggested that the greenhouse whitefly Trialeurodes vaporariorum could ingest and maintain Tomato yellow leaf curl virus after acquisition feeding on an infected plant; however, T. vaporariorum is a non-vector because of the presence of a selective transmission barrier at the luminal membrane surface of the epithelial cells of the midgut. The physiological differences in CLCuMuV and CLCuMuB transmission between Asia II 7 and the two invasive species need further study. Complete genomic information of Asia II 1 is studied (Hussain et al., 2019) and a similar study on Asia II 7 would be helpful to interpret the involvement of molecular factors in transmission capability of the newly identified vector in China.
In the present study, the transmission efficiency varied with the cotton variety. Asia II 7 was able to transmit CLCuMuV and CLCuMuB to the cotton varieties 112-2 and Xinhai-21, but not to Zhongmian-40. Furthermore, Asia II 7 transmitted the virus to variety 112-2 more efficiently than they did to Xinhai-21. Differences in transmission efficiency were assumed to be caused by cotton cultivars with different levels of resistance.
Twenty-two cotton varieties were screened for resistance to CLCuD by graft inoculation in the laboratory and whitefly-vectored transmission assays in the field cages in Pakistan (Rahman et al., 2005), 14 varieties were susceptible and 8 resistant. They found three genes being involved in G. hirsutum resistance to CLCuD, two for resistance and a suppressor of resistance. Akhtar et al. (2008); Akhtar et al. (2010) tested cotton resistance to CLCuMuV and cotton leaf curl Burewala virus (CLCuBV) by graft inoculation, the results showed variation in the resistance of cotton varieties. Similar results were obtained when these cotton species were tested using whitefly-vectored transmission. Experimental inoculation of CLCuMuV and CLCuMuB infectious clones in 46 Chinese cotton varieties indicated that 4 varieties were highly susceptible to CLCuMuV (Tang et al., 2017). In our study, the three varieties come from different cotton cultivation research institute in China, which were differences in genetic backgrounds, so they were different in resistance to CLCuMuV. Host-plant resistance in cotton cultivars is the best long-term strategy to protect the crop against CLCuD (Jones, 2001). With the rapid spread and emergence of new begomoviruses throughout cotton growing areas of the world, further detailed research is required to be done to identify resistance in some cotton varieties against both vector and virus.
CONCLUSIONS
Our results revealed that the native Asia II 7 was able to transmit CLCuMuV and CLCuMuB, the transmission ability of Asia II 7 varied with the cotton cultivars. We can quarantine and control whitefly vector and identify resistance in Gossypium species for slowing down the spread of CLCuMuV in China. | 2019-10-05T06:47:25.794Z | 2019-10-01T00:00:00.000 | {
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32778528 | pes2o/s2orc | v3-fos-license | Molecular and Biochemical Analysis of the Plastidic ADP-glucose Transporter (ZmBT1) from Zea mays*
Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous synthesis of ZmBT1 in Escherichia coli cells leads to the functional integration of ZmBT1 into the bacterial cytoplasmic membrane. ZmBT1 transports ADP-Glc in counterexchange with ADP with apparent affinities of about 850 and 465 μm, respectively. Recently, a complete ferredoxin/thioredoxin system has been identified in cereal amyloplasts and BT1 has been proposed as a potential Trx target protein (Balmer, Y., Vensel, W. H., Cai, N., Manieri, W., Schurmann, P., Hurkman, W. J., and Buchanan, B. B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 2988–2993). Interestingly, we revealed that the transport activity of ZmBT1 is reversibly regulated by redox reagents such as diamide and dithiothreitol. The expression of ZmBT1 is restricted to endosperm tissues during starch synthesis, whereas a recently identified BT1 maize homologue, the ZmBT1–2, exhibits a ubiquitous expression pattern in hetero- and autotrophic tissues indicating different physiological roles for both maize BT1 isoforms. BT1 homologues are present in both mono- and dicotyledonous plants. Phylogenetic analyses classify the BT1 family into two phylogenetically and biochemically distinct groups. The first group comprises BT1 orthologues restricted to cereals where they mediate the ADP-Glc transport into cereal endosperm storage plastids during starch synthesis. The second group occurs in mono- and dicotyledonous plants and is most probably involved in the export of adenine nucleotides synthesized inside plastids.
Cereal crops accumulate starch in seed endosperm plastids as main energy reserve. The pathway of starch synthesis in cereal endosperms is unique and requires enzyme isoforms that are not present in other tissues or non-cereal plants. The ability of heterotrophic plastids to utilize cytosolic precursors to support their biosynthetic and catabolic pathways depends on the presence of specific transporters in the plastid envelope. In cereal endosperms, the ADP-glucose pyrophosphorylase (AGPase), 2 which catalyzes the first committed and rate-limiting step in starch biosynthesis, is mainly localized in the cytosol with a total AGPase activity of about 85-95% (2). Therefore, ADP-glucose (ADP-Glc) is synthesized in the cytosol of cereal endosperms as the main precursor for starch synthesis and has to be subsequently imported into the storage plastids.
Several maize (Zea mays L.) endosperm mutants affected in starch quality or quantity were used to elucidate critical steps in amyloplast starch synthesis. The Waxy gene, which encodes a starch-granule-bound starch synthase involved in amylose synthesis (3) and the Shrunken-2 and Brittle-2 genes, which encode subunits of the AGPase (4 -6), were shown to be important for starch synthesis in maize. The Brittle-1 (BT1) maize mutant was identified in 1926 (7,8) and corresponding endosperm is severely reduced in starch content, which results in kernels with a collapsed angular appearance at maturity. The BT1 protein from Z. mays (ZmBT1) belongs to the mitochondrial carrier family and is located in the amyloplast envelope membrane (9,10). The absence of ZmBT1 correlates with a 12-fold higher level of ADP-Glc in the cytosol of BT1 mutant endosperm than in wild-type endosperm (11) and BT1 mutant kernels accumulate about 80% less starch than wild-type kernels (12). The incorporation of externally applied ADP-Glc into starch in amyloplasts isolated from BT1 mutant endosperm was reduced to about 25% compared with wild-type amyloplasts (13). These results indicate that ZmBT1 is involved in the transport of ADP-Glc into maize endosperm plastids (14), but up to now, no direct ADP-Glc transport mediated by ZmBT1 has been shown.
Recently, a BT1 homologue from a non-cereal plant, namely potato (StBT1 from Solanum tuberosum), was functionally characterized as a plastidic adenine nucleotide uniporter expressed in sink and source tissues (15). The physiological role of StBT1 is most likely the export of purine nucleotides that are exclusively de novo synthesized in plastids of autotrophic and heterotrophic tissues (15,16). These novel findings further urged for a comprehensive analysis of the biochemical transport features of the maize homologue ZmBT1. We present here direct transport properties of the heterologously expressed ZmBT1. For this we exploited an Escherichia coli expression system that was successfully used to analyze plastidic or mitochondrial carrier proteins (15,17,18). We also reviewed the endosperm-specific expression of ZmBT1 and proved the occurrence and expression pattern of further ZmBT1 homologues in maize.
EXPERIMENTAL PROCEDURES
Cultivation of Plants-Maize plants (Z. mays L.) were grown in a greenhouse at 22-26°C and watered once a day. The ambient light period was extended to 16 h/day with Philips Sont-Agro lights (200 mol quanta m Ϫ2 s Ϫ1 ). For RNA and genomic DNA isolation, maize tissues were collected and immediately frozen in liquid nitrogen until use.
Southern Blot Analyses-Genomic DNA was isolated from 4 g of frozen leaf tissue as described by Dellaporta et al. (19). About 10 g of genomic DNA were digested with high concentrated restriction enzymes (BamHI, HindIII, XbaI, and XhoI). DNA fragments were separated in 0.8% agarose gels, denatured, and neutralized according to standard procedures (20). After transfer to nylon membranes (Hybond-N membranes, Amersham Biosciences), DNA fragments were fixed to the nylon filters by optimal UV cross-link (254 nm, 120 millijoule cm Ϫ2 ) in a Spectrolinker. The membranes were prehybridized for 60 min at 68°C in 6ϫ SSPE buffer (20ϫ SSPE contains 3.6 M NaCI, 0.2 M sodium phosphate, 0.02 M EDTA, pH 7.7) supplemented with 1% SDS, 100 g/ml herring sperm DNA, and 5ϫ Denhardt's reagent. Hybridization was performed for 16 h at 68°C in the same solution plus the complete ZmBT1-cDNA fragment labeled by the random primer method with [␣-32 P]dCTP using the Ready-To-Go prime kit (Amersham Biosciences). Afterward, the membranes were washed once at 37°C for 15 min in 7ϫ SSPE, 0.5% SDS and twice at 60°C for 15 min in 0.1ϫ SSPE, 1% SDS. Blots were visualized by a Cyclon PhosphorImager (Packard).
Plasmid Construct for Heterologous Expression of ZmBT1 in E. coli-DNA manipulations were performed essentially as described by Sambrook et al. (20). The expression plasmid (pET 16b, Novagen, Heidelberg, Germany) encoding the recombinant ZmBT1 protein with an additional N-terminal tag of 10 histidine residues was constructed as follows: the cDNA coding the entire ZmBT1 was generated by PCR from first strand cDNA of maize endosperm tissue using Pfu DNA polymerase (Invitrogen). A sense primer including an NdeI restriction site and an antisense primer were used for the PCR (sense: 5Ј-GACT-GAcatATGGCGGCGACAATG-3Ј; the lowercase letters indicate the introduced base exchange to create an NdeI restriction site; antisense: 5Ј-CTACCTTCTTGGCCAAGAACTTTG-3Ј). The obtained PCR product was purified (Nucleospin Extract II, Macherey & Nagel, Düren, Germany), subcloned into the EcoRV restriction site of the plasmid pBSK (Stratagene), and checked by sequencing on both strands by chain termination reaction (MWG-Biotech, Ebersberg, Germany). For the construction of the E. coli expression plasmid (encoding His 10 -ZmBT1), the NdeI/BamHI DNA insert of the pBSK-plasmid was introduced in-frame into the corresponding restriction sites of the isopropyl -D-thiogalactopyranoside (IPTG)-inducible T7-RNA polymerase bacterial expression vector pET16b (Novagen, Heidelberg, Germany). Transformations of E. coli were carried out according to standard protocols. The nucleotide sequence of the ZmBT1 reported in this paper is available at the EMBL data base (www.ebi.ac.uk/embl/) under the accession number BT016796.
Heterologous Expression of ZmBT1 in E. coli-The E. coli strain Rosetta(DE3) was used for heterologous expression. The cDNA sequence encoding ZmBT1 under control of the T7-promoter was transcribed after IPTG induction of the T7-RNA polymerase (21). E. coli cells harboring the ZmBT1 expression plasmid (or control expression plasmid pET16b) were grown at 37°C in TB Amp/Clm medium (TB: 2.5 g/liter KH 2 PO 4 , 12.5 g/liter K 2 HPO 4 , 12 g/liter peptone, 24 g/liter yeast extract, 0.4% glycerin, pH 7.0). An optical density (A 600 ) of 0.5-0.6 was required for the initiation of T7-RNA polymerase expression by addition of IPTG (final concentration, 1 mM). Cells were grown for 1 h after induction and collected by centrifugation for 5 min at 4,000 ϫ g (room temperature, Sorvall RC5B centrifuge, rotor type SS34; Sorvall-Du Pont, Dreieich, Germany). The pellet was resuspended to an A 600 of 6 using potassium phosphate buffer (50 mM, pH 7.0) (22) and promptly used for uptake experiments.
Membrane integration of the recombinant full-length ZmBT1 in E. coli was confirmed by enrichment of the histidinetagged protein and Western blot analysis using a ZmBT1 specific antibody (10). E. coli cells (20 ml) harboring the ZmBT1 expression plasmid (or the pET16b control plasmid) were collected 1 h after IPTG induction (controls without IPTG) and transferred to liquid nitrogen to destroy cell intactness. After resuspension in a medium consisting of 10 mM Tris/HCl (pH 7.5), 1 mM EDTA, 0.1 mM Pefabloc, and 15% (v/v) glycerol, cells were further disrupted by ultrasonication (250 W, 3 ϫ 30 s, 4°C) and the suspension was centrifuged (10 min, 15,800 ϫ g, 4°C) to remove unbroken cells and inclusion bodies. Membranes extracted in the supernatant were sedimented for 45 min at 100,000 ϫ g (TFT 80 rotor, Kontron Instruments, Munich, Germany), resuspended in binding buffer A consisting of 5 mM imidazole, 300 mM NaCl, 50 mM Na 2 HPO 4 (pH 8.0, HCl), and 0.3% Triton X-100. After incubation on ice for 30 min, the not solubilized proteins were sedimented for 45 min at 100,000 ϫ g. The solubilized histidine-tagged ZmBT1 in the supernatant was purified by nickel-chelating chromatography according to the supplier's instructions (Qiagen). Histidinetagged protein was eluted with 500 M imidazole and desalted by Sephadex G50 centrifugation. For SDS-PAGE, a protein aliquot was added to concentrated SDS-PAGE sample buffer medium and incubated for 30 min at room temperature. Finally, the preparation was applied to a polyacrylamide gel (3% stacking gel, 12% running gel) for electrophoresis in the presence of 0.1% SDS. Proteins were transferred to a nylon Hybond P membrane (Amersham Biosciences) in transfer buffer (39 mM glycine, 48 mM Tris base, 20% methanol, pH 8.3) for 1 h at 300 V. Western blot analysis was preformed utilizing a ZmBT1-specific antibody as primary antibody that was raised against 56 amino acids from the C terminus of the ZmBT1. An anti-rabbit IgG-alkaline phosphatase conjugate was used as secondary antibody. The immunoreactive bands were visualized by 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium substrate.
Transport Assays with E. coli-IPTG-induced E. coli cells (100 l) harboring the ZmBT1 expression plasmid (or the given controls) were added to 100 l of potassium phosphate buffer (50 mM, pH 7.0) containing radioactively labeled ADP or ADP-Glc. [␣-32 P]ADP was enzymatically synthesized from [␣-32 P]ATP (PerkinElmer Life Sciences) as given in Tjaden et al. (17) and used at specific activities between 50 and 500 Ci/ mol. [ 14 C]ADP-Glc was used at specific activities between 20 and 250 Ci/mol. Uptake of nucleotides was carried out at 30°C in an Eppendorf reaction vessel incubator and terminated after the indicated time periods by transferring the cells to a 0.45-m membrane filter (mixed cellulose ester, 25 mm diameter; Schleicher & Schuell, Dassel, Germany) under vacuum (23). Cells were further washed to remove unimported radioactivity by addition of 3 ϫ 4-ml potassium phosphate buffer (50 mM, pH 7.0). The filter was subsequently transferred into a 20-ml scintillation vessel and filled with either 10 ml of water or 10 ml of scintillation mixture (Quicksafe A; Zinsser Analytic, Frankfurt/Main, Germany). Radioactivity in the samples was quantified in a Canberra-Packard Tricarb 2500 scintillation counter (Canberra-Packard, Frankfurt/Main, Germany). For efflux assays, E. coli cells were incubated with potassium phosphate buffer (50 mM, pH 7.0) containing 1 M [ 14 C]ADP-Glc as a transport substrate. After the indicated time periods, the uptake medium was diluted with several unlabeled nucleotides to a final concentration of 1 mM and the efflux of [ 14 C]ADP-Glc was monitored at the given time points. Efflux was measured by membrane filtration as described above. To analyze the degree of metabolic conversion of imported [ 14 C]ADP-Glc, we carried out a thin layer chromatography according to the method of Mangold (24).
Sequence Analysis-Multiple alignments of amino acid sequences from known BT1 homologues available at the EMBL data base (www.ebi.ac.uk/embl/) were obtained using Clustal X (25). The phylogenetic tree was created with PhyML (26) after aligning the sequences with Muscle (27).
RESULTS
Sequence Analysis of ZmBT1-The ZmBT1 cDNA clone was isolated from the first strand cDNA of maize endosperm as given under "Experimental Procedures." Six independent PCR products of ZmBT1 have been sequenced and found to exhibit 100% identity excluding failure of the Pfu polymerase. Compared with the open reading frame of the ZmBT1 sequence published by Sullivan et al. (28), our ZmBT1 cDNA sequence differs in the following positions: six additional nucleotides (G at bp position 246, 295, and 411, and GGA at bp position 1257-1259) and two nucleotide changes (GC instead of CG at bp position 338 -339). The resultant triple frameshift modified the deduced amino acid sequence ahead of the first predicted transmembrane domain (amino acid position 83-137) and the additional amino acid residue at the C terminus (position 419) led to a substantial higher similarity to other BT1 isoforms ( Fig. 1). Interestingly, the ZmBT1 cDNA sequence we determined was recently submitted by Lai et al. as well (2004, direct submission, accession number BT016796, www.ncbi.nlm.nih.gov). The deduced ZmBT1 protein sequence was aligned with putative BT1 homologues from wheat (Triticum aestivum), rice (Oryza sativa), barley (Hordeum vulgare), maize (Z. mays), barrel medic (Medicago truncatula), potato (S. tuberosum), and Arabidopsis thaliana (Fig. 1).
Southern Blot Analysis of ZmBT1 in Maize-In the light of the differences registered between BT1 gene copy numbers in mono-and dicotyledonous plants based on the rice and Arabidopsis genomes (35,36), we assessed BT1 gene copy numbers in maize by Southern blot analyses. We chose restriction enzymes that do cut either ZmBT1 or the recently published ZmBT1-2 homologue (accession number BT016800). The digestion of the genomic maize DNA with the restriction enzymes (XbaI, XhoI, and HindIII) led to two distinct bands on the Southern blot (Fig. 3, lanes 1-3). Only digestion with BamHI showed three bands, which is most probably due to a restriction site inside an intron of one of the two BT1 homologues. These results clearly indicate that two BT1 homologues occur in maize, in contrast to one homologue in dicotyledonous plants (15) (Fig. 2) and three homologues in rice (35).
Heterologous Expression of ZmBT1 in E. coli Cells-We showed previously that the heterologous synthesis of the plastidic StBT1 homologue in E. coli leads to the functional integration of StBT1 into the bacterial cytoplasmic membrane (15). Astonishingly, we identified StBT1 as a plastidic adenine nucleotide uniporter despite the attributed function to BT1 proteins to transport ADP-Glc in counterexchange with ADP and/or AMP (14,34,37). However, a high degree of sequence similarity for homologues belonging to the same protein family cannot be taken as proof for the same function (see Ref. 38), specifically when there are multiple homologues per genome indicating paralogy and potential functional differentiation between the homologues. The kinetic properties as well as the substrate specificity should be identified for each putative homologue.
The heterologous expression of ZmBT1 in different E. coli strains was analyzed using a peptide-specific antibody raised against a fusion protein including the 56 amino acid residues in the C-terminal sequence of ZmBT1. Substantial differences in the expression level of carrier proteins in E. coli have been reported (18,39), probably due to the unfavorable codon usage of several carrier proteins in E. coli (39). In fact, a screening of the ZmBT1 cDNA sequence revealed several codons rarely used in E. coli so that synthesized ZmBT1 protein in BL21, the most widely used expression host, was hard to detect (data not shown). In marked contrast, the use of E. coli Rosetta strains, which carry the pRARE plasmid and thus supply tRNAs for codons rarely used in E. coli, allows a high expression of ZmBT1. We confirmed the integration of the heterologously expressed ZmBT1 in the E. coli Rosetta cytoplasmic membrane by Western blotting by use of the above mentioned peptide-specific ZmBT1 antibody (Fig. 4).
We tested ADP-Glc as a putative transport substrate for ZmBT1. Uptake of [ 14 C]ADP-Glc into E. coli Rosetta cells harboring ZmBT1 in their membranes was linear with time for at least 15 min. The ADP-Glc uptake was strictly dependent on the membrane intactness and ZmBT1 protein synthesis (supplementary materials Fig. 1). Thus, we analyzed the affinity for ADP-Glc at different substrate concentrations. The results are given in Fig. 5A. Increased exogenous radioactively labeled ADP-Glc induced increased rates of ADP-Glc transport into E. coli cells harboring ZmBT1. Lineweaver-Burk analyses revealed an apparent K m value for ADP-Glc of 847.8 Ϯ 39.6 M and a V max of 191.5 Ϯ 28.7 nmol of ADP-Glc/mg of protein Ϫ1 h Ϫ1 (Fig. 5A).
To investigate the substrate specificity of ZmBT1, we measured the effect of various non-labeled metabolic intermediates on the rate of [ 14 C]ADP-Glc uptake (Table 1). Substantial inhibition of [ 14 C]ADP-Glc import could be observed with non-labeled ADP and ADP-Glc reducing the transport rate below 15 and 24% of the control (without effector), respectively. None of the other metabolic intermediates tested showed any substantial influence on [ 14 C]ADP-Glc uptake, which confirms ADP-Glc and ADP as the main substrates for ZmBT1 (Table 1). Furthermore, we analyzed the affinity of ZmBT1 for ADP (Fig. 5B). Increased exogenous radioactively labeled ADP induced increased rates of ADP transport into E. coli cells harboring ZmBT1. Lineweaver-Burk analyses revealed an apparent K m value for ADP of 465.2 Ϯ 36.2 M and a V max of 6.1 Ϯ 0.7 nmol of ADP/mg of protein Ϫ1 h Ϫ1 (Fig. 5B). The higher affinity for ADP compared with ADP-Glc is reflected in the competition experiments of ADP-Glc uptake ( Table 1). The substantial higher maximal velocity of ADP-Glc uptake seems to be a particular feature of this carrier (Fig. 5). Thus, the resulting relative catalytic efficiency (V max /K m ) of ZmBT1 is 18 times higher for ADP-Glc than for ADP.
Biochemical studies on isolated maize endosperm plastids led to the assumption that ADP-Glc is transported across the envelope membranes in counterexchange with ADP or AMP (37). AMP can be excluded as exogenous substrate for ZmBT1 because we could not detect any substantial inhibition of nonlabeled AMP on the ADP-Glc uptake or any direct uptake of radioactively labeled [ 14 C]AMP into E. coli cells harboring ZmBT1 (Table 1, data not shown). However, we could also exploit the E. coli system to investigate the counterexchange properties of the heterologously synthesized ZmBT1. The principle of this approach is to initiate a putative efflux of imported radioactively labeled [ 14 C]ADP-Glc through a high dilution (27). The PhyML phylogeny was created with 2 ␥-distributed rate parameters and the JTT model for amino acid substitutions. Bootstrap values above 85 out of 100 are indicated. A phylogeny created with Neighbor Joining using the identity matrix and correcting for multiple replacements had an identical topology. The phylogeny indicates a split between the AMP/ADP/ATP carriers and the ADP-glucose carriers, although the exact division between the groups as indicated in the tree is not very strongly supported. The extra sequences are a plant specific group (Arabidopsis and rice) of unknown function that is phylogenetically close to the Brittle1 group. The deoxynucleotide carrier group was used as outgroup because it is phylogenetically closest in a phylogeny of the 500 "best hits" to the Brittle1 group of proteins. At the right, experimentally determined substrate specificity of the carriers are indicated. Accession numbers of the BT1 proteins are given in the legend to Fig. 1 with the exception of TaBT1-2 (NCBI accession number BT009587), which is not a full-length clone.
(chase) with non-labeled substrates at a certain time point during an uptake experiment (15,22). To analyze whether imported [ 14 C]ADP-Glc is metabolized by E. coli cells harboring ZmBT1, we disrupted the cells at several time points after preloading with [ 14 C]ADP-Glc and analyzed the cytosolic fraction by thin layer chromatography. Most of the [ 14 C]ADP-Glc (about 90%) was found not to be metabolized by the E. coli cells over a time span of about 12 min, which suggests the involvement of [ 14 C]ADP-Glc in a putative nucleotide exchange (Fig. 6A). Fig. 6B shows a typical time course for [ 14 C]ADP-Glc uptake (at 1 M) into E. coli cells harboring ZmBT1. Right after the start of the chase with non-labeled ADP (1 mM), a rapid efflux led to a total release of labeled nucleotides of about 65% (7 min after the start of the chase). GTP, used as a control, is known not to be a substrate for ZmBT1 (Table 1) and, therefore, showed no influence on the uptake of radioactively labeled ADP-Glc after the chase. These results clearly reveal that ZmBT1 mediates a counterexchange of ADP-Glc and ADP. Non-labeled ADP-Glc (1 mM) inhibited significantly the [ 14 C]ADP-Glc uptake mediated by ZmBT1 but did not initiate any exchange of ADP-Glc (Fig. 6B).
Due to the fact that commercially available ADP-Glc is contaminated by other adenylates, namely AMP, ADP, and ATP (15), we determined the contamination of ADP-Glc by high performance liquid chromatography analysis (1.8% ADP; 0.6% ATP; 0.5% AMP) and carried out the same experiment using the calculated nucleotide contaminations as a non-labeled nucleotide mixture (18 M ADP, 6 M ATP, and 5 M AMP) for the chase during [ 14 C]ADP-Glc uptake. The chase with this nucleotide mixture did not lead to any competitive inhibition of [ 14 C]ADP-Glc uptake, so that the above mentioned competitive inhibition of non-labeled ADP-Glc is not influenced by the contamination (Fig. 6B). These results indicate a selective exchange of ADP-Glc with ADP. To further validate this mode of transport we preloaded E. coli cells harboring ZmBT1 with 500 M non-labeled exogenous ADP for 5 and 10 min, washed the cells, and performed [ 14 C]ADP-Glc uptake in comparison to not preloaded cells. Indeed, [ 14 C]ADP-Glc uptake into E. coli cells harboring ZmBT1 increased up to 174% compared with the control when the cells were preloaded with the counterexchange substrate ADP (Fig. 6C). To clarify whether endogenous AMP might have an influence on [ 14 C]ADP-Glc uptake into E. coli cells harboring ZmBT1 we preloaded these cells with 1 mM non-labeled exogenous AMP for 5 and 10 min, washed the cells, and performed [ 14 C]ADP-Glc uptake in comparison to non-preloaded cells (supplementary materials Fig. S2). In strong contrast to the experiment with ADP-preloaded cells (Fig. 6C), additional endogenous AMP has no influence on the ZmBT1 mediated ADP-Glc uptake (supplementary materials Fig. S2B).
We also determined the influence of several inhibitors on [ 14 C]ADP-Glc uptake ( Table 2). The highly specific inhibitors of the mitochondrial ADP/ATP carriers (AACs), bongkrekic acid and carboxyatractyloside (40,41), showed
TABLE 1 Effects of various metabolites on ͓ 14 C͔ADP-glucose transport activities of ZmBT1
Metabolic effectors were given at a concentration of 1 mM. ͓ 14 C͔ADP-glucose was present at a concentration of 200 M. Uptake into IPTG-induced E. coli cells harboring ZmBT1 was carried out for 8 min and stopped by rapid filtration (see "Experimental Procedures"). Data are the mean of three independent experiments with four replicates each. S.E. is less than 8% of the mean values. a ADP-Glc value is corrected by inhibition (0.5%) of the contamination with ATP, ADP, and AMP. b AMP value was determined at the presence of 1 mM adenosine (to prevent the cleavage of AMP by E. coli cells (15)) and corrected by the adenosine inhibition of 8.9%.
no inhibitory effect at the given concentrations ( Table 2). In addition, no considerable inhibition was observed with pyridoxal 5Ј-phosphate, a potential inhibitor of StBT1 and the plastidic phosphate translocators (15,42) (Table 2). Interestingly, the sulfhydryl reagent mersalyl, which is known to initiate a blockage of thiol groups (43), significantly inhibited the [ 14 C]ADP-Glc transport mediated by ZmBT1 below 12% of the control (Table 2). This indicates that SH-groups are of considerable importance for the activity of the ZmBT1. The activity of various chloroplast enzymes is known to be regulated by reversible thiol-disulfide interchange mediated by the ferredoxin-thioredoxin system (44). Recent studies on wheat starchy endosperm amyloplasts suggest also a thioredoxin (Trx) regulation of proteins in heterotrophic tissues. 42 amyloplast proteins were identified as potential Trx target proteins using a proteomic approach in combination with affinity chromatography and a fluorescent thiol probe (1). Interestingly, BT1, a major envelope protein of wheat amyloplasts, was also recognized as a thioredoxin target protein (1). To determine whether the activity of ZmBT1 is dependent on the redox state, we performed uptake of [ 14 C]ADP-Glc into E. coli Rosetta cells harboring ZmBT1 in the presence of diamide and/or dithiothreitol (DTT). In E. coli, the thioredoxin and glutaredoxin systems are known to keep reduced conditions for cytoplasmic proteins (45). This implicates that in ZmBT1 a disulfide bond formation is prevented under normal E. coli growth conditions. The addition of the membrane-permeant thiol-specific oxidizing agent diamide (diazenedicarboxylic acid bis(N,NЈ-dimethylamide) (45)) during the uptake experiments causes a strong inhibition of ADP-Glc uptake of E. coli Rosetta cells harboring ZmBT1 (Fig. 7A). At a concentration of 10 mM diamide the ADP-Glc uptake mediated by ZmBT1 is decreased up to 50%. In control experiments we confirmed the specificity of this effect on ZmBT1. The mitochondrial ADP/ATP carrier AAC2 from A. thaliana is not influenced by 10 mM diamide when heterologously synthesized in E. coli (Fig. 7A). Interestingly, the inhibition of ADP-Glc uptake into E. coli Rosetta cells harboring ZmBT1 was shown to be reversible. The decrease of ADP-Glc uptake mediated by ZmBT1 after preincubation with 10 mM diamide was mainly compensated by the addition of 40 mM reduced DTT (Fig. 7B). The addi-tion of 40 mM DTT (without diamide preincubation) does not affect any ADP-Glc uptake into E. coli Rosetta cells harboring ZmBT1, which indicates an optimal activation of ZmBT1 by reduced conditions in these E. coli expression cells (Fig. 7B). Expression Analyses of ZmBT1 and the BT1 Maize Homologue, ZmBT1-2, in Different Maize Tissues-To obtain further insight into the putative physiological roles of ZmBT1 and the recently identified ZmBT1-2 homologue, we analyzed the accumulation of the corresponding mRNAs in various autotrophic and heterotrophic tissues. Gene expression levels of low abundant proteins can be determined by real-time RT-PCR, which is more specific and sensitive than Northern blot analyses (15,46). The use of the total RNA mass (or 18 S rRNA) for normalization might be misleading because it consists predominantly of rRNA molecules that are often not representative for the mRNA fraction in different plant tissues (47). A reliable internal control should be similarly expressed in different cell types (48). Therefore, we chose the elongation factor EF-1␣, which catalyzes the first step of protein synthesis by binding the aminoacyl-tRNA to the aminoacyl site of ribosomes. EF-1␣ is described to be a suitable housekeeping gene commonly used in plants as an internal control for real-time RT-PCR (48 -50).
The real-time RT-PCR quantification shows an exclusive expression of ZmBT1 in maize endosperm tissues (15 days after pollination), which indicates an essential role for ZmBT1 in starch metabolism as reported earlier (10,14). In strong contrast, the ZmBT1-2 homologue is expressed in heterotrophic and autotrophic tissues indicating a general role in plant metabolism that was also observed for the BT1 homologues of potato (StBT1), Arabidopsis (AtBT1), and rice (OsBT1-2 and OsBT1-3) (15,35).
DISCUSSION
The ADP-Glc required for starch synthesis in plastids of cereal endosperm is mainly synthesized in the cytosol and has to be transported across the plastid envelope (2,51,52). Physiological studies on maize and barley mutants postulate that BT1 homologues are involved in the ADP-Glc transport (14,34,37). However, a conclusive proof that BT1 is the ADP-Glc transporter and a detailed analysis of this protein on the molecular level are still lacking.
In this paper, we have investigated the biochemical characteristics of the maize BT1 homologue ZmBT1 (Fig. 1). Heterologous synthesis of ZmBT1 in E. coli leads to the functional integration into the bacterial cytoplasmic membrane (Figs. 4 and 5). Uptake experiments on intact E. coli cells harboring ZmBT1 clearly reveal a high substrate specificity of ZmBT1 for ADP-Glc and ADP with affinities of about 850 and 465 M, respectively ( Fig. 5; Table 1). After preloading E. coli cells harboring ZmBT1 with radioactively labeled [ 14 C]ADP-Glc, efflux was initiated by addition of non-labeled ADP (Fig. 6B). Interestingly, a chase with 1000-fold non-labeled ADP-Glc did not cause any efflux of radioactively labeled [ 14 C]ADP-Glc but completely inhibited any further [ 14 C]ADP-Glc uptake. Furthermore, uptake of [ 14 C]ADP-Glc into E. coli cells harboring ZmBT1 was stimulated when cells were preloaded with nonlabeled ADP as a putative counterexchange substrate (Fig. 6C). These results characterize ZmBT1 as a carrier that mediates a strict counterexchange of ADP-Glc with ADP. Thus, ZmBT1 is here identified as an ADP-Glc transporter that enables ADP-Glc import into cereal endosperm plastids in exchange with ADP, which is generated during the ADP-Glc-dependent starch synthesis inside plastids (14,37).
Recently, efflux experiments on isolated wheat amyloplasts revealed that during the ADP-Glc-dependent starch synthesis ADP is the major adenylate exported from intact amyloplasts (53). The comparable low affinity of ZmBT1 for ADP-Glc (850 M) seems to be sufficient for effective transport due a highly active cytosolic ADP-Glc pyrophosphorylase (AGPase), which maintains high concentrations of ADP-Glc in the cytosol of maize endosperm (51)(52)(53)(54).
The components of a complete ferredoxin/thioredoxin system have been recently identified in amyloplasts isolated from wheat starchy endosperm. In cereal endosperm, ferredoxin is reduced not by light, as in chloroplasts, but by the metabolically generated NADPH via ferredoxin NADP reductase (1). Interestingly, the wheat BT1 was revealed as a potential Trx target protein (1). BT1 homologues possess several conserved cysteins, which is a prerequisite for the presence of a regulatory disulfide bond (Fig. 1). The inhibition and the reversible activation of ZmBT1 by diamide and DTT provide the first experimental evidence that the ADP-Glc carrier from cereal endosperm is in fact redox regulated as postulated for the wheat BT1 (Fig. 7). However, the specific cysteine residues that are involved in the redox regulation of ZmBT1 have still to be identified. Therefore, we aim at generating ZmBT1 Cys mutants by site-directed mutagenesis for further functional analyses.
The reducing power (NADPH) for the activation of the ADP-Glc carrier from cereal endosperm is derived from the oxidative pentose phosphate pathway residing in amyloplasts (55). NADPH is generated enzymatically by glucose 6-phosphate and 6-phosphogluconate dehydrogenases after import of Glc-6-P (56,57). In this context, the high expression of the glucose 6-phosphate/phosphate transporter in developing maize kernels makes sense. Glc-6-P imported by this carrier is used for the oxidative pentose phosphate pathway and not for the starch synthesis (57,58).
The regulation of BT1 gained prominence in subsequent studies showing that the cytosol, rather than the plastid, is the major site of ADP-Glc formation in cereal endosperm (2). Thus, the regulation of the BT1-mediated ADP-Glc transport into cereal endosperm plastids is of high physiological importance. The transport of sucrose into sink tissues such as endosperm would finally increase the NADPH/NADP ratio, which indicates a high energy status of the plant cells leading to a higher starch synthesis via the Trx-regulated ADP-Glc transport into heterotrophic plastids (1).
The occurrence of BT1 homologues in non-cereal plants such as A. thaliana, S. tuberosum, and M. truncatula was first unexplainable because in these plants ADP-Glc was not imported into the storage plastids for starch synthesis due to the plastidic localization of the AGPase. Recently, a BT1 homologue from a dicotyledonous plant, the StBT1 from potato, was characterized as a novel plastidic adenine nucleotide uniporter used to provide the cytosol and other compartments with adenine nucleotides exclusively synthesized inside plastids (15). Because the de novo purine nucleotide metabolism is of general importance in all plants, one should expect this novel type of plastidic carriers also in cereals. This implicates that maize should possess at least a second BT1 isoform. Indeed, Southern blot analyses revealed that the maize genome carries two distinct BT1 homologues (Fig. 3) and the second maize BT1 homologue (ZmBT1-2) was recently identified as well. Furthermore, the analysis of the rice genome revealed even three different BT1 homologues (35).
Interestingly, the phylogenetic analyses exhibit a subdivision of the BT1 protein family into two biochemically distinct groups (Fig. 2): the first group comprises BT1 homologues restricted to cereals where they are supposed to mediate the ADP-Glc transport into cereal endosperm storage plastids during starch synthesis (14,34) (Fig. 5; Table 1). The second group of BT1 homologues is present in mono-and dicotyledonous plants. These BT1 homologues are most probably involved in the export of adenine nucleotides synthesized de novo inside plastids (15,16). The phylogenetic classification of the BT1 family into two subgroups (Fig. 2) is further supported by the functional analyses of OsBT1-2 and OsBT1-3, which are identified as adenine nucleotide transporters that are unable to transport ADP-Glc (supplementary materials Table S1). The recently identified ZmBT1-2 homologue clearly belongs to the group of AMP/ADP/ATP carriers. The bootstrap value for the branch that contains OsBT1-3 and ZmBT1-2 is almost maximal (97/100) giving strong support for the notion that ZmBT1-2 is orthologous to an AMP/ADP/ATP carrier. However, the exact position of these two sequences relative to the other AMP/ADP/ATP carriers and the ADP-glucose carrier group is not well resolved (65/100). Nevertheless, based on the phylogeny and the functional information about the proteins, a single separation of the BT1 proteins into a monophyletic group of AMP/ADP/ATP carriers and a monophyletic group of ADP-glucose carriers appears the most parsimonious and therefore most likely scenario. Given the phylogenetic location of these carriers close to the deoxynucleotide carrier group, it is likely that the ancestor of the BT1 group was a nucleotide carrier rather than an ADP-glucose carrier (Fig. 2).
The biochemical classification of the maize BT1 homologues into two subgroups (Fig. 2) is also supported by the expression analysis. The ZmBT1 ADP-Glc carrier is exclusively expressed in endosperm tissues during starch synthesis, whereas ZmBT1-2 shows a ubiquitous expression pattern in heteroand autotrophic maize tissues as well, with the highest expression levels in silk and tassel (Fig. 8). The ubiquitous expression of ZmBT1-2 is in line with the proposed function of the recently identified plastidic adenine nucleotide uniporter (StBT1), which is supposed to have the general task to provide the cell with adenine nucleotides exclusively synthesized inside plastids (15). Similar observations were made for the expression patterns of the three BT1 homologues identified in the rice genome. In contrast to the endosperm-specific expression of the BT1 orthologue OsBT1-1, both BT1 paralogues (OsBT1-2 and OsBT1-3) exhibit a wide expression pattern comprising hetero-and autotrophic rice tissues, with the lowest expression level in seeds and roots (35). | 2018-04-03T02:15:38.989Z | 2007-08-03T00:00:00.000 | {
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216131657 | pes2o/s2orc | v3-fos-license | Maternal Allergy and the Presence of Nonhuman Proteinaceous Molecules in Human Milk.
Human milk contains proteins and/or protein fragments that originate from nonhuman organisms. These proteinaceous molecules, of which the secretion might be related to the mother’s allergy status, could be involved in the development of the immune system of the infant. This may lead, for example, to sensitization or the induction of allergen-specific tolerance. The aim of this study was to investigate the relation between maternal allergy and the levels of nonhuman proteinaceous molecules in their milk. In this study, we analysed trypsin-digested human milk serum proteins of 10 allergic mothers and 10 nonallergic mothers. A search was carried out to identify peptide sequences originating from bovine or other allergenic proteins. Several methods were applied to confirm the identification of these sequences, and the differences between both groups were investigated. Out of the 78 identified nonhuman peptide sequences, 62 sequences matched Bos taurus proteins. Eight peptide sequences of bovine β-lactoglobulin had significantly higher levels in milk from allergic mothers than in milk from nonallergic mothers. Dietary bovine β-lactoglobulin may be absorbed through the intestinal barrier and secreted into human milk. This seems to be significantly higher in allergic mothers and might have consequences for the development of the immune system of their breastfed infant.
Introduction
The human milk proteome comprises more than once thought. Besides a vast number of human proteins and peptides, it also includes nonhuman intact proteins, large protein fragments, and peptides (later referred to as proteinaceous molecules). The presence of such molecules in human milk has been demonstrated decades ago with immunochemical analysis [1] and has recently been confirmed with mass spectrometry [2].
According to studies using mass spectrometry, the main biological source of the nonhuman proteinaceous molecules in human milk seems to be bovine milk. A peptidomics study demonstrated the presence of two peptides originating from bovine β-lactoglobulin (BLG) and one originating from α S1 -casein [3]. In a later study, this was extended with peptides from α-lactalbumin (ALA), κ-casein, β-casein, and lactoferrin [4]. Evidence for the presence of intact bovine caseins and BLG has recently been provided [2,4,5]. In addition to the bovine proteins and peptides, Zhu et al. also identified several peptide sequences originating from other nonhuman species, which may include allergens [2]. So far, only peanut allergen has been identified with high sequence coverage by liquid chromatography tandem mass spectrometry (LC-MS/MS) [6]. The presence of egg, wheat, and house dust mite (HDM) allergens in human milk, which has been demonstrated using immunochemical methods [7][8][9], has not been confirmed yet with LC-MS/MS analysis.
The presence of these nonhuman proteinaceous molecules in human milk raises the question of how they end up there. It has been suggested that dietary proteins can be transferred through the intestinal barrier by both paracellular and transcellular pathways [10]. Furthermore, nonhuman proteins present in the mother's blood might also be transferred by these pathways through the mammary epithelia into the milk [11,12]. Nevertheless, it remains unclear which pathways are used for the transfer of nonhuman proteinaceous molecules into human milk.
Several of the studies that identified nonhuman proteinaceous molecules in human milk report a large interindividual variation in the levels of these molecules. An explanation for this variation has not been found yet, but it does not seem to be related to dietary intake [13]. Maternal asthma and allergy could be an important factor in this variation, since it is known that e.g., atopic eczema and asthma can have an influence on intestinal barrier integrity [14][15][16]. This could then lead to an increased passage of dietary proteinaceous molecules through the intestinal barrier. Research to date has not yet considered the relation between maternal allergic diseases and the levels of nonhuman proteinaceous molecules in human milk using LC-MS/MS. The purpose of the present study was therefore to identify nonhuman proteinaceous molecules in human milk and to investigate if the levels of these molecules were related to maternal allergy. This could be useful for further research into the mechanisms responsible for the transfer of these molecules and the effect of these molecules on the infant's immune system.
Milk Samples
We used data from a population-based Dutch birth cohort study: the Prevention and Incidence of Asthma and Mite Allergy (PIAMA) Study. Details of the cohort study are described elsewhere [17,18]. In short, pregnant women were recruited from the general population during their first antenatal visit. Their children (n = 3963) were born in 1996/1997. Pregnant women were identified as allergic or nonallergic through a screening questionnaire. House dust samples and breastmilk samples were collected in a subgroup of the population around the child's age of three months. Breastmilk collection was done by manual pressure or by use of a breast pump. Samples were stored in small plastic cups at −80 • C. Along with these samples, cat ownership and the frequency of consumption of milk and milk products by the mother was assessed using a questionnaire (Table 1). Maternal blood samples were collected at the child's age of one year. The study was performed in accordance with the ethical principles for medical research involving human subjects outlined in the Declaration of Helsinki. Therefore, the study protocol was approved by the Medical Ethics Committees of the participating institutes (Rotterdam MEC 132.636/1994/39 and 137.326/1994/130; Groningen MEC 94/08/92; and Utrecht, MEC-TNO oordeel 95/50). All parents gave written informed consent.
The current study is based on a data-dependent LC-MS/MS proteomics data set that was obtained in a previous study [19]. It comprises mass spectrometry data of human milk serum protein samples from 10 allergic mothers and 10 nonallergic mothers from the cohort study. The number of mothers included is based on a power calculation, aiming at finding a 5-fold difference, as detailed in Hettinga et al. [19]. The selection of the allergic mothers was based on (a) self-reported (history of) asthma, current hay fever, current allergy for pets, or current allergy for house dust or house dust mite in combination with (b) a high level of specific IgE against HDM (≥3.50 kU/L) and (c) exposure to HDM allergen in mattress dust ((Der p 1 + Der f 1) > 600 ng/m 2 ) (see Table 1). The selection of nonallergic mothers did not report any allergies or asthma. This group consisted of mothers with exposure to HDM allergen in mattress dust in the same range as the allergic mothers (600-2500 ng/m 2 ) as well as mothers with much higher exposures (≥24,000 ng/m 2 ). The nonallergic mothers were not tested for specific IgE against house dust mite. Table 1. Details on the mothers included in the sample collection, with allergy status, Der p IgE Rast-class of the allergic mothers, presence of a cat as pet, and consumption of milk and dairy products.
Characteristic Type Nonallergic Allergic
House dust mite allergy Self report 0 7 Doctor diagnosed 0 7 House dust allergy Self report 0 8 Doctor diagnosed 0 6 Allergic to pets Self report 0 9 Doctor diagnosed 0 8 From the milk samples, milk serum was obtained and serum proteins were prepared for analysis with filter-aided sample preparation. In short, full scan FTMS spectra were obtained (m/z 380 to 1400) in positive mode on an LTQ-Orbitrap system (Thermo electron, San Jose, CA, USA). The four multiply-charged peaks with the highest intensity were recorded in the linear trap in data-dependent mode (MSMS threshold: 5000). Further details of the sample preparation and LC-MS/MS analysis have been described before [19].
The data underlying the findings presented in this paper are available on request. Requests can be submitted to the PIAMA principal investigators. Their names and e-mail addresses are listed on the PIAMA website (http://piama.iras.uu.nl/index-en.php#collaboration).
Data Analysis
The raw MS/MS data was analysed using the Andromeda search engine of the MaxQuant software v1.6.1.0 [20]. Since the use of large databases in proteomic data analysis affects the sensitivity of the search [21], a complete but concise database was created for this study. This database contained human milk proteins (n = 2569), bovine milk proteins (n = 1006), and allergen proteins (n = 721). This database is provided in the Supplementary Materials, the fasta database. Allergens were added to the database because of their immunological relevance and bovine milk proteins because the majority of the nonhuman proteinaceous molecules in human milk was previously shown to originate from bovine milk [2]. The selection of human and bovine milk proteins was made based on previous data analysis of human and bovine milk protein samples (data not published) using databases with all human or bovine proteins available in UniProtKB (both downloaded from UniProt on 16-10-2018). This was complemented with data from reviews on the bovine milk and human milk proteome [22,23]. Allergen protein sequences were obtained from UniProt on 16-10-2018 by performing a search on all proteins annotated as allergen (search term: "annotation:(type:allergen)").
The search for peptide sequences was performed three times, in which the protein database was in silico digested with trypsin digestion, semi-specific trypsin digestion, or unspecific digestion. Maximum missed cleavages was set to two in the trypsin digestion mode. In all searches, a fixed modification was set to carbamidomethylation of cysteine. Variable modifications were set to acetylation of the peptide N-term, deamidation of the side chains of asparagine and glutamine, and oxidation of methionine, with a maximum of five modifications per peptide. The identified peptides were quantified using label-free quantification (LFQ). At both the peptide and protein levels, a false discovery rate of 1% was used. The peptide length was set from 6 to 35 amino acids. The precursor mass tolerance was set to 20 ppm, and fragment mass tolerance was set to 0.5 Da. Recalibration was carried out using a first search with a database containing common contaminants.
To remove all identifications that belong to sequences originating from human proteins, the MaxQuant output was subjected to a filtering consisting of six steps. First, all sequences originating from trypsin and keratin were removed as contaminants. Second, the reverse sequences from the decoy database were removed. Third, all sequences that had a full match with the human proteome were removed. Fourth, we removed all MS/MS scans that had a match in a separate search using only the whole human proteome database. Fifth, all sequences with an Andromeda score lower than 80 were removed to exclude low quality peptide spectrum matches (PSM). Sixth, PSMs with a second-best match to a human peptide sequence and an Andromeda score difference of <5 were removed.
Annotation
Protein entries containing an exact match with the identified and selected peptides were found using the Peptide Match service of the online Protein Information Resource [24]. This service makes use of an up-to-date UniProtKB database. Peptides were matched to this database without isoforms, where leucine and isoleucine were treated as equivalent.
Protein and organism annotation was added using a frequency of occurrence. All matching proteins and their corresponding taxonomic lineage were listed. A leading protein was selected for each peptide sequence based on the frequency of occurrence of this protein in the peptide match results. After this, a similar approach was used on the level of taxonomy, leaving the organisms with the highest number of matches to the identified peptides as leading organism or, in case of multiple organisms, the lowest common ancestor (LCA). With this approach, Bos taurus was preferred over e.g., Bos mutus as the leading organism because of a higher number of identified peptides that matched the Bos taurus proteins.
Statistical Analysis
Data analysis was carried out, and figures were made using R version 3.6.0 [25]. Missing values of LFQ intensities for the identified and selected peptide sequences were associated with levels below detection limit. Therefore, imputation was applied to 10 log transformed LFQ intensities, with values from a normal distribution downshifted from the sample mean with 1.8 and with a standard deviation of 0.3.
Differences between the allergic and nonallergic group were tested using a two-sided unequal variances t-test and a Benjamini-Hochberg correction was applied on the resulting p-values. Significantly different peptides were selected with a p-value < 0.01. An additional threshold of 0.75 was set on the difference between the means of the sample groups ( 10 log transformed intensity values) in order to select only significant sequences with a large between-group difference.
Confirmatory Analysis
Bovine caseinate (prepared in-house), lactoferrin, BLG, ALA and bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in a 100 mM Tris solution and digested with trypsin. For confirmation of the nonhuman, non-bovine peptides, 12 peptides were acquired through synthesis by Royobiotech Co., Ltd. (Shanghai, China). Protein digests and synthetic peptides were analysed one by one on the same LC-MS/MS system and with the same parameters as used for the analysis of the human milk samples [19]. A summary of the workflow and confirmation of MSMS spectra is visualised in Figure 1. After LC-MS/MS analysis, experimental MSMS spectra that matched theoretical nonhuman peptide sequences were selected when there was no close peptide sequence match (PSM) with a human peptide sequence. Spectra with a PSM score < 80 or a full match with the human proteome were removed. The final remaining spectra were confirmed with retention time and MSMS spectra of bovine milk, pure proteins, or synthetically acquired peptides.
Results
In this study, data-dependent shotgun proteomics data of human milk serum from 10 allergic and 10 nonallergic mothers was analysed. In a search for nonhuman proteins and protein fragments, the identified peptides were filtered and LFQ data was used for quantification.
Identification of Exogenous Peptides
Trypsin-digested human milk serum protein data was analysed using a database containing human milk, bovine milk, and allergen protein sequences. The identified peptide sequences were filtered to remove all human peptides and as many false positives as possible. In total, 78 nonhuman peptide sequences were identified in 20 samples. From these, 62 sequences had Bos taurus as leading organism (Table 2) and 16 sequences were assigned to non-bovine allergens ( Table 3). Most of the identified peptide sequences (n = 48) were from trypsin-digested proteins. In addition, 10 peptides were semi-trypsin digested and 20 were not digested by trypsin. Table 2. All identified nonhuman peptide sequences that were assigned to bovine proteins, with the corresponding UniProt protein id, name of the leading protein, and in silico digestion mode. Per group of allergic and nonallergic mothers, the number of samples in which the peptide sequence was identified is listed.
Sequence
Leading semi-specific a confirmed by analysis of acquired synthesized peptide; b last common ancestor (LCA) in case of multiple leading organisms.
Peptide sequences of 29 different bovine proteins were identified. From these proteins, the major bovine milk allergen, BLG, was identified with the highest sequence coverage (67%). To confirm the identification of the bovine sequences, tryptic digests of the major bovine milk proteins, BLG, BSA, α S1 -casein, and ALA were analysed. This led to confirmation of 20 sequences based on MS/MS spectrum and retention time. The identification of another 16 sequences was confirmed by MS/MS spectra and retention times of these sequences in a bovine milk protein data set (data set not published). The protein with the second highest sequence coverage is bovine serum albumin (BSA). The identified peptide sequences (n = 14) correspond to a sequence coverage of (22%). In contrast to studies from other groups that removed these peptides from their data sets because of the use of BSA as quality control in their studies [2,4], we did not remove these peptides from our results. No evidence was found for carryover of BSA peptides in the LC system. Several BSA peptide sequences identified in human milk were not found in a trypsin-digested BSA standard solution that was used in our laboratory, indicating that the BSA-derived peptides are genuine. Considering these findings, it is likely that BSA or its proteolytic fragments are present in human milk.
From the non-bovine allergen proteins or protein fragments, proteins from Felis catus (domestic cat), Equus caballus (horse), and Triticum aestivum (common wheat) were identified with two or more peptide sequences. To confirm the identification of these peptide sequences, one synthesized sequence of each identified protein was acquired and analysed. From these nine peptides, two sequences were confirmed based on MS/MS spectrum and retention time. These sequences had cat and horse serum albumin as leading protein. The remaining seven sequences could not be confirmed. In several cases, the PSM of the synthesized peptide resembled the PSM of the human milk samples, but the retention time differed significantly. These PSMs are likely false positives, showing that the search for low abundant peptide sequences in shotgun proteomics is prone to finding PSMs with artefacts or co-eluted peptides. This confirms the importance of the used method in which synthesized peptides were used for confirmation.
In addition to the 16 sequences that were assigned to non-bovine allergens, 11 peptides were annotated with Hevea brasiliensis (Rubber tree) as leading organism. Two of these sequences were confirmed with MS/MS spectra and retention time of synthesized peptides. Nevertheless, data analysis of three other data sets of human milk shotgun proteomics did not show the presence of these peptides (data not published). Therefore, these sequences were considered as contaminant and removed from the results.
Another possible source of false-positive identifications could be the presence of unknown human protein variants due to point mutations. Out of the 78 identified nonhuman peptide sequences, 26 have one amino acid different from their human homologue. As an example, the sequence LVNELTEFAK with Bos taurus as leading organism has LVNEVTEFAK as homologue in Homo sapiens. The V → L could therefore be the result of a point mutation. Nevertheless, for all these 26 sequences, no research was found that confirmed the occurrence of these point mutations in Homo sapiens.
MS/MS spectra of the identified peptides and their confirmation can be found in the Supplementary Materials (Supplementary Figures S1-S38).
Differences between Allergic and Nonallergic Mothers
Out of the 78 nonhuman peptide sequences, 15 sequences were only identified in milk from allergic mothers, whereas in milk from nonallergic mothers, 11 unique sequences were identified. This difference can be largely attributed to sequences that match to bovine proteins ( Table 2). After imputation of the LFQ data and performing a t-test with maternal allergy as grouping variable, 16 peptide sequences appeared to be significantly different in intensity between the two groups ( Figure 2).
As shown in Figure 2, nine sequences were found to be significantly higher in intensity in milk from allergic mothers. These sequences were annotated to BLG (n = 8) and alpha-2-HS-glycoprotein (n = 1) as leading protein. The seven sequences that were significantly higher in intensity in milk from nonallergic mothers were annotated to BSA (n = 6) and to an uncharacterized protein (n = 1), with semi-specific trypsin digestion. All the significantly different sequences were annotated to proteins that originate from Bos taurus. As can be seen in Figure 3, there is a consistent difference between the two groups, indicating that the significant differences are not caused by outliers. Figure 2. Volcano plot with the ratios of the group means of the 10 log transformed label-free quantification (LFQ) intensities of the identified peptide sequences. Significantly different peptides (false discovery rate < 0.01 and difference between groups > ±0.75) are represented by filled red circles and labelled with the corresponding amino acid sequence. On the right side of the plot, the peptides with a higher level in allergic mothers are presented, and on the left side, the peptides with a higher level in nonallergic mothers are presented.
Discussion
The goal of this study was to identify nonhuman proteinaceous molecules in human milk and to investigate differences in these molecules between milk from allergic and nonallergic mothers. Out of the 78 resulting nonhuman peptide sequences identified in this study, 11 sequences were reported previously in human milk studies using LC-MS/MS [2,5]. Contrary to these studies, we focused on milk serum, discarding the caseins by ultracentrifugation. This could explain the major difference with Zhu et al. when it comes to the number of identified sequences matching with bovine caseins [2]. The relatively high levels of BLG peptide sequences that we found in milk from allergic mothers explains the high sequence coverage of BLG in the current study when compared to Zhu et al. [2]. Because we removed small peptides by filter-aided sample preparation (10-20 kDa cutoff), no comparison could be made with previous peptidomics studies [3,4]. Other qualitative differences with these previous studies can be attributed to the more strict filtering on false positives that we applied, the inclusion of serum albumins, the inclusion of semi-trypsin and non-trypsin-digested sequences, and to the inclusion of milk from allergic mothers.
The transfer of proteinaceous molecules from the mother's intestinal tract to the mammary gland is still poorly understood, especially when it comes to intact proteins or large protein fragments. In the current study, trypsin was used to digest the proteins before analysis. It can be expected that the majority of the identified peptides was digested by trypsin. Nevertheless, some peptide sequences were identified that were not, or partly, digested by trypsin. This might indicate that there are also nonhuman protein fragments present in human milk that were digested by other proteases than trypsin, probably before these fragments even entered the milk. The high sequence coverage for BLG, with all but one sequence digested by trypsin, is an indication for the presence of intact BLG in human milk. This is supported by the findings of Zhu et al., who, although with a lower sequence coverage, also identified BLG in human milk. In addition, several other studies reported the identification of intact BLG in human milk using immunochemical analysis [2,26,27]. The presence of intact BLG may be due to its relatively small size and its high resistance to pepsin digestion, as previously shown by the presence of intact BLG in jejunum samples [28]. From our results, it appears that especially proteinaceous molecules from bovine milk end up in human milk. This might relate to the high consumption rate of dairy products in the Netherlands, considering that 23% of the average dietary protein intake originates from milk and dairy products [29]. In line with this, the highly abundant bovine milk serum proteins (BLG and BSA) were identified with the highest sequence coverage. Bovine ALA, another major milk serum protein, was identified with only two peptide sequences. This low sequence coverage was expected because of its high digestibility and high homology with human ALA. This high homology reduces the number of unique peptides that can be identified from this protein. Besides the bovine peptide sequences, peptide sequences of cat and horse serum albumin were identified and confirmed by the analysis of synthesized peptides. No relation was found between the presence of cat serum albumin peptides in milk and the ownership of a cat as pet by the respective mothers. Nevertheless, it is known that mammalian serum albumins are present in animal dander and exposure to this is not limited to direct contact [30,31]. The serum albumins of cat or horse could end up in the human digestive system by ingestion or inhalation and could subsequently be transferred to the milk. Whether these proteins are present as intact proteins or in large fragments remains unclear because of the relatively low sequence coverage that was found. Several other studies reported the detection of other dietary allergen proteins in human milk, such as egg ovalbumin, peanut allergen, and wheat gliadin [6,9,13]. Peptide sequences of these three proteins were initially detected in the current study but were filtered out due to a low PSM score or to not being confirmed by analysis of synthesized peptides. This could still mean that these proteins are present in human milk but in too low concentrations for positive identification.
Several previous studies have investigated a possible difference in nonhuman proteins between milk from allergic (maternal history of atopic diseases) and nonallergic mothers. Høst et al. [26] and more recently Matangkasombut et al. [32] did not find a difference in BLG levels in milk between the two groups. Another study, also investigating BLG levels, found BLG in the milk of all allergic subjects involved and not in the milk of all nonallergic subjects [27]. Sorva et al. found that BLG levels in milk of allergic and nonallergic mothers were similar after 24 h on a milk-free diet [33]. Nevertheless, the levels of BLG tended to be higher in milk from allergic mothers one hour after consumption of 400 mL of bovine milk. Surprisingly, the current study shows a significant difference concerning peptide sequences originating from BLG and BSA. A similar finding has not been reported before. The difference between our results and the aforementioned studies can possibly be explained by the characteristics of the allergic subjects and a difference in methodology. The current study has, for example, a strict selection on both HDM-specific IgE and allergy symptoms, whereas previous studies did not elaborate on the definition they used for atopy or allergy, and in some cases, the selection of allergic subjects was based on symptoms only. In addition, immunochemical analyses have shown to be influenced by cross-reactivity between human and bovine proteins, which make them less reliable than LC-MS/MS analysis [34,35]. For the current study, only an indication of the frequency of consumption for milk and milk products was available (Table 1). Nevertheless, it seems that the allergic mothers even consumed less milk or milk products when compared to the nonallergic mothers. Therefore, our findings indicate that there is a difference between the allergic and nonallergic mothers when it comes to the transfer of bovine proteinaceous molecules from the intestinal tract to the mammary gland.
From the intestinal tract to the blood, proteins can be absorbed by both paracellular and transcellular pathways. In reviewing the literature, Reitsma et al. suggested that a difference in intestinal absorption of proteins between non-sensitized and sensitized persons can take place by both pathways [10]. Which of these two is involved in the transfer of dietary proteins into human milk is not known. One option for a transcellular pathway concerns transport of intact antigens with specific IgE via the CD23 receptor [36]. With regard to the current study, this would suggest an increased level of BLG-specific IgE in HDM allergic mothers, which seems unlikely and has not been mentioned in literature. Another transcellular pathway is through enterocytes and involves degradation of the protein in lysosomes [37]. A recent study using CaCo-2 cell monolayers showed that casein fragments survive transfer by this pathway but that BLG seems to be completely degraded [38]. Therefore, this pathway seems unlikely. A third transcellular pathway is via M cells, and it has been suggested that BLG can be transferred through this pathway without degradation [39]. Nevertheless, there is no evidence that transport through these pathways is increased in allergic mothers. A prerequisite for the uptake of proteinaceous molecules through the paracellular pathway is an impaired intestinal barrier. Reitsma et al. pointed out that sensitized persons have an increased level of mast cells that release IgE-induced tryptase [10]. The tryptase affects the tight junctions in the intestinal barrier, leading to an increased permeability, which could allow the passage of proteinaceous molecules. Nevertheless, this pathway is linked to food hypersensitivity reactions where the location of sensitization is in the intestinal tract itself. In patients with HDM allergy, sensitization occurs primarily in the respiratory tract. However, Calderón et al. suggested that HDM sensitization can be systemic and could cause reactions in other parts of the body [40]. Such a lung-gut cross-talk is plausible, considering the evidence showing that the mucosal immune system can be considered as a system-wide organ [41]. In reviewing the literature, Zhu et al. suggested, in line with this hypothesis, the role of a thymic stromal lymphopoietin-mediated pathway that is induced by HDM allergen sensitization, which might promote the breakdown of the epithelial barrier in the intestinal tract [42].
Several other factors could be involved in the disruption of the intestinal barrier. Firstly, it has been shown that, independent of atopy, asthma can be associated with an increased intestinal barrier permeability [14,43]. In the current study, seven out of 10 of the allergic mothers reported asthma (Table 1). Secondly, Tulic et al. showed that HDM is often present in the gut and that its cysteine protease Der p 1 causes disruption of the epithelial barrier [44]. This disruption appeared to be similar for HDM-sensitized and HDM-non-sensitized subjects. Nevertheless, due to an inflammatory response, it might be possible that recovery of the intestinal barrier dysfunction is delayed or incomplete in allergic subjects. This might then explain the permeability of the intestinal barrier in the allergic subjects in the current study, the majority of whom have HDM allergy. it should be noted that the majority of the in vivo studies on intestinal permeability make use of small inert molecules and their passage through the intestine. it has been shown that an increased transfer of these molecules through the intestinal barrier does not necessarily correlate with the transfer of antigens [45]. In addition, a previous study showed, using ELISA, that levels of BLG in human milk were not related to intestinal barrier permeability [33]. Therefore, more research is needed to elucidate whether the increased barrier permeability caused by these factors indeed leads to an increased passage of proteinaceous molecules.
After passage through the intestinal barrier, it is expected that the nonhuman proteinaceous molecules enter the blood stream and are subsequently transferred through the mammary epithelium into the alveolar lumen. This transfer seems to take place through a one-way transcytotic pathway. Monks et al. showed the role of this pathway in the transfer of extracellular serum albumin in mice and suggested that this is the same pathway that is involved in the transfer of IgA [12]. After transfer to the milk, the nonhuman proteinaceous molecules end up in the digestive system of the infant. Worth noting is that Hettinga et al., in analyzing the same data but focusing on human proteins, found significantly higher levels of several protease inhibitors in human milk from allergic mothers [19]. These protease inhibitors (cystatin C, inter-alpha-trypsin inhibitors, and serine-protease inhibitors) are potentially active against enzymes that hydrolyse BLG. Consequently, the human milk composition of allergic mothers might reduce the hydrolysis of BLG in the infant's intestinal tract.
Since the current study does not include absolute quantification, the exact level of these molecules in human milk remains unclear. Regardless of their level, it is known that bovine milk proteins in human milk can have an effect on the infant. Several reported cases described non-IgE-mediated food protein-induced enterocolitis syndrome caused by bovine milk proteins in exclusively breastfed infants [46,47]. In all these cases, the infant had a positive family history for atopy and clinical manifestations were resolved after the mother strictly eliminated cow's milk from her diet. It remains unclear whether nonhuman proteinaceous molecules in human milk can have an effect on the development of the immune system of the breastfed infant beyond causing allergic symptoms. Verhasselt et al. showed, using a mouse model, that antigen transfer through breastmilk induced tolerance and protection from allergic asthma [48]. Translating this to BLG, it is known that BLG-derived peptides can be HLA-DRB1-restricting, a characteristic that might support oral tolerance development [49]. In line with this, Peters et al. showed recently that early introduction of cow's milk was associated with a reduced risk of cow's milk allergy [50]. The presence of higher levels of BLG or its derived peptides in human milk of allergic mothers might therefore have a protective effect on further allergy development. Nevertheless, evidence remains speculative and a direct relation needs to be investigated.
Interestingly, BSA peptide sequences were found in significantly lower levels in milk from allergic mothers. Previous research with rats showed that intact BSA can pass the intestinal epithelium [51]. Nevertheless, the difference found between the two groups is difficult to interpret. The most likely but speculative explanation is a specific pathway that is activated in healthy mothers but that is negatively regulated in allergic mothers.
Conclusions
In conclusion, in the present study, a significant difference in levels of nonhuman proteinaceous molecules in human milk of allergic and nonallergic mothers has been observed. Sequences from BLG appeared in higher levels and sequences from BSA appeared in lower levels in milk from allergic mothers when compared to milk from nonallergic mothers. These findings suggest that there is a difference in transfer of proteinaceous molecules through the intestinal barrier of allergic mothers, allowing dietary proteins to enter the bloodstream and ultimately the milk. This study has raised important questions about the role that these proteinaceous molecules might play in the development of the immune system of infants. | 2020-04-23T09:07:20.772Z | 2020-04-01T00:00:00.000 | {
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262026909 | pes2o/s2orc | v3-fos-license | Aging attenuates the memory advantage for unexpected objects in real-world scenes
Across the adult lifespan memory processes are subject to pronounced changes. Prior knowledge and expectations might critically shape functional differences; however, corresponding findings have remained ambiguous so far. Here, we chose a tailored approach to scrutinize how schema (in-)congruencies affect older and younger adults’ memory for objects embedded in real-world scenes, a scenario close to everyday life memory demands. A sample of 23 older (52–81 years) and 23 younger adults (18–38 years) freely viewed 60 photographs of scenes in which target objects were included that were either congruent or incongruent with the given context information. After a delay, recognition performance for those objects was determined. In addition, recognized objects had to be matched to the scene context in which they were previously presented. While we found schema violations beneficial for object recognition across age groups, the advantage was significantly less pronounced in older adults. We moreover observed an age-related congruency bias for matching objects to their original scene context. Our findings support a critical role of predictive processes for age-related memory differences and indicate enhanced weighting of predictions with age. We suggest that recent predictive processing theories provide a particularly useful framework to elaborate on age-related functional vulnerabilities as well as stability.
Introduction
We continuously accumulate knowledge about the world.From repeated experiences we learn that our environment follows compositional rules.For instance, objects underlie physical constraints (a toothbrush does not float in space) and are usually found within a certain context (a toothbrush belongs in the bathroom).By abstracting and storing these regularities as schemata, our brains can predict future encounters with similar environments and objects [1].This prior knowledge of scene structure is particularly useful for object identification, when searching for specific objects or guiding attention towards goal-relevant information [2][3][4].Schemata do not only support perception, but are also key for memory processes [5][6][7].Given pronounced age-related changes in memory capacities, the functional role of schemata for age effects on object memory might be critical.However, so far studies have rarely addressed how prior knowledge and expectations affect older adults' memory for context-embedded objects.
While many cognitive functions, such as processing speed, memory capacities, and inhibitory control are subject to age-related decline, world knowledge remains stable or even improves [8].An amplified impact of prior knowledge has been suggested to contribute to older adults' decline and stability in memory performance [9].Increased reliance on predictions could serve as a compensatory mechanism to optimize memory decisions.However, this advantage might fail and even turn to a disadvantage when predictions are violated and an overreliance on them becomes detrimental.Although our environment is generally predictable, deviations from expectations are essential for knowledge updating and adjusting behaviour appropriately [10].A specific disadvantage for processing unexpected information would critically limit adaptivity to changing environments and affordances.
Several studies have been concerned with the role of schema expectations for object memory in older and younger adults, however, quite heterogenous result patterns have been reported.In order to provide an overview of the pervious findings, it is important to clarify that the generic use of the term congruency effect should be avoided since it can refer to contrary effect directions.The term subsumes performance benefits from schema-congruency as well as from schema-incongruency.This ambiguity can be resolved by distinguishing between a congruency advantage, i.e., a memory advantage for objects presented in congruent scene contexts, and an incongruency advantage, i.e., a memory advantage for objects presented in incongruent scene contexts.Whether an object memory advantage arises from schema congruency or schema incongruency, respectively, has been found to substantially depend on instructions during encoding.Notably, studies consistently provided evidence for only minor overall age-related differences in object memory, indicating robust resources in healthy aging [11][12][13][14][15][16][17].However, memory advantages that emerged from object-scene (in) congruencies were subject to specific age effects.
When explicit instructions drawing attention to possible object-scene (in)congruencies were provided, observers consistently, i.e., older and younger adults, showed a memory advantage for objects that are congruent with schema-based expectations about a given scene [11][12][13].Importantly, this congruency advantage was found to be more pronounced in older adults.The age-related benefit was observed in absolute object recognition performance as well as in memory for object details.These findings suggest an enhanced schema bias in older adults.While boosting recognition performance, though, the overreliance on prior knowledge could induce increased false alarm rates, qualifying a putative age-specific advantage.
In contrast to evidence for congruency advantages, memory studies that refrained from giving explicit instructions about objectscene (in-)congruencies yielded a reversed result pattern.Here, memory advantages were observed for objects that are incongruent with schema-based expectations about a given scene, i.e., incongruency advantages [14][15][16][17].Performance benefits were robustly present across age groups, however, age effects on the magnitude of these incongruency advantages have remained ambiguous.In a paradigm using schematic line drawings of scenes, older adults showed more pronounced advantages than younger adults [16].In contrast, studies focusing on objects embedded in real-world scenes yielded similar benefits across age groups [18] or even an opposite trend, i.e., less pronounced incongruency advantages in older than in younger adults [14,15,17].In summary, the latter studies provided evidence that object memory can reliably benefit from object-scene incongruencies across the adult lifespan, but the advantage seems limited in older adults, indicating that prioritized processing of incongruent objects might be weakened.
We here aimed to specifically focus on naturalistic scenarios in which memory for objects might be required in everyday life.We particularly considered three criteria for the memory task to be close to real-world affordances that have not been combined in the previous studies described above.First, we presented objects naturally embedded in real-world scenes in contrast to a detached presentation [11][12][13]16].In addition, photographs were supposed to ensure a standardized and controlled presentation of scenes, refraining from real-world settings [14,15,18].Second, we presented the scenes in a free-viewing paradigm without additional task, e. g., a search task or an explicit object memory task [13,16,17].Tasks per se affect how context information in scenes is processed [3,19] and might confound age effects [20,21].Finally, we did not provide explicit instructions on possible object-scene inconsistencies [11][12][13], avoiding strategic processes that are especially vulnerable during ageing [22].This tailored approach allows us to scrutinize how prior knowledge and expectations shape age effects on object memory in real-world scenes under free-viewing conditions.
Participants
A total of 23 older (11 males, age [years]: M = 69.3,SD = 8.0) and 23 younger adults (11 males, age [years]: M = 26.8,SD = 6.1) participated in this study.Older adults were part of our local database and screened for cognitive impairment using the MoCA [23].Younger adults were recruited by calls for participation and matched in terms of educational background and reported gender.Older adults reported slightly fewer years of school education than younger adults, 11.7 years (SD = 1.7) and 12.7 years (SD = 0.9), respectively.Overall our sample is characterized by a bias towards higher educational levels with 52.2% of our participants having completed a higher academic degree.Neurologic or psychiatric disorders were screened out by self-report.Procedures and methods conformed to the Declaration of Helsinki [24] and were approved by the local ethics committee at Justus Liebig University Giessen (LEK 2017-0025).All participants provided informed written consent prior to the experiment.
Setup
Data was collected via an online platform (https://www.testable.org/).Participants carried out the experiment using their own stationary computers.Given the online setting of our study, we had to tolerate variance in the actual setups our participants used.However, basic technical parameters were logged and we were able to confirm that there were no systematic setup differences between our both age groups.With regard to operating systems, equivalent proportions of older and younger adults run macOS (13.0% vs. L. Klever et al. 26.1%) or Windows (87.0% vs. 73.9%),indicating similar distributions, χ 2 (1, N = 46) = 1.24, p = .265.Screen resolution varied in width between 810 px and 2560 px, in height between 614 px and 1440 px, and in the diagonal between 1249 px and 1809 px.Most importantly, we observed for neither parameter significant differences between both age groups (all two-sample t-tests yielded ps > .08).
Stimulus size was standardized by a calibration procedure so that stimuli scaled with the screen resolution of the participants' screens.At the start of the experiment, participants were asked to match the length of a line shown on their screens to the length of a bank card.General instructions ensured undisturbed, quite conditions and a fixed viewing distance from the screen, i.e., a distance approximately equal to the length of an arm (~60 cm).
Stimuli
We used indoor scenes photographs taken from the SCEGRAM database [25].Based on the consistency ratings for every object-scene condition that are provided with the database, we derived two groups of object-scene combinations with which we maximized the difference between scenes with regard to supposed violated expectations.Given the above described calibration procedure, scenes were displayed at an approximate size of 18.9 • × 14.3 • .The scenes gave context information by six different room types (e.g., kitchen, bathroom).Target objects were naturally embedded in these scenes and were either congruent or incongruent with the given context (see Fig. 1A for examples).Target objects without scene context and distractor objects, taken from another database [26], were used for recognition.For recognition individual objects were displayed at an approximate size of 12.0 • × 7.4 • .Distractor objects were carefully chosen to match the target objects with regard to actual size, color, and semantical congruency with the given six room types.In addition, scenes without target objects were presented for matching recognized objects to their remembered context.A choice of three different scenes next to each other was presented, each with an approximate size of 8.6 • × 6.5 • .
Procedure
Participants first freely viewed 60 scenes, of which half contained target objects congruent or incongruent with the context, respectively.Participants saw each object and each scene, respectively, only once.There were no repetitions.Instructions called for memorizing the scenes in general and did not point to objects.The scenes were presented in randomized order, 3000 ms each with an ISI of 800 ms.Subsequently, participants filled out some unrelated questionnaires, introducing a delay of M = 14 ± 1 min.Finally, participants were presented with the 60 target and 60 distractor objects after one another in randomized order.They had to label each object as old or new, i.e. whether they have seen it in the scenes or not.If labelled as old, objects had to be matched to the scene presented in previously.Three alternative scenes were offered, one scene giving a congruent, two scenes giving an incongruent context (see Fig. 1B).This configuration was chosen according to similar procedures in previous work [11], focusing in particular on congruency biases in scene associations for objects encoded in an incongruent context.When the valid incongruent scene context is erroneously not selected, participants either opt for the second incongruent scene or the congruent scene.Given these two options the Fig. 1.Illustration of encoding and recognition procedures.(A) Scenes for encoding contained naturally embedded objects that were either congruent or incongruent with the given context.(B) Recognition procedure for a congruent target (upper row) and an incongruent target object (lower row).Note: Colored frames and labels are here used for illustration and were not used in the actual procedures.
L. Klever et al. congruency bias in associative scene memory can be evaluated.Responses were entered via the keyboard.All participants were familiarized with the procedure by two preceding demonstration trials based on additional scenes and objects, not included in the main experiment.
Data analyses
All measures we used for evaluation of memory performance were calculated for each individual participant and were than submitted to statistical analyses.Recognition performance was assessed in terms of the well-established d prime index (d') [27].D′ is a measure of discriminability based on signal detection theory that is unaffected by response biases.It is given by the difference between the z-transformed standardized proportions of correct detections and false alarms, characterizing the ability to discriminate targets from distractors.In our memory task, higher d' values indicate better recognition performance.To evaluate how well participants remembered the scene context of objects, we calculated the proportion of scene selection errors relative to the overall number of correctly recognized objects.Data were analyzed using mixed ANOVAs with the between-subject factor age group (older vs. younger) and the within-subject factor context (congruent vs. incongruent).In order to explore whether scene selection errors for incongruent objects were driven by a congruency bias, i.e., a bias to associate those objects erroneously with congruent scene contexts, we additionally calculated which proportion of these errors were made in favor of the congruent scene.Response times for old and new decisions were explored using median values.Since response times tend to be skewed and are prone to outlier data, median values provide a more robust measure than mean values.Data were submitted to a two-factorial ANOVA with the between-subject factor age group (older vs. younger) and the within-subject factor object type (distractor, congruent, incongruent).If appropriate, main analyses were followed by post hoc paired comparisons with Bonferroni-Holm correction.Significance level was set to α = .05 in all statistical analyses.If not stated otherwise, descriptive values are given as means ± SEMs.
Object memory
We first determined whether object memory systematically varies between the two context conditions and two age groups (Fig. 2).We submitted d' values to a two-factorial ANOVA with age group as between-subjects factor and repeated measures on the factor L. Klever et al.
Scene memory
Participants' ability to remember the correct scene context of target objects was evaluated by the error rates in matching correctly recognized objects to corresponding scenes (Fig. 3).We ran a two-factorial ANOVA with age group as between-subject factor and repeated measures on the factor context.The main effects for age group, F(1, 44) = 10.48,p = .002,η p 2 = 0.19, and context, F(1, 44) = 69.24,p < .001,η p 2 = 0.61, reached significance, but were qualified by a significant interaction effect, F(1, 44) = 9.12, p = .004,η p 2 = 0.17.Follow-up t-tests indicated that older and younger adults made considerably more errors assigning incongruent than congruent objects to the correct scenes (t(22) = 6.95, p < .01,d = 1.45, and t(22) = 4.59, p < .01,d = 0.96, respectively).However, the increase in error rates was more pronounced in older adults.Whereas error rates of both age groups did not differ for congruent target objects, t (44) = 0.25, p = .804,d = 0.07, older adults were especially prone to errors when assigning incongruent objects to their corresponding scenes, t(44) = 3.99, p < .01,d = 1.18.
Response times
We analyzed how response times in the object recognition task varied across age groups and object types, i.e., distractor, congruent, and incongruent objects.Median response times were submitted to a mixed ANOVA with the between-subject factor age group and the within-subject factor object type (Fig. 4).We determined a significant main effect for age group, indicating age-related slowing, F(1, 44) = 25.99,p < .001,η p 2 = 0.37.The main effect for object type was also significant, F(2, 88) = 10.45,p < .001,η p 2 = 0.19.There was no interaction between both main effects, F(2, 88) = 0.50, p = .607,η p 2 = 0.01, suggesting that response times were similarly affected across age groups.The main effect of object type was followed up by paired comparisons.Responses were faster for distractors than for congruent objects, t(45) = 4.54, p < .01,d = 0.67, and incongruent objects, t(45) = 3.50, p < .01,d = 0.52), target objects.Response times for congruent and incongruent target objects did not differ, t(45) = 0.67, p = .502,d = 0.10.
Discussion
Prior knowledge has been suggested to be a key factor in understanding older adults' memory performance [9].However, it is not well understood how violations of expectations affect memory for context-embedded objectsespecially under naturalistic viewing conditions.Object-scene inconsistencies challenge prior knowledge and provide substantial informational gain which could aid memory [28].This benefit might be attenuated in older adults.Using well-controlled photographs of real-world indoor scenes containing object-scene (in)consistencies, we addressed the question whether violations of scene context expectations affect memory for embedded objects differentially in older and younger adults.
We found that schema violations are overall beneficial for object memory in older and younger adults.As target objects were embedded in a rich, natural environment, we suggest that object-scene relationships were automatically processed, requiring minimal processing resources [29,30].This might have facilitated to observe robust memory benefits in older adults although they face reduced Fig. 3. Effects of age group and encoding context, i.e., congruent and incongruent, on error rates during scene selection.Filled dots show the mean across observers and semi-transparent lines provide individual data.Older adults are plotted in orange, younger adults in blue.Error bars give 95% confidence intervals.Exploration of scene selections for incongruent objects showed that older adults erroneously favored the congruent scene in 72% of the time, whereas younger adults showed a rate of 37%.These rates support a more pronounced congruency bias in older adults.
L. Klever et al. processing capacities and processing speed [31,32].In addition, associative demands, being especially challenging in old age [33], were minimized.However, we determined an age-related reduction of the incongruency advantage.This finding corroborates previous results showing less pronounced incongruency advantages in older than in younger adults [14,15,17].In addition, older adults' memory representations of scene context were biased towards congruent information.This was reflected in a greater number of schema-congruent errors when participants were asked to match correctly recognized objects encoded in an incongruent context [see also [11,12]].Older adults tended to erroneously associate a congruent scene context.Such a congruency bias has been observed for memory of object locations in scenes [17], but here we showed that it is of even broader relevance, affecting memory for the whole scene context.Analysis of response times overall corroborated age-related slowing.Retrieval processes, though, were similarly shaped in both age groupswith faster responses for distractors and no difference for objects encoded in a congruent or incongruent context.
Our findings are consistent with recent models on how prior knowledge shapes perception [10].Information processing is supposed to be first biased towards prior knowledge.But when an event is greatly unexpected, it elicits surprise, which, in turn, could signal the necessity to update existing knowledge, leading to enhanced processing.Although rooted in perception, this model may be applied to memory and is in principle compatible with previous accounts [5,28].Our data indicate that older and younger adults weigh unexpected information differently, but we can only speculate at which memory stages, i.e., encoding or retrieval, age-related differences emerge.In addition, it remains to be explored whether predictive processing might be specifically hampered by age-related general slowing of information processing [32].
Given the crucial role of active vision for memory [34], age-related differences in encoding could explain why the memory advantage for incongruent objects is attenuated in older adults.It is well documented that younger adults fixate incongruent objects earlier, longer, and more frequently [e.g., 35,36], while processing of congruent objects is reduced [37].We suppose that this fixation pattern also holds for older adults, leading to an overall augmented encoding of incongruent information.However, it might be less pronounced due to greater viewing of congruent regions [17] and possibly a reduced motivation for exploring novel information [38].Alternatively, retrieval processes might be biased toward congruent information due to age-related vulnerabilities in critical functional neural networks, involving in particular the ventromedial prefrontal cortex and the medial temporal lobes [5,7].
Our findings provide novel insights into the role of expectations for object memory in naturalistic scenarios and age-related vulnerabilities.We have shown that incongruent context efficiently boosts object memory across the adult lifespan and that the principle mechanisms are quite similar in older and younger adults.Schema violations can be beneficial for stabilizing memory performance in older age.However, the advantage is significantly attenuated and qualified by an overall congruency bias in older adults.We suggest that our memory data reflects critical age-related changes that can be related to recent predictive processing theories [compare [10,37]].These have put forward that functional efficiency of predictions crucially depends on the precision of the information input.While high precision is supposed to go along with a particularly high sensitivity to unexpected information, low precision is supposed to bias processing towards predictions.Given reduced sensory precision with increasing age, it appears consistent that, although violations of expectations remain a powerful way to enhance information processing, this mechanism is weakened.At the same time, an increased bias towards predictions, e.g., a more pronounced congruency bias, can be expected.To conclude, predictive processing theories seem well suited to contribute to our understanding of age-related functional changes and allow to consider vulnerabilities as well as stability within a coherent framework.
Fig. 2 .
Fig. 2. Effects of age group encoding context, i.e., congruent and incongruent, on object memory.Filled dots show the mean across observers and semi-transparent lines provide individual data.Older adults are plotted in orange, younger adults in blue.Error bars give 95% confidence intervals.
Fig. 4 .
Fig. 4. Mean response times as a function of age group and object type, i.e., distractor, congruent, incongruent.Error bars correspond to 95% confidence intervals. | 2023-09-18T15:05:12.024Z | 2023-09-01T00:00:00.000 | {
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251564014 | pes2o/s2orc | v3-fos-license | Development of Augmented Reality Application for Made-to-Order Furniture Industry in Pampanga, Philippines
The focus of the study was to develop a mobile application utilizing marker-less augmented reality for specific made-to-order products to support furniture and fixtures businesses. The study implemented mixed-methodology to properly identify the various stakeholders' considerations in developing the application. Interviews with key informants were conducted to ensure that the features were appropriate for the intended user needs, and selected ISO standards were used as evaluation criteria. The results indicate that the mobile application with marker-less AR technology was found to be highly acceptable by three evaluators (i.e., customers, owners, and IT experts). The study also highlighted the use of AR-related technology in this case, where marker-less has the potential to improve customer purchasing experience even further. Future studies may include using newer technologies to further improve the application. The study suggests that Augmented Reality technology could be used to connect specific businesses directly to consumers regardless of setting or context.
INTRODUCTION
The COVID-19 pandemic helps us appreciate the role of technology more than ever, particularly the things we take for granted, such as the way we interact with things and with one another. Rapid technological progress is a highlight during this period, particularly in multiple industries. Businesses, for example, are looking for new and existing technologies to enhance their business processes and practices. In recent years, technological innovations have also played pivotal roles and shifted paradigms in how businesses integrate and engage their customers about their services and products (Muratovski, 2015). E-commerce offers a great opportunity for any business because it delivers flexibility in terms of time and location. Customers use e-commerce applications to buy stuff online where there is a vast range of products and services (Geetha & Rathi, 2020). Also, it provides convenience and saves time since customers can buy any product from their offices, in the comfort of their homes, or anywhere in the world through the use of the internet (Rahman et al., 2018).
Augmented Reality (AR) is one of the emerging technologies for ecommerce in the 21st century (Yamin, 2019;Young & Smith, 2016). The popularity of AR on mobile devices is due to advancements in smartphone technology (Sudarshan, 2018). Also, AR has matured significantly over time, from the conceptual belief that augmented reality answers the important technical obstacles by creating applications that augment virtual objects in the real world that are entertaining and usable (Aljojo et al., 2020;Gjøsaeter, 2015;Young & Smith, 2016). With the number of devices that AR is capable of, as reflected in billions of downloads of Google Play Services, AR could change the way we do business online. For example, AR can bring life to a product and provide immersive content (Berryman, 2012;Yim et al., 2017;Oh et al., 2008). It also creates a sense of presence for objects that are not really there (Jung et al., 2015;Krevelen & Poelman, 2010;Yim et al., 2017). As a result, virtual objects merged with AR affect the decisions of possible customers a lot more than the images and videos usually used in traditional ecommerce experiences (Abrar, 2018;Flavián et al., 2019). Applications with AR offer a great experience and improve customer engagement by enabling the ability to customize 3 customers' preferences (Baytar et al., 2020;Berryman, 2012;Flavián et al., 2019;Richter & Raška, 2017;Yaoyuneyong et al., 2016;Yim et al., 2017;Oh et al., 2008) while allowing customers to explore their options while buying through an online application (Richter & Raška, 2017). Another example is that, AR-integrated advertisements are favored over traditional advertisements among potential customers (Bilgili et al., 2019).
Likewise, AR allows the improvement of the real world by placing a virtual object in real-time (Baytar et al., 2020;Carmigniani & Furht, 2011;El-Seoud & Taj-Eddin, 2019). Such innovations could also evoke higher purchase intentions (Abrar, 2018;Adam & Pecorelli, 2018;Baytar et al., 2020;Richter & Raška, 2017), provide new opportunities (Carvalho et al., 2011;El-Seoud & Taj-Eddin, 2019;Richter & Raška, 2017) for businesses to reach out to customers (Carmigniani & Furht, 2011), develop possibilities for the customer's engagement (Abrar, 2018;Baytar et al., 2020;El-Seoud & Taj-Eddin, 2019;Yaoyuneyong et al., 2016), add value to their products (Flavián et al., 2019), and stay ahead of the competition (Abrar, 2018;El-Seoud & Taj-Eddin, 2019;Oh et al., 2004). In addition, Lu and Smith (2008) said that AR can be used to provide more accurate information on products like furniture, jewelry, accessories, and other decorative products that need space and use volume (Carvalho et al., 2011;Oh et al., 2008). AR technology also provides the user the power to make better decisions when purchasing these kinds of products. Accenture discovered in a more recent survey on the use of AR in e-commerce that 88% of customers indicated that the use of AR in furniture businesses will likely increase their intention to purchase a furniture product (Accenture, 2014). This was supported by Flavián et al. (2019), which said that by incorporating AR technology, businesses can optimize the user experience for purchasing things (Oh et al., 2004;Oh et al., 2008). For example, AR technology can enhance the customer's buying experience in pre-purchase scenarios, wherein the customer may view a product in real-time located in their chosen places based on the color of the paint on their walls and ceiling and other decorations in their house. As mentioned by Baytar et al. (2020), AR could be used to improve the online shopping experience, which can lead to higher revenues (Bucko et al., 2018).
This prompts the study to develop a mobile application with a product preview using AR for made-to-order products. It aims to provide a platform for furniture and fixtures businesses to promote their products by giving customers the power to personally feel and evaluate the product at their own time and place, which in turn enhances customers' engagement and buying experience. Specifically, the study focused on 1) designing and developing a website; 2) developing and integrating a mobile-based application using AR technology specifically for furniture and fixture businesses; and 3) evaluating the compliance of the mobile application to International Organization for Standardization (ISO) 25010.
METHODOLOGY
The study used a mixed-method for identifying the design considerations of the AR mobile application. The study adopted the contextual design approach. This method was followed to ensure that it supports and meets the needs of the intended users (i.e., furniture business owners and potential customers) (Holtzblatt & Beyer, 1997). In the qualitative part of the study, an interview with three informants (i.e., production staff, secretary, and store manager) was initiated. These informants were selected due to their experience, involvement, and existing knowledge of the made-to-order furniture business. The informants were asked about the existing processes regarding their ordering procedures and the issues they usually faced during inquiries related to the made-toorder products. Questions related to the possible functionalities that might be helpful to their business were also asked. All the interviews lasted about 10 to 30 minutes. A clarification interview was also conducted during the development process in order to ensure that the application caters to the needs of the intended users. Video recordings were utilized with the proper consent of the informants to document all of their responses.
For the quantitative study, the developed application was evaluated by 35 customers (i.e., 10 potential and 15 existing customers), five staff, and five IT experts using a 4-point Likert scale (4 being the highest) and selected ISO 25010 quality standards (i.e., Functional Suitability, Usability, and Portability) (see Table 1). Responses from the questionnaire are coded as "strongly disagree", "disagree", "agree" and "strongly agree". A verbal interpretation ranged from 4 as highly acceptable to 1 as not acceptable. The evaluation was purposive in nature and was conducted in Pampanga in different locations over one week. The overall mean rating of the evaluations was then reported.
Development of the E-commerce Website
Based on the series of interviews among the key informants, the website should have the following main features: 1) display available furniture and services; 2) improve customer interaction; and 3) provide automation for processing customer orders and furniture delivery. Figure 1 showcases the main categories of furniture, namely: living room, dining set, garden set furniture, and day bed. According to the informants, these four (4) furniture types are their main products.
Figure 1. Home page
According to the informants, the website should also be able to showcase featured furniture ( Figure 2). This way, the future customer will be able to see what kind of furniture the business usually creates. The website also has the ability to purchase products online, track the production status of made-to-order furniture, and customize and personalize furniture before purchase. This is consistent with the study of Lu and Smith (2008) that found that these kinds of features would greatly increase the customer's comfort in buying and purchasing furniture online.
Development of Mobile Application and Integration of AR Technology
One of the main problems on an e-commerce website was that the videos and pictures that advertise certain products like furniture are different from the actual product. Specifically, size and color that complement the interior of the house. This warranted the study to use AR technology to help both the customer and the furniture business owner ease the problem mentioned. Suggestions based on the interviews related to these served as a guide in the development of the mobile application with AR technology. The prototype was developed in Unity 3D, while the AR technology integration utilizes the Vuforia engine. The 3D drawings initially used in the prototype were provided by the informants and recreated and modeled using the SketchUp software. This integration paved the way for the application to be capable of previewing the products directly to the desired location using the actual sizes and designs before purchase. According to the informants, this feature is needed as it can help both the customer and the business owner visualize the desired product, thus enhancing the customer's buying experience (Figure 3). 7 Figure 3. Screenshots of the mobile AR application Table 2 shows that the application is rated as highly acceptable among the respondents of the study. This rating was observed in several aspects of the ISO 25010 quality standards. For functionality, the product was able to provide real-time experiences in terms of the product position (Mean = 3.84, n = 35) and scale (Mean = 3.84, n = 35), which was in previewing the actual product. In terms of its product preview feature, the application was also rated as being consistent with real-world (Mean = 3.80, n = 35). Additionally, the ability to monitor the product status and development was evaluated as highly acceptable (Mean = 4.0,n = 35). This indicates that the AR mobile application provides a real-time experience for both customers and business owners, making the process of purchase and visualization seamless in terms of customer engagement and buying experience. For usability, both the website and the application were user-friendly (Mean = 3.72, n = 35), provided clear instruction, particularly in terms of its navigation features (Mean = 3.76, n = 35), and the ability to showcase and preview the product in a desired location and track production status. The usability of the application 8 was evaluated as highly acceptable in terms of its ability to visualize products in real-time that improve the buying experience for both the customer and the business owner. For portability, the application can easily be moved to another location and can be accessed anytime using an internet connection. This indicates that the application was readily available anytime and anywhere whenever the customer felt the need to evaluate the product inside the house, in the garden, or any other place where they desired.
CONCLUSIONS AND RECOMMENDATIONS
The mobile application was evaluated as highly acceptable across three selected ISO 25010 standards. It showed that AR technology provides a way for businesses to enhance their customers' buying experiences. The study has also shown that real-time status updates and product previews based on the desired location of the made-to-order product were the most sought-after features in the furniture business. The study also highlighted the use of AR-related technology in this case, which has the capability to further improve the customer buying experience. Future studies may include using newer technologies (e.g., mixed reality) to improve the application, as well as using the same technology on other business entities that require product visualization.
PRACTICAL IMPLICATIONS
The study showed that AR-related technologies could be helpful to local industries in the Philippines. It also shows that such technology can be used by specific business models like made-to-order furniture, where instantaneous replies and spatial information are needed. Moreover, the study also shows that businesses, regardless of context, can connect with their potential consumers using emerging technologies like AR. | 2022-08-16T01:16:00.432Z | 2022-08-13T00:00:00.000 | {
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222473674 | pes2o/s2orc | v3-fos-license | THE TRANSLATION OF PHRASAL VERBS IN THE SUBTITLING FROM ENGLISH TO PORTUGUESE IN THE SERIES BEWITCHED
This study aims to determine how the phrasal verbs used in English in the TV series Bewitched convey the same meanings of the translation to Portuguese in the subtitles. The research follows the model of Vinay and Darbelnet (1989) with focus on part of their theory called Oblique Translation. The study is exploratory and the episodes of Bewitched series selected were from the first and the last season, aiming at comparing the English phrasal verbs with the respective translation in the subtitles to Portuguese. The software AntConc was used to locate phrasal verbs through scripts; the software Notepad++ was used to compare the English and Portuguese versions; and the Excel software enabled the researcher to organize the data found. The results are in accordance to Vinay and Darbelnet’s theory (1989, 2004), emphasizing that the phrasal verbs with the particles up, away and off found in the corpus were translated using the processes of Oblique Translation instead of literal translation.
Linguagens -Revista de Letras, Artes e Comunicação -ISSN 1981-9943
Blumenau, v. 13, n. 2, p. 289-300, maio/ago. 2019 DOI: http://dx.doi.org/10.7867/1981-9943.2019v13n2p289-300 290 There are many languages in the world, each one with its own particularities. For people who speak Portuguese as a first language, the use of phrasal verbs when learning English as a foreign language may sometimes be challenging to understand because this type of structure does not behave like any other structure in Portuguese. According to the Talmyan typology (TALMY, 1991;2000), English is a Satellite-Framed language: most of the verbs expressing some sort of location idea operate and function around a ''satellite" structure, in other words, a prepositional or adverbial particle; whereas Portuguese is part of the group of languages which are Verb-Framed, in which verbal structure regarding location is expressed through a single verbal unit. Phrasal verbs follow the Satellite-Framed structure, therefore their translation into Portuguese may fit what Cappelle and Loock (2017, p 4.) series or film subtitling. The search was conducted through Base de Dados da Capes. One example of study is the dissertation of Moraes (2015). Her study analyses how subtitlers attempt to maintain the cultural expression in the translation of collocational patterns, phrasal verbs included. Thus, this study aims at determining how the translation of English phrasal verbs in the first and the last season of the series Bewitched has been featured to the target language in Portuguese. It is an exploratory research study, encompassing the following methodological procedures: a) selecting the episodes to be analyzed in Bewitched in English (original) and in Portuguese (target language); b) using a Corpus Linguistics method to find the phrasal verbs in each episode through ANTCONC 3 (ANTHONY, 2004) software; and c) carry out a case study to compare the original scripts with phrasal verbs with the translation in the subtitles to Portuguese, checking how they were translated: literally or using oblique translation, according to Vinay and Darbelnet's studies (1989;2004) .
The article is divided into the following parts: a) a description of the theory in which the study is grounded, followed by some key information regarding phrasal verbs and subtitling; b) the description of the methodology used; c) the analysis and discussion of the results; c) the concluding remarks. Campos (1986) argues that translation is an action of transferring from a language to another a specific written text. The original language can be referred to as "source language" and the final destination of the translation as "target language". Barbosa (1990) points out that there are two kinds of translation: literal translation, that is, word-by-word translation; and free translation, which is the transmission of the context itself.
THE UNIVERSE OF TRANSLATION AND PHRASAL VERBS
Oustinoff (2011) elicits the three kinds of translation coined by Jakobson: Intralingual translation: a reformulation of the speech; Interlingual translation: the translation from a language to another, properly said, and Intersemiotic translation: an interpretation of the linguistic signs (words) using non-linguistic signs (images, for example).
This study will be grounded by the techniques proposed by Vinay and Darbelnet (1989;2004), working with Interlingual translation. According to Vinay and Darbelnet (1989;2004), there are two axes in which the translation can be distributed: direct (literal) translation and oblique translation (not literal or free). Direct translation implies that the isolated words in the original language are translated one by one into the target language. It should only be used if the translation has the same meaning as in the original language. That may also be the case where the original language may borrow some expressions of the translated language when there is no equivalence (BARBOSA, 1990).
Oblique translation, on the other hand, makes use of a number of resources that assist in the process of achieving a better result in the translating process (VINAY; DARBELNET, 1989;2004) After, we have Modulation: changes the point of view of the construction of a clause or phrase; used when a translation, even if seeming appropriate in grammatical terms, is considered unsuitable or awkward in the target language. For example, the sentence in Following, Equivalence: maintains the same situation in both languages, even using different grammatical structures; in other words, whenever a linguistic approach is no longer suitable to carry out a translation, the translator can rely on other procedures such as loantranslations, neologisms, idioms, metaphors, etc. The translation of proverbs is an example.
Finally, Adaptation: modifies the translation to create a version of the text according to the cultural context of either language. For example, names of foods may sometimes undergo adaptations, because food is intrinsically a cultural concept. It is important to state that, according to Vinay and Darbelnet several of these processes may occur within the translation of a same sentence.
PHRASAL VERBS IN THE ENGLISH LANGUAGE
Phrasal Verbs are part of a larger category known as Multi-Word Verbs (BIBER et al 2010). Multi-Word verbs include also Prepositional Verbs, formed by a verb and a preposition, but, unlike phrasal verbs, the adding of the preposition does not change the meaning of the combination.
According to Eastwood (2005), a phrasal verb is a set consisting of a verb plus a prepositional or adverbial particle (e.g., in, down, off, up). A similar definition is brought by Harmer (1998, p.39): "[…] they are formed by adding an adverb or preposition (or adverb and a preposition) to a verb to create new meanings". Cowan (2008, p. 171) categorizes phrasal verbs in two types: "transitive phrasal verbs" and "intransitive phrasal verbs". He explains that transitive phrasal verbs need an object and have the possibility to change position within the sentence. When change of position occurs, it is named a "particle movement rule". Cowan shows the example below, where it is possible to observe that the particle "up" goes to the end of the clause without changing the meaning. On the other hand, Intransitive phrasal verbs do not need an object. An example is "The plane took off". Cowan (2008, p. 173) calls the case in this example as a "pure intransitive phrasal verb" category because the adverb cannot be separated from the verb.
HOW THE SUBTITLING PROCESS OCCURS
According to Nobre (2002), for the subtitling process to be performed, it is necessary to follow a few steps. The first thing to do is to compare the original script with the target language and do the transcription of the lines. Then, it is necessary to mark how long each speech lasts. After marking the time, the composition of the subtitles is made and they are inserted inside the film or series. When everything is approved by the client, the subtitles will finally be recorded over the film or series. Luyken (1991) and Nobre (2002) also describe some responsibilities attributed to the subtitle translator: 1) Taking the context into account while translating one language to another; Gorovitz (2006) points out that the translator should be loyal both to a will and to the expectations of its recipients; 2) Transcribing a text from oral to written, remembering that this text does not have the approval of the author (GOROVITZ, 2006); 3) Creating subtitles accounting for the necessity of a textual reduction due to the time as well as the number of characters allowed, the addition or removal of information, the font and size to be used for the subtitles.
METHODOLOGY
Before conducting an analysis regarding whether the phrasal verbs present in the episodes are translated literally from English to Portuguese or if they convey other meanings, the first step is recognizing which phrasal verbs are present in Bewitched. For this, the AntConc program, a software used to analyze the corpus was used. It is compatible with txt. From the data found, the table created for each episode contains a column with the phrasal verb found from the software; a second column, with the meaning of the verb phrasal searched for phrasal verbs dictionaries one by one, manually; in the third, its official literal translation in Portuguese, based on bilingual dictionaries (English-Portuguese) and the websites Word Reference 6 and Cambridge Dictionary 7 ; and in the last column, as it was translated to subtitling (in this case, it was done manually too, reading the scripts in Portuguese and reading the scripts in English to find the same event and notice the differences among them). The objective of these tables is to compare the data in English and Portuguese and investigate how the translation is presented.
The particles searched for in AntConc were up, off and away. They were chosen because tests conducted with the software showed that these were the particles more frequently used in the original dialogues. After that, a second document in Excel was created to separate the original script with each phrasal verb in English, its context and its translation to Portuguese. A first column was created to represent the number of the episode; the second, the phrasal verbs found by AntConc; the third, the clauses or phrases with the context of the phrasal verb selected to be analyzed, and the last one the same context translated in Portuguese. A sheet was created for the first season and another one for the last season.
ANALYSES OF THE PHRASAL VERBS FOUND
The analysis of the phrasal verbs found take into account the processes described by Vinay and Darbelnet (1989;2004): transposition, modulation, equivalence and adaptation.
According to Kövecses (2003, p.6), metaphors are "[…] extremely simplified words, but it is exactly the simplified nature of these words that enables us to make use of parts of it in 6 Available at https://www.wordreference.com/. 7 Available at http://dictionary.cambridge.org/pt/dicionario/ingles-portugues.
Linguagens -Revista de Letras, Artes e Comunicação -ISSN 1981-9943
Blumenau, v. 13, n. 2, p. 289-300, maio/ago. 2019 DOI: http://dx.doi.org/10.7867/1981-9943.2019v13n2p289-300 295 creating more abstract ones". Some emotions, for example, are also better understood through metaphors because, sometimes, there is not a word that sums up that situation in which the person is living. Because of that, she or he uses metaphorical expressions. The phrasal verbs turn off , get off and tie up occur in the corpus in a metaphorical way, being translated using the Equivalence and Modulation processes, respectively.
Let us regard the following examples taken from the series:
(1) Why don't you turn off the Kravitzs' door until we check it for shorts?
Desligue a porta dos Kravitz para que a gente a cheque.
(2) Whatever Serena turned on, I am gonna turn off.
3) You ready? I am just waiting for you to get it off your chest.
Está pronto? Estou só esperando que você fale.
O Sr. Stephens está ocupado.
In (1), we can observe that the phrasal verb has been translated literally, that is, according to the signified, switch off. However, if we look at the meaning of the translation applied in (2) we will see that the idea of switch off is used as erase, in the sense of the person named Serena has started some confusion that should soon be controlled. The metaphor here was used as a representation of what is being said and assists in the result of the conversation that would involve romantic feelings. In Portuguese, a word for romantic feelings is "flame".
If this word had been applied literally, the correct word to remove the flames would be the word "apagar" in this target language.
In (3), the phrasal verb "get off" is combined with "your chest" forming an idiomatic expression in the English language. This idiomatic expression is metaphorical, using the image of 'removing' something from your chest to externalize an idea or thought. However, the metaphor is not carried into the target language, and the translator chose to modulate the expression into something more appropriate in Portuguese.
Another phrasal verb, tied up (example 4), has suffered modulation in the translation.
In the English language, it is a metaphor in which the idea is to represent that Mr. Stephens is Linguagens -Revista de Letras, Artes e Comunicação -ISSN 1981-9943 Blumenau, v. 13, n. 2, p. 289-300, maio/ago. 2019 DOI: http://dx.doi.org/10.7867/1981-9943.2019v13n2p289-300 296 really occupied with doing another thing and the translation of the metaphor "amarrado" would be a possibility for Portuguese speakers. However, it would not fit the formality of the situation expressed by the contextualization of the politeness marker 'I'm afraid'.
The phrasal verb pick up appears to have undergone some different translation processes in the series subtitling. Examples 5, 6 and 7 illustrate that the metaphorical meaning of pick up was used in the original, not the literal meaning of "lifting from the ground". The translation in example 5 uses literal/direct translation; the translation of example 6 makes use of the Equivalence process, using the rhetoric figure known as metonymy.
(5) I'll pick you up at the office at […]
Eu o pegarei no escritório às […] (6) One of the cars will pick him up.
Onde vamos buscá-las?
In example (7,) the translation undergoes the process of Modulation, since if using the verb that was used in example (5) ("pegá-las") the idea of the sentence would change. The verb "buscar" in Portuguese entails the idea of picking someone up using a vehicle, which was what the characters were going to do in the context.
Another noticeable feature is that in all these examples (5-7) the phrasal verbs were translated in a diachronic way, to reflect how people spoke in the 1960s. Back then, people had the habit of conjugating Portuguese verbs correctly using the object pronouns more formally. This accounts for the use of the Transposition process in the three examples.
Another process that appears to be used in the translation of some phrasal verbs in the scripts of the series is the Adaptation. Examples 8 and 9 illustrate the use of the phrasal verb give up: (8) I love you, and I can't give you up.
(9) You mean you're willing to give him up? Well, you could put it that way.
Quer dizer que está disposta a abrir mão dele? Bem pode-se dizer assim. The original meaning of the phrasal verb give up is to stop doing or having something.
In example (8), the Adaptation process used the concept of "losing" someone, a collocation very commonly used in the culture of Brazilian Portuguese when speaking about breaking a romantic relationship. Just as in examples (5), (6) and (7) the pronoun " -la" adapts to the time the series was produced. In the current days it may have been translated into "perder você".
The use of the pronoun in the Portuguese version also signals that the Transposition process has been used by the translator.
Example (9) refers to the adaptation process in which the original has been translated into a more updated version, using an idiomatic expression in the target language. One translation that could have been used would be "desistir", but the translator decided to use "abrir mão", probably better suited for the situation.
Another example of a phrasal verb that can be found metaphorically is go away (example 10). The usual meaning of it is to leave; to run away.
Juro que ela não sumirá.
In this part of the conversation, Samantha and James talk about being late to visit the Tower of London ("it" refers to the tower). To keep the idea of the comic part of the scene the translators chose to translate as " it will not disappear"; the adaptation of the translation into Portuguese carried the intrinsic meaning of "sumir" in Portuguese, which has a definite idea of disappearing for good.
Translations are created thinking about the meaning and the feeling of the message. In the case of example 11, we have the literal meaning for tapper off, that, in Portuguese, is "se estabilizar". But, sometimes, this expression is more formal than informal. Because the scene was more informal, the translators adapted another idea for this phrasal verb, using the Transposition process.
Vou me adaptando aos pouquinhos.
This was a way for people who were watching to get acquainted with the scene and at the same time be able to experience what was happening and feel what the character was trying to get across with the expression. Here is also a case of the Adaptation process, using a diminutive expression " aos pouquinhos", which means little by little, so that the informal tone could be achieved.
The phrasal verb hurry up, in example 12, is another instance of Transposition. The translator chose to use a different word class to express the meaning of the phrasal verb, an adverb of time instead ("logo" means "soon").
(12) Well, get rid of him! And hurry up, it's cold in here.
The Phrasal Verbs before analyzed are a good instance of what Oustinoff (2011) predicted about the necessity of speeches to be reformulated for a better understanding in the target language. The processes described by Vinay andDarbelnet (1989, 2004) were present in the occurrence of these multiword constructions, playing an important role in the quality of the subtitling presented to the audience; as well, it is a possibility that the processes were also applied so that the subtitles would fit not only the meaning intended but also the time frame the subtitles feature.
CONCLUSION
This article reports a study of phrasal verbs found in the TV series Bewitched, examining how the translation of English phrasal verbs in the first and the last season of the series Bewitched has been featured to the target language in Portuguese. The methodology used to search for the phrasal verbs was a Corpus Linguistics tool, AntConc, and the scripts of the first and the last season were used. The phrasal verbs with the particles up, off, and away were chosen following the frequency criteria.
The results of this study confirm that the phrasal verbs were not translated literally from the English Script into the Portuguese subtitles, but could be analyzed into what Vinay and Darbelnet (1989;2004) call Oblique Translation processes: adaptation, modulation, equivalence and transposition.
It is believed that this work contributes to the area by making people aware of the process of subtitling for the Portuguese language, as well as for any other language, that is much more complex than one imagines and that it surely requires a deep analysis. Further developments of this study could analyze the translation of all phrasal verbs in the series script, to check if the processes are being used instead of the literal translation, characterizing the subtitling process of this series more thoroughly. | 2020-10-15T16:39:27.557Z | 2019-08-31T00:00:00.000 | {
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257795201 | pes2o/s2orc | v3-fos-license | “Just Standing Still”: A Qualitative Study on Adolescents’ Experiences of School Closures Due to Emerging COVID-19 in Bissau, Guinea-Bissau
The COVID-19 pandemic affected the lives of children in a myriad of ways across the world. It exposed and aggravated existing inequalities between children within countries and across continents and hampered education. In Guinea-Bissau, school closure was one of the first restrictions implemented to confront the emerging pandemic. The aim was to describe and analyse the experiences of adolescents of school closures in the capital Bissau, their concerns about their future and manifestations of inequality. Data were collected by semi-structured, open-ended interviews with 30 adolescents aged 15–17 years three months into the pandemic during an enforced state of emergency. A thematic analysis identified five themes: appreciation of education, feeling left behind, being stuck in confinement, suggestions for support, and a disrupted future. The results highlight global rather than local inequalities in the demographic, manifested by a lack of targeted educational support for public and private school students; they knew about such efforts elsewhere. The school-attending participants suggested ways to mitigate disruptions in their education, while those out of school aiming to return saw their possibilities fading away. They appreciated education for personal and national benefits, and participants worried about the long-term effects of the pandemic. The study highlighted education loss for all and disrupted future expectations.
Introduction
The COVID-19 pandemic has affected, to a varying extent, all countries across the globe since its start in late December 2019. It has resulted in disruptions in services and the daily activities of people across all layers of societies, albeit unequally [1]. Initially, children were considered to have a relatively low risk of developing severe symptoms of COVID-19 and lower case fatality rates than other age groups [2]; moreover, children were not seen as the main drivers of community transmission [3]. Nonetheless, children's lives were affected unprecedentedly, with existing inequalities being spotlighted and exacerbated nationally and across continents [1,[4][5][6][7].
Many governments implemented school closures early on to curb the rapid propagation of the pandemic. Rapidly, evidence was accumulated that such measures harmed children's health and well-being, including their physical and mental health and life satisfaction [8,9]. Additionally, the school closures hampered education, while efforts in increased online teaching exposed the existing digital divide within the demographic [10,11]. In sub-Saharan Africa (SSA), with adolescents aged 10-19 years comprising 23% of the total population [12], school closures resulted in higher dropout rates, with disadvantaged students being driven into labour, and subsequent dampened future aspirations [13]. Diverse approaches were applied for remote learning in the sub-continent, including class lessons on radio, television and online platforms to avoid education loss during school closures [14]. Yet, the learning gap is estimated to grow due to the unequal access to such tools and cuts provided by education budgets [4,9,[14][15][16][17]. Furthermore, school closures increased the frequency of child marriages, child pregnancies, and violence against children and resulted in worse overall health [7,8,[18][19][20].
This research takes notice of childhood and youth studies which seek to allow children and youth to express themselves to understand the myriad of interrelationships that daily reinforce their social practice [21][22][23]. Childhood and youth studies are undergoing a theoretical and conceptual revision, creating fertile ground for critical thinking aiming at decolonising respective fields of study [24][25][26][27]. Conceptualisations of child and youth agency flourish with increased emphasis on interdependency and relational aspects [21,28], and concepts like waithood and stuckness reflect how underprivileged children and youth deal with their challenges [29][30][31][32]. Further, scholars pay increased attention to the future aspirations of the young and their emotional expressions of anticipation and hopefulness [33,34]. The commonly used binary division between children and youth into groups belonging to the Global North and Global South, respectively, risks homogenisation and a lack of differences within each group and the exaggeration of differences between groups [35,36]. Such conceptualisations are noted to be related to practices that propagate the colonisation of knowledge, namely that of the Global North deciding epistemology in terms of what kind of knowledge is of value and in what way that knowledge should be gained [37].
During the first year of the COVID-19 pandemic, most published research on children's experiences stemmed from high-income settings, primarily relying on digital platforms [38]. As part of a decolonising effort to disseminate knowledge, we have elsewhere reported on the impact of the emerging COVID-19 pandemic in Guinea-Bissau on the lives of begging Quranic school boys [39], and how adolescents understood, and to what extent, the pandemic had affected their lives [40]. School closure was one of the governmental measures taken during the early phase of the pandemic, even before the diagnosis of the first case. Here, the aim was to describe and analyse adolescents' lived experiences of school closure in the capital Bissau as a preventive measure in the early phase of the pandemic. Further, the aim was to hear their opinions on education, if and how school closures manifested existing inequalities within the demographic, and how the pandemic caused concerns about future aspirations.
Setting
Guinea-Bissau is a fragile low-income country in West Africa characterised by political instability, and it ranked 177 out of 191 on the Human Development Index 2021/2022 [41,42]. Since 2000, school enrolment has been progressing; however, the quality of education is deplorable due to the lack of school materials and inadequate teacher training [43][44][45]. Economic inequality marks access to education, and repeated teacher strikes keep public schools closed for months each year [46,47].
Like other countries in the West African region, Guinea-Bissau has been confronting the COVID-19 pandemic. In response to the threat, the government implemented strict restrictions on movement on 17 March 2020, while the first two cases of COVID-19 were confirmed on 25 March 2020. The government declared a state of emergency on 27 March 2020. Free movement and market activities were initially allowed between seven and eleven o clock but gradually extended until eight o clock in the evening as the pandemic unfolded. Further, there were restrictions on the number of people on public transport, which was initially banned but later allowed if people respected the measure of two metres of physical distancing. Furthermore, private cars were not to have more than three passengers. These restrictions were valid until 10 June 2020 and then gradually lifted. During this period, schools were closed, both public and private schools, and it was not clear when schools would be allowed to reopen. At this initial stage of the pandemic, the Bissau-Guinean population generally complied with the lockdown measures, despite its severe impact on daily lives, including those of adolescents [40]. At the time of the study, the number of confirmed COVID-19 cases was 1614 nationwide, but these were mainly in Bissau (91%), with 21 deaths being reported [48].
Research Design
Two authors (ZJ and BI) collected data on 20-24 June 2020 in 5 out of the 47 geographic areas of Bissau, the capital of Guinea-Bissau. Their selection was based on data from the national 2009 population census, but they are among the most populated ones in the capital area. Local knowledge of the setting and attention to the diverse ethnic background characterising each urban area also guided the selection. Collaborators, known to be influential people linked to associations in each neighbourhood, established contacts and permission to interview adolescents in each area. They helped to identify participants aged 15-17 years with a keen focus on gender and school attendance. In each neighbourhood, six adolescents were selected, three boys and three girls; of them, two attended public school, two attended private schools, and two had not enrolled for the current academic year. The purposive sampling aimed to maximise the diversity of participants' experiences, not to have a representative sample [49].
The two authors interviewed 30 adolescents, giving due attention to government recommendations on COVID-19 preventive measures, such as using masks and keeping their distance. They conducted, individually, 15 interviews each, face-to-face, at a place chosen by the participants. Before the interview, they informed the participants about the study, its aims and the approximately 20-30 min interview format. The research team contextualised the semi-structured, open-ended interview guide formulated to facilitate the data collection. It was designed around eight themes, with several sub-questions per theme. The themes were: general, background, education, family, neighbourhood, friends, internet and future. The interviewers translated the guide into the participants' lingua franca, Kriol, to enable constructive discussion, particularly on issues of concern to the respondents. It was also conducted to standardise the usage of language and the meaning of words, taking into account Kriol's various ways of expression.
The two interviewers transcribed the audio-recorded interviews and translated them into Portuguese. In collaboration with one of the co-authors (JE), the first author (FNB) listened to all the interviews and translated the Portuguese version into English; subsequently, a content analysis of the data was performed in Atlas.ti using inductive thematic coding, also referred to as open coding [50]. In line with the grounded theory theoretical approach, the authors derived codes from coherent parts of the text rather than searching for preconceived ones. Thereafter, the codes were sorted using axial coding and merged into more extensive categories or themes [51]. After that, the key themes related to school closures and education were analysed to understand the adolescents experiences of school closures.
Ethical Considerations
Children's rights regarding the preventive responses to the COVID-19 pandemic include keeping them visible and listening to their voices [7,38,52]. Collaborators identified adolescents who fulfilled the study criteria in the five urban areas. Considering their mature age, they were invited to participate, and came to an agreement on a date and place for an interview with two authors (ZJ and BI). At the beginning of the interview, respondents gave their verbal consent for participation. Parental agreement was not requested, considering that the participants were 15-17 years old.
The study is one component of a larger research project that aims to articulate and explore manifestations of inequality among Bissau-Guinean adolescents in and out of school [10,39,40,46,53,54]
Results
At the time of the interviews, no participant knew somebody who had become sick with COVID-19. The lockdown period was coming to an end while the schools were still closed. Five interlinked central themes emerged when considering the effect of school closures on their lives: (1) appreciation of education; (2) feeling left behind; (3) being stuck in confinement; (4) suggestions for support; and (5) a disrupted future.
Appreciation of Education
Although none of the questions raised inquired directly about participants' views on the value of education, both in-school and out-of-school adolescents explained how greatly they regarded education. The out-of-school participants wanted to return to school if given the chance and considered education essential for the general development of young people. One out-of-school boy (aged 17 years) said: "When you don't have school, what are you like: a branch, a tree without roots, because a tree without roots, . . . it can never develop if you don't have school, you can never develop in society". Others were more concerned with employment and their possibilities for future survival; for instance, an out-of-school girl (17 years) said: "I consider school important because it is the school that will help me tomorrow [in the future]." Furthermore, a few underlined that education was crucial for their country. "I don't like to see anyone standing still," a boy (aged 17 years) attending a public school stated, and he added "I want that we all have a good future . . . so that everyone can work for their country. That everyone can have an education . . . and be worthy citizens."
Feeling Left Behind
Independent of the form of schooling, public or private, the participants were upset about the school closures. The participants who had enrolled before the school closures claimed they had not received any assistance from their schools. A girl (aged 17 years) enrolled in public school, said: "We are just standing still. The school has not found any other way to teach us." A boy (aged 16 years) also attending public school had the same complaint: "I feel the lack of support from the school and teachers. Now, there is no support from the school to the students." The participants were aware that alternative online platforms were used elsewhere to provide education; however, they were deprived of such solutions due to a lack of internet access and suitable devices. A private school boy (aged 15 years) explained that "in other countries, people study through the internet, but we in Bissau do not have that, we don't have it . . . lack of internet, lack of mobile, computer . . . we lack these things." A girl (aged 16 years) attending a private school wanted access to online classes: "If the internet in Bissau were good, I would like the teachers to do online classes . . . even if they don't teach new subjects, we could be active in those subjects that we had already received." Most participants were unaware if any schools in Bissau were using alternative approaches to support their students. A public school girl (aged 17 years) mentioned she had heard about students who received some support, "but our school, they don't give anything. If you go to our school now, you see people sitting there, but they don't give us any tasks." In rare cases, if someone mentioned support, it was thanks to a particular teacher. A girl (aged 16 years) in a private school gave the information that some teachers had tried to support their students through social media, but student participation was limited because of the lack of internet. Her classmates created a group on WhatsApp, but she did not participate much because she lacked internet: "I don't know if other people have it, but my classmates created a group with the biology and ecology teacher to help us, like, if we had doubts, we send audios, to take our doubts away." A few participants mentioned that they had received some support with their studies from family members. A boy (aged 17 years) who attended public school noted "my uncle sometimes takes the board out and takes the initiative to exercise the subject with us." Another boy (aged 17 years) attending public school said "I stay at home and read books on my initiative. I had my brother who used to tutor me, but now he has stopped [helping me]."
Being Stuck in Confinement
The lockdown, including the school closures, changed the adolescents' daily routines, with the participants mainly staying at home. Due to police violence, the participants and their families were obliged to remain home except for a few hours daily [40]. Due to the restrictions, most participants who had been employed within the informal sector had stopped working. Several had the same story to tell, as did a girl (aged 17 years) in public school: "I used to go to school in the morning; when I left school, I studied; when I finished studying, I would go out to sell on the road. Now I don't do anything; I don't sell; I only sit at home." A few participants said they had taken up new employment and skill training during the school closures, and several mentioned that they or their friends had travelled to rural villages to help with farming activities. Nonetheless, most participants reported mainly spending their time at home during the school closures, during which some increasingly took part in family chores. Some explained that they had taken on new tasks, such as educating younger siblings and informing them about how to protect themselves from the virus.
A few participants mentioned that thanks to the school closures, they had the opportunity to spend more time with their families and strengthened their family ties. A girl (aged 16 years) enrolled in a private school argued "we didn't have those family ties before, but today, when there is nowhere to go, we sit, we stay having fun at home, going less out on the street." However, most participants discussed being bored and having nothing to do except stay home during the lockdown. Many mentioned that they missed their friends and classmates or, as one participant, a girl (aged 16 years) enrolled in a private school, said, "I used to go to church and play with my classmates, but now, nothing. I just stay at home," Likewise, a boy (aged 17 years) attending a private school complained "I practically stay at home without doing anything. I felt more comfortable when I went to school because I read subjects and did all the school tasks. But these times, I only stay at home doing nothing. That is very complicated." Despite being stuck at home, they imagined alternatives.
Suggestions for Support
The participants who had enrolled before the school closures wanted support from their schools. Most of them presented ideas on how the schools could assist them in continuing their education during the lockdown; e.g., they suggested the schools could reopen with some social distancing measures in place. A girl (aged 17 years) enrolled in public school stated "I think they should reduce the number of students per class and make the use of masks compulsory; maybe that could help. Place buckets of water for handwashing at any time and avoid crowding." A boy (15 years) enrolled in a private school argued that the school could help students and staff "to maintain hygiene in the school, to monitor the students entering the school, to bring that thermometer to see those who have that disease and those who don't have the disease." Many participants also suggested that they would have liked to have received some homework from their teachers. One girl (aged 17 years) enrolled in a public school said "they were supposed to go to school and give us some homework for us to do at home and if we finished the homework, we should bring it back to them." Another suggested that the class leaders could act as intermediaries, bringing the homework between the teachers and the students. A girl (aged 15 years) attending a private school argued against studying at home "because we, when we stay at home, we don't manage our time, we stay playing." A few participants mentioned distance learning through the radio as a possibility; for instance, a girl (aged 17 years) enrolled in a private school argued "I would like the teachers to go to the radio stations to give classes. We can tune in and follow if they indicate the radio station." When suggesting how to continue with their studies despite the pandemic, some students mentioned that the school fees needed to be lowered to secure school access for all adolescents. A private-school boy, aged 15 years, said "there are others who can't afford to go to school because of money. They should reduce the price of tuition and fees so that all of us can go to school." Others pointed out that their parents or guardians had already paid the school fees and, therefore, they should be allowed to finish the school year. A private school girl (aged 17 years) said "I want them to help us finish the school year. After that, they can continue with the restrictions. Our parents paid the enrolment fees; it's bad if we don't finish the school year."
Disrupted Future
Many participants were worried that the school closures would result in a delay in their education. "It [the pandemic] has delayed me because if we didn't have coronavirus, I would be studying right now; we would have already taken the exams," explained a boy (aged 15 years) enrolled in a private school. A girl (aged 17 years) attending a public school argued "we are not going to school; we are sitting here and standing still for a long time. This causes us delays because time waits for no one. Age is passing-and we are standing still". It was nothing new for the public school students that their school was closed; before the pandemic, their schools had been annually closed for long periods because of teacher strikes. However, everything else was closed this time, with the risk of additional delay in education. A girl (aged 17 years) enrolled in a public school was worried: "I'll have to repeat grade eight for the third time in a row, not because of successive failures, but because of the instability of the system." Most of the out-of-school students had hoped for the possibility to enter school again; however, with the devastating economic effects of the emerging pandemic on the family economy, that dream seemed far-fetched. A participant argued that the pandemic would not change anything at all for the future; everything would return to the same conditions.
Discussion
This qualitative study aimed to understand to what extent the school closures during the state of emergency due to the unfolding COVID-19 pandemic affected the daily life and the future dreams of 15-17 years old adolescents in a low-income sub-Saharan country here in the capital Bissau, Guinea-Bissau. How does it manifest and expose existing inequalities? Key findings of the investigation include the participants' broadly shared appreciation of education, a lack of educational support during the lockdown independent of attendance in public school vis-à-vis that in private schools, their numerous suggestions for ways of assistance and almost universal worries about disrupted futures.
The participants, including the out-of-school ones, appreciated education's multiple roles. They underlined its importance in securing future employment for personal development and national prosperity. While one literally praised education as a route to "worthy citizenship", others expressed similar thoughts. Like their peers in South Africa and Uganda [13,55], the study participants in Bissau appreciated education as a route out of poverty and to a better future. Scholars have noted the high esteem attributed to education among general citizens in Guinea-Bissau, resulting in efforts to establish community schools to navigate the frequent strikes in public schools [43,56].
Independent of enrolment in private or public schools, the students lamented the lack of support for their studies during the lockdown. The COVID-19 pandemic reinforced the digital divide [11,15]; yet, for a successful implementation of digital solutions, the school, teachers, and students must be adequately equipped and prepared [57]. Although private school students in Bissau have some advantage in terms of access to digital technologies and the internet compared to public school students [10], there was, in fact, no difference between the support provided in these schools in Bissau. Occasionally, the in-school participants reported attempts to offer support which were made by an individual teacher or a group of students; in all cases, these were unsuccessful for the respective interviewee.
None of the in-school participants received practical support during the lockdown from their school, which is in line with the worst scenarios [6,11,15]. In contrast, in Ghana, some schools facilitated remote learning, yet depending on family resources, students benefitted unequally [58]. The Bissau-Guinean adolescents were aware of alternative online education platforms used elsewhere. Despite the challenging circumstances, they suggested myriad ways to mitigate the consequences of the pandemic on their education. While upset about the lack of support, several participants talked in terms of rights, claiming they were entitled to support; instead, they were stuck.
Almost all participants felt they were standing still, missing their former routines of life, not least going to school. The notion of stuckness is highly contingent on the passing of time, as was reflected in a girl's lament that she and her peers were getting older while they were "standing still." According to Alcinda Honwana [32], the concept of waithood refers to the "prolonged period of suspension when young people's access to social adulthood is delayed or denied." Marjoke Oosterom [30] points out that "waithood is not about waiting or lack of engagement with the labour market, but rather about hard work, negotiation and claim making". Before the pandemic, the study participants were busy; along with their studies, many worked within the informal sector, were engaged in house chores and enjoyed the company of peers [40]. Thus, one may argue that their everyday lives before the pandemic were characterised by active, busy waithood; in contrast, lockdown life was more like being stuck in confinement [31].
Almost all participants were concerned about their disrupted futures, including those out of school. Considering their general appreciation of education for individual and collective prosperity, they were upset about the expected delay in their studies, yet this was nothing new to public school students. For some, their educational level would no longer correspond to their age, while others lamented the additional delay. Out-of-school adolescents saw their dream of returning to school as being at risk of petering out. Most recognised that the pandemic posed additional threats to further education with worsened financial circumstances; thus, several participants mentioned reducing school fees as a requirement for education [6,15,59]. Ann Mische points out that "utopic end-states can be richly and lovingly imagined, while neglecting short term contingencies or alternative middle-term pathways toward realizing those futures" [60]. The eagerness of out-of-school adolescents to return to school might be realistic and rational because some manage to do so, as reflected in the high level of over-age students, particularly in public schools [46,56]. At the time of the interviews, the participants' abilities to evaluate the short-term actions were heavily compromised, even more so than middle-or long-term ones.
Like many other children around the globe [8], the adolescents in Bissau suffered from school closures and lost valuable time for education to secure future opportunities. Highlighting the need for education, school closure due to the COVID-19 pandemic is almost an insult to injury to the participants in an already inadequate educational system plagued by frequent teacher strikes [46,61]. School closures may have a knock-on effect if policymakers do not promptly rectify the education gap caused by the pandemic and the problems of school closure and unequal access to education [62]. Our study reveals that the prospects of young people in Bissau have been negatively impacted by the school closures caused by the pandemic, which is in line with studies from other settings worldwide [13]. However, due to a lack of educational support, access to digital technology and the internet, and other appropriate support, adolescents in Bissau were hit harder than many of their more fortunate peers elsewhere. Their voices need to be heard and acted upon to mitigate school closures' negative impact on their future health and well-being. Further, they need to be better integrated in the development of policies concerning them and they need to be given space for active participation in decision-making processes, applying a child rightsbased approach [7,38,63,64]. In the spirit of decolonising efforts, adolescents' involvement in the co-production of knowledge on issues of concern to them could constructively contribute to the rupture of adulterish, ethnocentric and racist assumptions [25,65].
This research was conducted early in the COVID-19 pandemic in Guinea-Bissau, with lockdowns and school closures for public and private schools. Previously, school closures had been 'reserved' only for those attending public schools due to repeated teacher strikes. School closures became a global practice to confront the COVID pandemic. The consequences of that policy are emerging, indicating a negative impact on student achievement, particularly younger children and those of a low socioeconomic background [66]. Further, reliance on digital platforms and remote learning brings additional challenges that must be addressed for successful student learning [47,57]. In contrast to most countries, Sweden kept schools open with no reported loss in reading skills among primary school students in grades 1-3 [67]. Studies have also highlighted the negative impact of closures on children s mental health and general well-being [8]. In settings such as Guinea-Bissau, with the frequent closures of public schools because of teacher strikes [46], globally gained experience during the pandemic, alongside our findings, should result in efforts to keep all schools open at all costs to safeguard children's learning outcomes and their future health and well-being.
A strength of this research is that it captures the voices of adolescents on school closures in an emerging pandemic with no end in sight at the time of the study. The opinions of the enrolled students and of those out of school highlight the volatile nature of that status; all groups struggled to continue schooling and conveyed the notion of education as a route to worthy citizenship but were threatened by the school closures. It is a limitation that the sampling of participants was purposive, aiming at diversity in the adolescents' voices regarding gender, school enrolment, habitation, and ethnicity, and it did not provide representative data. Due to the complexity of the topic, a participatory approach would have been desirable but impossible because of the general lockdown measures in place. Lastly, this study leaves out the experiences of adolescents living in urban areas outside the capital and in rural settings.
Conclusions
This research captures the diverse voices of adolescents living in the capital city of Guinea-Bissau. The data collection took place after the participants had experienced three months of a state of emergency, including the closures of all schools. Despite the generally favourable situation of adolescents enrolled in private schools compared to that of students enrolled in public ones, all the schools left their students without functioning educational support during the lockdown. Thus, rather than manifesting inequality between private vs. public school adolescents in Bissau, our study highlighted education loss for all.
The voices of adolescents in Bissau need to be heard and acted upon to potentially mitigate some of the negative impacts the school closures have on them, including postpandemic. Such mitigation measures must be framed within a child rights-based approach to succeed and need monitoring and further research. Of particular interest is if and how adolescents will be engaged in the recovery process, for instance, in the co-production of knowledge on matters of interest to them. Also, how much attention will be given to their worries about disrupted educational prospects and future aspirations? In the global inequality and colonial history context, will the pandemic stand out as a worldwide trigger to improve the quality and functionality of an educational system for all, or will it locally become remembered as a new, additional burden? Informed Consent Statement: Verbal informed consent was obtained from all subjects involved in the study.
Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy considerations. | 2023-03-29T15:26:10.246Z | 2023-03-27T00:00:00.000 | {
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221114200 | pes2o/s2orc | v3-fos-license | Tolerance analysis of non-depolarizing double-pass polarimetry
Double-pass polarimetry measures the polarization properties of a sample over a range of polar angles and all azimuths. Here, we present a tolerance analysis of all the optical elements in both the calibration and measurement procedures to predict the sensitivities of the double-pass polarimeter. The calibration procedure is described by a Mueller matrix based on the eigenvalue calibration method (ECM). Our numerical results from the calibration and measurement in the Mueller matrix description with tolerances limited by systematic and stochastic noise from specifications of commercially available hardware components are in good agreement with previous experimental observations. Furthermore, by using the orientation Zernike polynomials (OZP) which are an extension of the Jones matrix formalism, similar to the Zernike polynomials wavefront expansion, the pupil distribution of the polarization properties of non-depolarizing samples under test are expanded. Using polar angles ranging up to 25$^{\circ}$, we predict a sensitivity of 0.5% for diattenuation and 0.3$^{\circ}$ for retardance using the root mean square (RMS) of the corresponding OZP coefficients as a measure of the error. This numerical tool provides an approach for further improving the sensitivities of polarimeters via error budgeting and replacing sensitive components with those having better precision.
I. INTRODUCTION
Polarimeters characterize the polarization properties of materials. They find application in, for instance, optical samples [2], cancer non-invasive screening tools [3] in clinics, hyper-numerical-aperture lithography [4][5][6][7][8] where controlled polarization enhances the contrast and thus enabling smaller structures to be written on the wafer.
Inherited from standard interferometry [9], the doublepass configuration detecting the phase shift between its two arms has been developed for sensing applications such as dilatometric measurement [10] and pH monitoring [11]. In polarimetry, a double-pass layout enables angle-resolved measurements, whereby the polarization response of a sample for a range of polar angles and all azimuths can be measured in a synchronous approach. This simplifies the measurement setup and saves time compared to, otherwise, an apparatus with a function of rotating a solid angle over a certain range. Since the light is transmitted through the sample being tested twice, each ray nominally picks up the same polarization properties in both the outgoing and return paths. Given the same apparatus errors outside the sample being tested, the double-pass configuration offers double the sensitivity of the polarization properties. The interferometric merit of the double-pass, on the other hand, is utilized in aligning the optical components in the angle-resolved polarimetry. While experimental demonstrations have validated the concept of double-pass polarimetry in angle-resolved polarization measurements [2], repeatability analysis to tolerances of the double-pass polarimeter has not been * yuym3133@smee.com.cn; all authors contributed equally. studied systematically. The present work attempts to fill this gap by providing a detailed sensitivity analysis of the polarimeter repeatability.
An example of the operation of a double-pass polarimeter includes the calibration and measurement procedures. In the calibration apparatus as illustrated in Fig. 1a, a coherent laser illuminates a polarizer (P1) and a quarterwave plate (Q1) successively before being reflected by a non-polarizing beam splitter (BS). The coherent laser, the polarization components P1 and Q1, together with the reflective path of the BS, form the polarization state generator (PSG). The laser beam then passes through the calibration sample in the forward and reverse directions with the help of a mirror. The change of the polarization state of the beam caused by the calibration sample and the mirror is analyzed by the polarization state analyzer (PSA) and readout from the CCD. The PSA consists of the transmission path of the BS, the quarter-wave plate Q2 and the polarizer P2. The goal of the calibration setup is to characterize the polarization properties of the PSG and PSA accurately using calibration samples and the eigenvalue calibration method (ECM) [1]. The polarization properties of the calibration samples can be extracted using the same setup. In the measurement procedure the calibration samples and the mirror are subsequently replaced with an objective lens, the sample under test (SUT) and a hemispherical mirror as shown in Fig. 1b. The focus of the laser beam from the objective is aligned to coincide with the center of the curvature of the hemispherical mirror, to ensure that the beam is reflected back along the incoming optical path. The SUT is placed away from the focus for the laser beam to cover its pupil.
In this work, we break down the angle-resolved measurement of a SUT into 3 steps. In step 1, the trans- Step 2 Step 1 Step mittance amplitude for the two orthogonal polarization eigenstates and the retardance of the calibration samples are extracted from the calibration apparatus by comparing the intensities with and without the calibration samples.
Step 2 is an algorithmic procedure used to obtain the polarization properties of the PSG and PSA. This algorithm depends not only on the measured intensities with and without the calibration samples, but also on the polarization properties extracted in step 1. In step 3, the measurement setup employs the calibrated PSG and PSA to measure the polarization of the objective, SUT and hemispherical mirror together. Because the PSG and PSA are maintained unchanged during calibration and measurement, they cause no systematic change in the errors in measuring the SUT. Tolerance analysis of the components affecting the polarization from steps 1 to 3 results in the polarization measurement sensitivities.
We characterize the polarization of a non-depolarizing sample in terms of its diattenuation and retardance, which quantify the transmission amplitude difference between the two orthogonal brightest and darkest axes and the phase difference between the two orthogonal fastest and slowest axes, respectively. For non-depolarizing samples, the Jones matrix representation of the polarization is all that is required and is simpler than the Mueller matrix representation, in that the Jones matrix uses fewer parameters, only 4 complex elements compared to 16 real elements for the Mueller matrix. The diattenuation and retardance across the pupil can be expanded in terms of the orientation Zernike polynomials (OZP) based on the Jones matrix formalism [12][13][14], and the RMS of the coefficients quantifies the diattenuation or retardance across the entire pupil by analogy with Zernike polynomials for wavefront expansion. By inputting tolerances of available commercial products into the numerical model, we predict a sensitivity of 0.5% RMS OZP (a unit stands for the RMS of the corresponding OZP coefficients) for a diattenuation pupil, equivalent to a pupil with a mean diattenuation of 1%. Likewise, the prediction of the sensitivity for a retardance pupil is 0.3 • RMS OZP corresponding to a pupil with a mean retardance of 0.6 • .
This numerical tool takes the systematic and stochastic errors of each component in the system for both the calibration and measurement as inputs, and derives the sensitivities of diattenuation and retardance to errors in the measured values in a bottom-up approach. Whereas double-pass polarimeters can find application for characterizing incident-angle dependent variable attenuators [15], wide-view-angle polarizers and retarders [6,8,16] in lithographic equipment, this numerical tolerance analysis paves the way for predicting the sensitivity of the polarization properties for those optical components. Furthermore, this numerical tool can help to improve sensitivity via error-budgeting [17]. Depending on the relative contribution of each tolerance error, targeted hardware could be replaced to improve the sensitivity.
A.
Step 1: Determining the properties of calibration samples Classical calibration procedures usually rely on standard samples with well-known properties [18] or similar devices with higher accuracy. The former approach requires strict sample fabrication, while the later one limits the accuracy of the polarimeters to be calibrated to, roughly, that of the calibrating polarimeter. The ECM developed by Compain et al. [1] largely relaxes the requirement for special calibration samples, and is able to extract the polarization properties of the calibration sample from the polarimeter itself, hence nominally guaranteeing measurement accuracy. The ECM uses linear dichroic polarizers and retarders with retardation far from 180 • [1,19]. These polarization elements need to be homogeneous [20]. That is their eigen polarization states of polarizing elements are orthogonal. Here we extend the ECM to double-pass polarimetry. Due to the flat mirror in the double-pass layout sketched in Fig. 1a, wave plates with retardance of 90 • are excluded from use as calibration samples. A dichroic polarizer and a 1/6-wave plate are selected as calibration samples in this work.
Intensities modulated by the PSG and PSA are recorded. The calibration sample is first retracted from the optical path in the setup in Fig. 1a, leaving only the mirror. This results in the intensity matrix i 0 Here matrix a is the calculated PSA matrix from the intensity measurement. It is constructed from the 1 st to the u th configuration of the PSA, using the first row of the Mueller matrices of the PSA. The calculated PSG matrix w is formed by v different configurations of Stokes vectors. The middle term m mirror on the right hand side (RHS) of Eq. (1) is the measured Mueller matrix for the mirror. We then insert the dichroic polarizer and the 1/6-wave plate separately to obtain the intensity matrices in which the subscript stands for the i th calibration sample. Matrices m f i and m b i can be further decomposed to is the rotation matrix corresponding to azimuthal rotation angle θ of the calibration samples. The superscript f and b denote that the light passes through the calibration sample in a forward path and a backward path after reflection from the mirror, respectively. The measured Mueller matrix of the dichroic polarization elements m i , with zero azimuthal angle, can be expressed as [1] in which t X and t Y are the measured transmittance amplitudes of the sample along the two orthogonal directions, X and Y. We define the Z direction of the coordinate to be aligned with the ray propagation direction, the X direction to be pointing inside, and the Y direction to be pointing upwards at the start of the beam near the laser as demonstrated in Fig. 1b. The measured retardance difference between the X and Y directions is φ.
The quotient matrix c i is defined as the product of the inverse of the intensity matrix i 0 and the matrix i i , which gives The Mueller matrix of the mirror m mirror is in the form of Eq. (3), where the non-identity of the reflectance is expressed by the transmittance amplitudes t X and t Y , and the retardance φ of the mirror in Eq. (3) is taken to be the sum of 180 • and noises. The last relation (≈) becomes an equality when no noise is present in the measured intensity matrices i 0 , i 1 or in the control of the azimuthal angle θ of the calibration samples. To ensure the uniqueness of the solutions, the full rank of the PSG's w matrix is required for the inversion in Eq. (4) and the PSA's matrix a has the same requirement. The true combinations of the azimuthal angles of the polarizing elements in the PSG are chosen to maximize the absolute value of the determinant of the true PSG matrix W in order to minimize the inversion error of W in calculating the Mueller matrix for the calibration sample. Here, we use the convention that matrices with uncapitalized and capitalized letters symbolize the measured (or calculated) values and the actual (or true) values, respectively. The true PSA matrix A is optimized in the same way. The coherent laser source beam in the PSG is modeled as a linearly polarized electrical field of E in = [1; 1]/ √ 2. The PSG uses 4 configurations in our simulations for convenience in performing the inversions, i.e., set v = 4 in Eq. (1). Each configuration is obtained by varying the azimuthal angles of the polarizer P1 and the quarter-wave plate Q1. The true values of the polarization properties of the PSG and PSA used in the simulation are summarized in Tab. I.
The maximum absolute value of the determinant of the PSG is optimized to |detW |=0.58 and that of the PSA is |detA|=0.06. The reflectance and transmittance amplitudes of the BS are idealized to be √ 0.5 in this modeling. Note that the Mueller matrix of the BS only affects the optimization of the azimuthal angle configurations for P1, Q1, Q2 and P2. It has no influence on the calibration error for the PSG ∆W = w − W or that for the PSA ∆A = a − A in step 2. In the experiments, the Mueller matrix for the transmission and reflection paths through the BS could be measured in advance using a single pass polarimeter in transmission [19,21] and reflection [1] to ensure the calibration accuracy. The quotient matrix c i in Eq. (4) is similar, in the linear algebra sense, to the square of the measured Mueller matrix of the calibration sample [m i ] 2 given that matrix [R(θ)w] is invertible. Therefore, the quotient matrix c i and [m i ] 2 share the same eigenvalues. While the transmittance amplitudes can be calculated from the two real eigenvalues λ 1 and λ 2 , as t X = 4 √ λ 1 and t Y = 4 √ λ 2 , the retardance of the calibration sample φ is a function of the two complex eigenvalues λ 3 and λ 4 , as Error sources depending on the measurement time scale are categorized into stochastic noise and systematic errors. Characteristic time scales are the total measurement time for intensities without the calibration samples i 0 , those with the calibration samples i i and the sampleswitch time in between. Stochastic noise with a time scale shorter than the total measurement time, comes from the laser source, the CCD, vibration of the rotatory positioners and the mechanical mounts of the optical elements. Each pixel of the CCD has a fluctuation of ±0.3% in the measured intensity which is modeled as statistically the same, and comes primarily from the repeatability of the laser source [22] and the random spatial non-uniformity in the CCD [23]. Both cross-talk between neighboring pixels and electrical shot noise contribute to the spatial non-uniformity. Cross-talk is simulated via the correlation length of these noise sources across the CCD. The correlation length is taken to be 1 pixel for simplicity, i.e. no cross-talk is assumed. For longer correlation lengths, filtering algorithms may be applied to reduce the noise influence. The impact of electrical shot noise on the signal to noise ratio decreases as the number of photons increases (assuming the photon-to-electron conversion rate of 1). By carefully selecting the measurement conditions so that the CCD is near saturation, electrical shot noise buried in the signal controlled by the power of the laser and the integration time of the CCD can have less than 1/10 of the influence on diattenuation and retardance caused by the quantization noise due to the analogueto-digital conversion (ADC) of the CCD. Electrical shot noise can therefore be safely neglected under the assumption of near CCD saturation, 10 14 photons per pixel in the model. The stochastic vibration of the rotatory positioners attached to polarization elements P1, Q1, Q2, and P2 in axial direction is taken to be 0.01 • . This follows from the Thorlabs' motorized rotator K10CR1 [24] specifications. Tilted variation of the PSG and PSA on the other hand is allocated to the polarization properties of the mirror and the BS in addition to the stochastic noise of the retardance and reflectance across the mirror. The pre-measurement of the mirror can be performed by a single pass polarimeter in reflection mode using analysis [1] similar to this step to obtain those stochastic noise. The difference lies in that for the double-pass layout the light probes a SUT in both forward and backward directions, while in the single pass polarimeter the light incidents on a SUT (mirror here) only once. To calibrate the mirror under normal incidence, an additional BS is required to deflect the beam from reflection in the single pass polarimeter and should be calibrated in advance. The tolerance types for stochastic noise and their values are summarized in Tab. II. Elements of the measured intensity matrix in Eq. (1) equal to the true values plus errors, i x,y 0 = I x,y 0 + ∆I x,y 0 (where x = 1, 2, ..., u; y = 1, 2, ..., v). The measured azimuthal angle θ = Θ + ∆Θ in Eq. (4) is the sum of true value Θ and precision ∆Θ of rotatory positioners. The reflectance error of the mirror ∆R X/Y = r X/Y − R X/Y , the retardance error ∆Φ = φ − Φ and the transmittance amplitude error ∆T X/Y = t X/Y − T X/Y all follow the same convention.
Although the PSG and PSA nominally have systematic errors, with the settings of the PSG and PSA being the same between the calibration (Fig. 1a) and measurement (Fig. 1b) setups there is no systematic change in the errors for the PSG and PSA matrices w and a. Therefore, the systematic error introduced in the modeling comes from the mirror in steps 1-2, the objective and the hemispherical mirror in step 3. The CCD is a common element in both the calibration and measurement layout, nevertheless, information loss in the process of ADC cannot be calibrated out. Hence the systematic error from the CCD must be included in all 3 steps. Systematic errors are listed in Tab. III.
With the stochastic and systematic errors of each component of the polarimeter listed above, we simulate both the stochastic noise and systematic errors in the properties of the calibration samples using a bottom-up approach. To reduce the rotational asymmetrical noise such as the tilt angle of the calibration samples, we rotate the calibration sample azimuthally and take the average over the -90 • to 90 • range. Figure 2a shows the calibrated transmittance amplitudes t X , t Y and retardance φ as a function of the azimuthal angle of the 1/6-wave plate sample. The average measurement value is displayed as a red line with the true values of the transmittance amplitudes being T X = 0.98, T Y = 0.97 and that of the true retardance being Φ = 60 • . The stochastic noise is defined as the difference between the average measurement over all azimuths and the true value. A 1000-trial simulation in Fig. 2b indicates that the calibration stochastic noise for transmittance is ∆T X < ±0.0002, ∆T Y < ±0.0002 and that for retardance is ∆Φ < ±0.012 • as shown in Tab. II. For the calibration sample polarizer, this step is sufficient to determine the transmittance amplitude of the bright transmission axis, but not the dark axis. Using two 40 dB polarizers in series could ensure a stochastic error ∆T Y < ±0.0002. The calibrated polarization properties of the 1/6-wave plate and polarizer, together with their stochastic noise determines the calibration accuracy for the PSG w and PSA a matrices in step 2.
B. Step 2: Calibration of the PSG and PSA matrices
In this subsection, we calculate the PSG w and PSA a matrices as well as expand the working range of the azimuthal angles of the calibration samples from those used in the past [25]. To compute the PSG matrix w, w in Eq. (4) is first replaced with an unknown matrix x. This results in m DP i x − xc i = 0, where the sample matrix for the double-pass polarimeter is defined as The linear operator h i is a 16×16 matrix, the elements of which are detailed in Eqs. (A3) and (A4) in Appendix A. The calibrated PSG matrix w is then the non-zero solution to Eq.
In this way, all 16 calculated eigenvalues λ(1) < λ(2) < . . . < λ(16) of k must be positive and real. The eigenvector with eigenvalue closest to 0 is the calculated PSG w matrix, after the 16×1 eigenvector being reshaped into [19], and the first sample is a polarizer with azimuthal angle Θ = 0 • . To find suitable combinations of azimuthal angles that guarantee calibration accuracy, we plot the error estimator log[λ(2)/λ(1)] as a function of the azimuthal angles for calibration sample 2 (a polarizer with different azimuthal angle to sample 1) and sample 3 (a 1/6-wave plate) in Fig. 3a. Both of their azimuthal angles are varied from -90 • to 90 • .
Stochastic noise contributions to the error estimator include the rotational repeatability of the calibration samples as limited by mechanical positioners, intensity fluctuations, stochastic polarization noise of the calibration samples in step 1 and that of the mirror. Systematic errors come from the quantization error of the ADC, and the polarization properties of the mirror. Values of these errors are given in Tabs. II and III. Each combination of azimuthal angles in Fig. 3a is averaged over 100 trails to reduce the influence from stochastic noise. The larger the value of the error estimator, the closer the smallest eigenvalue of k in Eq. (6) is to 0, and consequently the more accurate the calculated PSG w matrix will be. We observe that the error estimator is relatively small, log[λ(2)/λ(1)] < 9, when the 1/6-wave plate has the same azimuthal angle (Θ ≈ 0 • , the middle horizontal reddish line in Fig. 3a) as that of the first polarizer. It is likely that the lack of calibration accuracy is due to the azimuthal angle overlap of the two orthogonal eigenstates of the first polarizer and the 1/6-wave plate, blurring the precision of the eigenvalue-based calibration method.
We further calculated the calibration error between the calibrated and the true PSG matrices ∆W and that of the PSA error matrix ∆A. The calculated PSG w matrix is normalized by its transmission before the comparison with the true PSG matrix W , because the eigenvector of Eq. (6) can be scaled with any real number. As the calculated PSA matrix a is derived from the measured intensity using Eq. (1), it will give an inverse scaling factor to the calculated PSG matrix w if the normalization is not done. Consequently, normalization of transmission only serves for obtaining the error for the PSG ∆W and the PSA ∆A. The PSG w and PSA a matrices without normalization will not affect the measurement accuracy of a SUT in step 3. The logarithm of the error of the PSG ∆W and the PSA ∆A as a function of the azimuthal angles of the 1/6-wave plate and the polarizer are plotted in Fig. 3b and Fig. 3c, respectively. The first element (1,1) of the 4×4 error matrices can be chosen without loss of generality. The other 15 elements of the error matrices ∆W and ∆A share roughly the same calibration error. The cross areas in the middle of the error matrices for the PSG and PSA display a relatively worse accuracy, and are aligned with the error estimator map, log[λ(2)/λ(1)] in Fig. 3a. As a result, the requirement for alignment of the calibration samples can be relaxed to all the yellowish areas in Fig. 3a, corresponding to the error estimator log[λ(2)/λ(1)] > 10. Former experimental observations reveal the calibration accuracy of the PSG and PSA matrices, where an average of a standard deviation over all 16 Mueller matrix elements is employed for quantification [19]. In those experiments, the averaged standard deviation is 6.7×10 −4 for the PSG matrix and 6.0×10 −4 for the PSA matrix over 38 calibrations. We simulate the pixel-based PSG and PSA matrices for 10000 trails, and obtain the averaged standard deviation of 5.9×10 −4 for the calibrated PSG and that of 3.6×10 −4 for the calibrated PSA, which is in line with the experiments, verifying our tolerance analysis for the calibration.
C. Step 3: Angle-resolved measurement
The alignment of the objective to the center of the hemispherical mirror can be monitored by adding an interferometer arm to form an interference pattern on the CCD. This added arm would extend horizontally from the laser and the PSG, and have a mirror at the end.
The simulation flow leading to the prediction of the sensitivities for the angle-resolved measurements is depicted in Fig. 4a. The simulation uses a generated Jones matrix covering the whole pupil (in short Jones pupil matrix) as the true Jones pupil matrix of a SUT J true . It is synthesized by the RMS of the coefficients of up to order 72 in an expansion using the OZP for diattenuation and retardance [12][13][14].
The Jones pupil matrix is converted to a Mueller pupil matrix to be compatible with the Mueller matrix description M true of the PSG and PSA in step 1-2. The true PSG matrix W , PSA matrix A, stochastic noise from the hemispherical mirror (whose values are listed in Tab. II), combined with the systematic errors from the objective and hemispherical mirror (whose values are listed in Tab. III), result in the true intensity I true . Objectives usually contain multiple lenses to ensure a specific image quality over the field of view. As polarization relies on the order of the components the light passes through, the polarization of the light traveling through the objective in the forward direction, from collimated space to the focus as sketched in Fig. 4b tom, differs from the light transmitted by the objective in the backward or return direction. As an example, we choose a Japanese patent 61 2925 860129 in the CODE V database [26] with a half incident angle of 25.4 • to investigate its polarization properties. Without applying optical coatings to the objective, we trace the polarization of the objective in both the forward and backward directions. The backward beams exiting the object have maximum deviation angles of 0.29 • along the periphery due to the imperfect wavefront of the objective. Systematic errors of diattenuation and retardance of the objective considering the retrace error in the backward direction are listed in Tab. III. Random intensity noise at each pixel and information loss from the ADC are added to the intensity as error sources to form the measurement intensity I measure . The calibrated PSG matrix w, PSA matrix a, and idealized Mueller matrix [1 0 0 0; 0 1 0 0; 0 0 -1 0;0 0 0 -1] are employed to calculate the Mueller matrix of the SUT in the forward path M measure . The Mueller pupil matrix is converted to the Jones pupil matrix afterwards. This procedure removes the information about depolarization contained in the Mueller matrix to obtain the Jones matrix. Depolarization in the measured Mueller matrix M measure comes from overlap of incoherent electromagnetic fields [3]. To convert the Mueller matrix with limited depolarization to the Jones matrix J measure , the non-depolarization condition for the conversion trace(M T M ) = 4m 2 11 [27] is approximated as |trace(M T M ) − 4m 2 11 | < 0.01. For measurements that meet this condition, the Jones matrix can be derived from the Mueller matrix via expressions given in Ref. [28].
The RMS of the OZP coefficients for either diattenuation or retardance is a single number used to quantify the goodness of a Jones pupil via the relative transmittance amplitude difference between the brightest and darkest axes or retardance delay between the fastest and slowest axes across the pupil of a SUT, respectively. Mathematical details of the OZP can be found in Appendix B. For 72 terms the highest power in the radial direction of the OZP is 10, corresponding to the highest radial power of the 36 th term of the fringe Zernike polynomials [26].
Though the true reflectance amplitude of the hemispherical mirror is not unity, only the difference between the reflectance in the X and Y directions affects the diattenuation and retardance of the pupil. This is because the measured Jones pupil matrix J measure is further decomposed into a product of apodization, a partial polarizer, a retarder and two other physically meaningful matrices [5], and only the diattenuation pupil in the partial polarizer and the retardance pupil in the retarder will be further expanded by the OZP. Writing the reflectance in the X and Y directions of the hemispherical mirror as r X = r Y + ∆R XY , the average of the reflectance in the X and Y direction contributes only to the apodization of the SUT. The difference of the reflectance amplitudes ∆R XY will be counted in the first term of the OZP expansion (see Eqs. (B6) and (B7) for the mathematics). Since rotating the hemispherical mirror azimuthally for 90 • swaps the reflectance values r X and r Y , taking the average of the fitting coefficients to the OZP expansion, measured with 0 • and 90 • hemispherical mirror rotation, improves the accuracy of the OZP coefficients for the diattenuation and retardance pupils.
We decompose both J true and J measure into an OZP description of retardance and diattenuation, using the first 72 terms. The RMS of the coefficients are calculated as RMS = 72 j=1 coe j /(j + 1), with coe j denoting the j th OZP coefficient. Comparison of the true Jones pupil matrix for the SUT and the measured value is made by running the simulation through the flow in Fig. 4a for 100 trials. The repeatability in terms of the RMS of the OZP coefficients replaces the mean value in the standard variance [29] with the true value, defined as Before predicting the sensitivity presented by RMS OZP in the Jones matrix description, we apply our tolerance analysis to the SUT in terms of the Mueller matrix in the measurement procedure similar to that reported experimental observations in Ref. [1]. We compare the true Mueller matrix M true with the measured Mueller matrix M measure in the simulation flow as sketched in Fig. 4a. Both of the two matrices are normalized to their (1, 1) elements, so that the relative error of the (2, 2), (3,3) and (4,4) elements of the matrices can be calculated under the condition of a non-identity Mueller matrix of the mirror. Off-diagonal elements of the Mueller matrices M true and M measure are small due to the generated weak polarization properties of the SUT, leading to unphysically large relative errors, and thus they are safely disregarded in the comparison. We obtain a maximum 0.4% over all three Mueller matrix pupils, in good agreement with the 0.5% in the reported experiment.
III. RESULTS AND DISCUSSION
Sensitivity is defined in terms of a boundary. In Fig. 5, the boundary where repeatability equals the true value is the line with a slope of 1 through the origin (0,0). Away from the gray shadow areas, the repeatability (i.e. the measurement uncertainties) are smaller than the true values. The sensitivity of the diattenuation pupil depends on the corresponding retardance. Larger retardance leads to better sensitivity for diattenuation in general. The same phenomenon applies to the sensitivity of retardance as well. It is likely that the measurement is more sensitive when the SUT exhibits strong polarization properties, and the retardance and diattenuation are not decoupled in calculating the repeatability of either of them. To reduce the sensitivity from a set of values to a single value, we quantify the sensitivity of diattenuation with an additional requirement: the corresponding retardance of the pupil should be of the same order of magnitude as the diattenuation. This results a 0.5% RMS OZP sensitivity for diattenuation. With the same requirement, the predicted sensitivity for retardance is 0.3 • RMS OZP.
Visualization of the pupils for the true diattenuation and retardance when their sensitivities are reached (labeled with black arrows in Fig. 5), i.e. 0.5% RMS OZP for diattenuation and 0.3 • RMS OZP for retardance, is shown in Fig. 6a and Fig. 6d, respectively. The mean of the measured pupils of diattenuation and retardance comes from the measured Jones pupil J measure , and a decomposition of the measured Jones pupil in terms of diattenuation and retardance thereafter. Reconstruction of the pupils for diattenuation and retardance is based on the first 72 terms of the OZP expansion, where each pixel of the pupils for diattenuation and retardance is averaged over 100 trials. The sensitivity of diattenuation shows an average of 1% over all pixels of a pupil displayed in Fig. 6b with repeatability around 1/3 of that displayed in Fig. 6c. For the sensitivity of retardance, the average Fig. 4a. Black arrows point to the pupils that meet our definition of sensitivity, and polarization properties of these pupils are visualized in Fig. 6. of the pupil is 0.6 • as shown in Fig. 6d with a repeatability around 1/3 of that as well, as shown in Fig. 6f. Directional lines on the diattenuation pupils denote azimuthal angles for the partial polarizer, while they denote those for the retarder on the pupils of retardance. The azimuthal angle pupil reconstructed from the OZP coefficients may have a 90 • shift, due to the limitation of the inverse trigonometric functions described in Eqs. (B14) and (B15) in Appendix B. Horizontal lines represent an azimuthal angle of 0 • , while vertical lines represent 90 • . White lines with directions other than vertical or horizontal represent error, the larger the error of the direction, the farther away the direction of the white line is from either vertical or horizontal. When treating the 90 • shift to be error-free, the repeatability of the azimuthal angle is 5 • for diattenuation and 3 • for retardance averaged across the pupil, lower than 1/10 of the mean values of that for diattenuation and retardance. Concentric circles with different radii on the pupil correspond to different incident angles of the laser beam away from the focal plane of the objective. In Fig. 6, from inside to outside the concentric circles correspond to angles at the objective of 5 • , 15 • and 25 • . Pupils of diattenuation and retardance provide a visualization of the azimuthal anisotropy and polarization response of a refractive sample under non-normal incidence.
IV. CONCLUSION
In conclusion, we have performed a detailed tolerance analysis of the calibration and measurement procedures for a double-pass polarimeter, and have predicted the sensitivity of the polarimeter to systematic errors and stochastic noise. The eigenvalue calibration method ECM [1] is used in the polarimeter calibration, resulting in the Mueller and Stokes description of the PSG and PSA characteristic matrices. The Mueller pupil matrix of an arbitrary non-depolarizing SUT is predicted before it is converted to a Jones pupil matrix. Our tolerance model for the calibration of the PSG and PSA, as well as the measurement of the Mueller matrix pupil are consistent with previous experimental observations [1,19]. Thanks to the Jones pupil decomposition and the OZP expansions of diattenuation and retardance, the whole pupil of the SUT can be described by two values, diattenuation and retardance in terms of the RMS of the OZP coefficients. The sensitivity prediction for diattenuation is 0.5% and that for retardance is 0.3 • . The double-pass polarimeter offers a platform to measure angle-resolved SUTs, revealing the azimuthal inhomogeneity of retardance and diattenuation. The ECM, tolerance analysis and the subsequent conversion of the measured Mueller pupil matrix of the SUT to a Jones pupil matrix in terms of the OZP expansions to predict sensitivities and visualize retardance and diattenuation pupils can also be applied to a single pass polarimeter. Though the incident angle would not be resolved in the single pass polarimeter, without a BS and a mirror fewer noise sources are included. The singe pass polarimeter can achieve better sensitivity of diattenuation and retardance as well as resolve the small inhomogeneity of the pupil under normal incidence.
V. ACKNOWLEDGMENTS
The authors are thankful to Zejiang Meng for introducing the ECM algorithm, Vladimir Nikishkin for coding assistance, and Wei Wang for the initial contact with vendors for the specifications of the components used in the modeling as well as his exuberant personality. Last but not the least, we acknowledge an extended vacation due to COVID-19 outbreak.
Appendix A: Error propagation
We have derived a simplified theory of error propagation for double-pass polarimetry to cross-check our numerical simulations with tolerances. By employing perturbation theory to the first order, we theoretically calculate the error of the PSG matrix ∆W given the measurables without any calibration samples for i 0 and with the calibration sample for i i .
The noise propagation of the quotient matrix c i combining Eqs. (A2) and (4) to the first order results in the expression Letting the unknown x = W + ∆W , the linear operator h i (x) in Eq. (5) is expanded to the first order with the quotient matrix from Eq. (A2), as Factoring the first term on the RHS in Eq. (A3), we operate on the elements of the matrices. It follows that by applying the relation for the least square fit ∆W p,q = δ p,F δ q,G ∆W F,G , where δ is the Kronecker delta, p, q, F and G are summed from 1 to 4, we have The single indices µ and ν label all possible combinations of F, G and p, q. The last two terms on the RHS in Eq. (A3) are influenced by the intensity with the calibration sample ∆(AM b i M mirror M f i W ) and without it ∆(AM mirror W ). Hence, the intensity error is defined . Assuming G i,µ,ν is invertible, Eq. (A3) can be simplified to This expresses the linear relationship between one element of the PSG error matrix ∆W ν and the sum of the stochastic noise of the calculated calibration sample ∆(M DP i ) times the true PSG matrix W and the noise of the measured intensity ∆I i,µ .
To verify the validity of our numerical tool for the tolerance analysis, we simulate the PSG error ∆W matrix as a variation of the intensity error. The stochastic noise of the calibration sample ∆(M DP i ) in Eq. (A6) is idealized to be 0. Modeling results show that the error across the pupil of one element of the 4×4 PSG matrix ∆W 3,1(β=9) increases linearly with the intensity noise as expected as shown in Fig. 7. The intensity noise normalized by the intensity of the PSA and PSG varies from ≈ I(r, ω) + j coe j OZ j (r, ω) (B6) J ret (φ, β, r, ω) = cos φ(r, w) 2 I(r, ω) cos 2β(r, ω) sin 2β(r, ω) sin 2β(r, ω) − cos 2β(r, ω) ≈ cos φ(r, ω) 2 I(r, ω) + i j coe j OZ j (r, ω), where the approximation sin φ(r,ω) 2 ≈ φ(r,w) 2 is used. The term OZ j (r, ω) is further decoupled into a position (r) dependent term and an orientor matrix depending on the azimuths ω, as where n indexes the highest power in radial direction and =0, 1 for the 2 orientor matrix in Eq. (B5). The order label j represents combinations of the OZP indices m, n, with the relation n−m = 2l, l = 0, 1, ...n, n ∈ Z + . Corresponding relation between j and m, n, up to the first 16 terms of the OZP is displayed in Tab. IV. The OZP expansion of diattenuation and retardance are approximations. To test the accuracy of these approximations, we use the first 72 orders of the OZP. A to-be OZP expanded and reconstructed diattenuation pupil consists of 4 pupils of elements, among which two pupils are independent. We label the upper-left element in the matrix as Jinput dia xx = d 2 cos 2γ and that in the upper-right as Jinput dia xy = d 2 sin 2γ. Similarly, two independent matrix elements for retardance are Jinput ret xx = sin φ 2 cos 2β and Jinput ret xy = sin φ 2 sin 2β. As shown in Fig. 8, the pupils of the two independent elements in diattenuation or retardance matrices are compared between the reconstruction from the coefficients to the OZP and the inputs. Two independent elements of the matrix j coe j OZ j (r, ω) are reconstructed from the OZP coefficients, as Jreconst xx (r, ω) = Fig. 6a. b The input pupil for retardance is from Fig. 6d. The difference between the reconstructed and the input original pupil based on the first 72 orders of the OZP is around 1/10 of either of them.
Differences from the input Jinput and reconstructed Jreconst Jones matrix pupils are an order of magnitude less than either the input Jinput or the reconstructed Jreconst. Diattenuation and retardance pupils used for comparison comes from those in Fig. 6a and Fig. 6d, respectively. This difference is around 1/3 of that between the mean of the measured pupil and the repeatability pupil for either diattenuation (in Figs. 6b-6c) or retardance (in Figs. 6e-6f), demonstrating that the OZP expansion well represents diattenuation and retardance pupils. Therefore, errors contributed from the reconstruction with the first 72 orders of the OZP is negligible in calculating the sensitivities for diattenuation and retardance. The reconstruction of the diattenuation and retardance pupils as well as the direction of the partial polarizer and the retarder from the two independent elements Jreconst xx and Jreconst xy is given by | 2020-08-13T10:05:14.730Z | 2020-08-12T00:00:00.000 | {
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244732611 | pes2o/s2orc | v3-fos-license | The Second Study of Clinical and Immunological Findings in Anti-laminin 332-Type Mucous Membrane Pemphigoid Examined at Kurume University—Diagnosis Criteria Suggested by Summary of 133 Cases
Background Recently, we published an article retrospectively summarizing the results in 55 anti-laminin 332 (LM332)-type mucous membrane pemphigoid (MMP) cases examined at Kurume University, which were diagnosed by strict inclusion criteria, including positive reactivity in direct immunofluorescence and absence of antibodies to non-LM332 autoantigens. However, indirect immunofluorescence using 1M-NaCl-split normal human skin (ssIIF) is also valuable for diagnosis of anti-LM332-type MMP. Methods In this second study, we selected 133 anti-LM332-type MMP cases, which were diagnosed by our different inclusion criteria: (i) immunoglobulin G (IgG) deposition to basement membrane zone (BMZ) by direct immunofluorescence or IgG reactivity with dermal side of split skin by ssIIF, (ii) positivity for at least one of the three subunits of LM332 by immunoblotting of purified human LM332, and (iii) the presence of mucosal lesions. Clinical, histopathological, and immunological findings were summarized and analyzed statistically. Although these cases included the 55 previous cases, the more detailed study for larger scale of patients was conducted for further characterization. Results Clinically, among the 133 patients, 89% and 43% patients had oral and ocular mucosal lesions, respectively, 71% had cutaneous lesions, and 17% had associated malignancies. Histopathologically, 93% patients showed subepidermal blisters. The sensitivities of ssIIF and direct immunofluorescence are similar but are significantly higher than indirect immunofluorescence using non-split human skin (both p < 0.001). In immunoblotting of purified LM332, patient IgG antibodies most frequently reacted with LMγ2 subunit (58%), followed by LMα3 (49%) and LMβ3 (36%). Thirty-four percent patients recognized additional non-LM332 autoantigens. Statistical analysis revealed that autoantibodies against non-LM332 autoantigens might stimulate the production of anti-LMγ2 antibodies. Conclusions This retrospective study further characterized in more detail the clinical and immunological features of 133 cases of anti-LM332-type MMP, in which the new diagnostic criteria without positive direct immunofluorescence reactivity were useful for the diagnosis. Higher frequency with anti-LMγ2 antibodies suggested more significant pathogenic role of this subunit. Additional autoantibodies to non-LM332 autoantigens detected in one-third of the patients may contribute to complexity in anti-LM332-type MMP, including the induction of anti-LMγ2 antibodies.
INTRODUCTION
Basal keratinocytes adhere to connective tissue at basement membrane zone (BMZ) of the epidermis (1). The interaction between keratinocytes and extracellular matrix proteins regulates many cellular behaviors, including cell adhesion, migration, proliferation, differentiation, and apoptosis (2). Laminins (LMs) are major extracellular matrices at BMZ. LMs are heterotrimeric glycoproteins consisting of three subunits, which are covalently linked by disulfide bonds, and are composed of many isoforms (1).
Laminin 332 (LM332) (previously called as epiligrin and laminin 5) is the most important LM isoform for the skin integrity (1) and is composed of a3, b3, and g2 subunits (3). LM332 is a ligand of integrin a6b4, which is a major transmembrane component at hemidesmosome, a cell-matrix junction at BMZ (4). LM332 also adheres to integrin a3b1, which locates at focal adhesion, another adhesion device (4). LM332 is a target protein both in hereditary disease, i.e., Herlitz or non-Herlitz types of junctional epidermolysis bullosa, and in autoimmune disease, i.e., anti-LM332-type mucous membrane pemphigoid (MMP) (abbreviated as LM332-MMP in the present study) (4).
MMP (previously called as cicatricial pemphigoid) is a heterogeneous subepidermal autoimmune bullous skin disease (AIBD), which affects mainly various mucous membranes and occasionally skin (5)(6)(7). Although oral mucosa is most commonly affected, ocular, nasal, pharyngeal, laryngeal, esophageal, and genital mucosae are also involved (6). The clinical course and prognosis of MMP are affected by the specific autoantigen targeted, the titer and bioactivity profile of corresponding autoantibodies, and the specific mucosal sites of disease activity (8).
In MMP, direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) tests show in vivo bound and circulating anti-BMZ autoantibodies of immunoglobulin G (IgG) and/or IgA subclasses, and various biochemical analyses detect a number of autoantigens (5). MMP is subdivided into two major types: anti-BP180-type MMP (BP180-MMP) and LM332-MMP. Approximately 90% and 10% of reported MMP cases are the former and the latter, respectively (1). BP180-MMP patient show IgG and/or IgA autoantibodies reactive mainly with BP180 C-terminal domain, although LAD-1, soluble BP180 ectodomain, and BP180 NC16a domain are also occasionally recognized (5,6). In contrast, LM332-MMP patients have IgG antibodies reactive with the 165 and 145 kDa LMa3 subunits, the 140 kDa LMb3 subunit, and the 105 kDa LMg2 subunit in immunoblotting (IB) of purified human LM332 (3). Recently, an IIF using recombinant LM332 was also developed for the detection of autoantibodies against LM332 in MMP sera (9).
Recent studies showed that LM332-MMP patients had an increased relative risk of cancer (7,10,11). However, the significance of these results is still obscure because of a limited number of patients with LM332-MMP.
Recently, we have reported a retrospective study of the clinical and immunological findings summarized for 55 LM332-MMP cases, which were diagnosed with very strict inclusion criteria, including positive DIF and the absence of other autoantigens (12). This study indicated that IIF using 1M-NaCl-split normal human skin (ssIIF) is also valuable for the diagnosis of LM332-MMP (12).
As the second version to the previous study (12), in the present study, using new inclusion criteria with positive ssIIF and concurrence of other autoantigens, we selected 133 cases of LM332-MMP from our large AIBD cohort at Kurume University, which included the 55 previous cases. Then, we further assessed in more detail both clinical features and immunological findings in the 133 cases, and extensive statistical analyses were also performed, which suggested that the new criteria are useful for the diagnosis of LM332-MMP.
Patients and the Information for the Clinical Features
As one of the centers for diagnosis of AIBDs in Japan, we have collected sera and information for 4,547 patients with various AIBDs, which were sent for our tests from other institutes for 14 years (2001-2014). Diagnosis of LM332-MMP was made based on our new inclusion criteria: (i) IgG deposition to BMZ by DIF or IgG reactivity with dermal side of split skin by ssIIF, (ii) positivity for at least one of the three subunits of LM332 by IB of purified human LM332, and (iii) the presence of mucosal lesions.
The information, including age, gender, medical history, and clinical features for mucocutaneous lesions, was obtained from consulting letters sent from other institutes. In addition to oral and ocular mucosal lesions, we collected information for lesions on nasal, pharyngeal, laryngeal, esophageal, and genital mucosae. Furthermore, to evaluate the severities of LM332-MMP, "oral score" (0-5) was calculated by the numbers of involved parts on oral mucosae (i.e., lip, tongue, cheek, gingiva and palate), and "mucosal score" (0-7) was calculated by the number of involved mucosae (i.e., oral, ocular, nasal, pharyngeal, laryngeal, esophageal, and genital mucosae), as we previously reported (12).
However, for some patients, information for clinical and histopathological features could not be obtained from the consulting letters. In addition, results of some serological tests could not be obtained, mainly because of the shortage of sera due to large and long-lasting nature of the present study. Therefore, assessments of most parameters were performed only for patients in whom the information of the parameters was available. This study was performed following the guidelines of Kurume University School of Medicine and Declaration of Helsinki Principles and was approved by the ethics committee of Kurume University School of Medicine.
IF Assays
We performed DIF using biopsy specimens from patients for depositions of IgG, IgA, IgM, and C3 to epidermal BMZ. For the diagnosis of MMP, we routinely performed two IIF assays including IIF using normal human skin and ssIIF (13)(14)(15)(16)(17). Normal human skin was obtained from our hospital. Both IgG and IgA autoantibodies were examined by the IIF assays. The representative result of ssIIF for a LM332-MMP case is shown in Figure 1A. In the result description, "DIF-not detected (ND)" indicates that DIF was not done, and therefore, the result of DIF was unknown; "DIF-" indicates that DIF was done, and the result was negative.
Statistical Analysis
We compared differences among various clinical parameters and immunological results with chi-square test, Fisher exact test, Mann-Whitney rank-sum test, Student's t-test, and Pearson's correlation by SigmaPlot 12.0 soft (Hulinks, Inc., Tokyo, Japan). p values <0.05 were considered as statistically significant. cases, 55 patients (41%) showing positive IgG deposition of BMZ in DIF and exclusive reactivity with LM332, whose clinical and immunological findings had been summarized in the previously published paper (10), were defined as DIF+/LM332 group in the present study. According to the results of DIF for IgG deposition to BMZ and ssIIF for IgG reactivity with skin dermal side, the 133 cases were divided into four groups as "DIF+/ssIIF+," "DIF+/ ssIIF-," "DIF-/ssIIF+," and "DIF-not detected (ND)/ssIIF+". According to the results of IIF using normal human skin for IgG reactivity to BMZ, the 133 cases were divided into two groups as "IIF+" and "IIF−", excluding 3 cases without IIF results.
Diagnoses and Grouping
According to the autoantigens detected, the 133 cases were also divided into two groups as "sole LM332," which reacted only with LM332, and "multiple-antigens (Ags)," which reacted with LM332 and other antigen(s).
The Results of All the 133 LM332-MMP Patients
The 133 LM332-MMP patients consisted of 66 male and 58 female, although gender was unknown in 9 patients. The average age of patients with information for age was 66.45 years (male, 68.03 years and female, 64.60 years).
In the 133 patients, 71% had cutaneous lesions with blisters as the major symptom ( Table 1). In addition, 17% patients had associated malignancies with lung cancer as the most frequent one ( Table 1). For various immunofluorescence detection methods, the positive rates of DIF for IgG deposition to BMZ, DIF for C3 deposition to BMZ, IIF of normal skin for IgG reactivity to BMZ, and ssIIF for IgG reactivity with dermal side were 88%, 77%, 56%, and 87%, respectively. The sensitivities of DIF for IgG deposition to BMZ and ssIIF for IgG reactivity with dermal side were similar but were significantly higher than that of DIF for C3 deposition to BMZ (both p < 0.05) and that of IIF of normal skin for IgG reactivity to BMZ (both p < 0.001). The sensitivity of DIF for C3 deposition to BMZ was also significantly higher than that of IIF of normal skin for IgG reactivity to BMZ (p < 0.05). By IB of purified LM332, the positive rates of reactivity with the 165-kDa LMa3, 145-kDa LMa3, LMb3, and LMg2 were 49%, 45%, 36%, and 58%, respectively. These results indicated that autoantibodies in the sera of our cohort of LM332-MMP patients most frequently reacted with LMg2 subunit of LM332.
Because of the lack of detailed information for the treatments in most cases, we could not analyze the treatments.
Statistically, no clinical parameters were found to be correlated with autoantibodies against any subunits of LM332, suggesting that autoantibodies against each subunit of LM332 contribute similarly to clinical and pathological features of LM332-MMP.
The Results of "IIF+" and "IIF−" Groups According to the results of IIF using normal skin for IgG reactivity to BMZ, the 133 cases were divided into two groups as "IIF+" (73 cases) and "IIF−" (57 cases), excluding 3 cases without IIF results.
The clinical, immunological, and histopathological features of IIF-group are also summarized and shown in Tables 1 and 2.
The Results of "Sole LM332" Group and "Multiple-Ags" Group According to the autoantigens detected, the 133 cases were divided into two groups as "sole LM332" (88 cases) and "multiple-Ags" (45 cases).
In the 45 patients in multiple-Ags group, the numbers and rates of patients positive with various non-LM332 autoantibodies are summarized in Table 3. Eight different non-LM332 autoantigens were detected, with the highest positive rate (67%) for BP180 ( Table 3). The average number of non-LM332 autoantigens recognized by patient sera was 1.6, while one patient reacted with 5 non-LM332 autoantigens, i.e., BP180, BP230, envoplakin, periplakin, and Dsg1. Thirty (67%) cases reactive with BP180 may be diagnosed as concurrence of LM332-MMP and BP180-MMP.
In addition, among the 30 LM332-MMP cases with anti-BP180 autoantibodies, ssIIF showed IgG-positive reactivities with both skin epidermal and dermal sides in 17 patients, positive reactivity with only epidermal side in 6 patients, positive reactivity with only dermal side in 5 patients, and negative reactivity with both epidermal and dermal sides in 2 patients. The five LM332-MMP cases with anti-Dsg 3 autoantibodies and four cases with anti-Dsg 1 autoantibodies did not show the staining with keratinocyte cell surfaces in the epidermis in IIF.
Comparative Analyses of the Results in Various LM332-MMP Groups
The results in the previously reported 55 DIF+/LM332 group patients, who showed positive IgG deposition to BMZ in DIF and reacted exclusively with LM332 (12), are also listed in Tables 1 and 2 for comparison. We performed comparative and statistical analyses of the results in all the LM332-MMP groups shown in Tables 1 and 2.
Compared with the DIF+/LM332 group (55 cases), all of the LM332-MMP group (133 cases) showed no significant differences on clinical and immunological results. Compared with DIF +/LM332 group, DIF−/ssIIF+ group had higher rates on oral blister and nasal lesion (both p < 0.05) and lower positive rate of autoantibodies against the 145-kDa LMa3 in IB of purified LM332 (p < 0.05) ( Table 4). Compared with the DIF+/LM332 group, multiple-Ags group had higher rate on oral blister (p < 0.05), accordingly lower rate on oral erosion (p < 0.05), higher positive rate of IgG reactivity to BMZ in IIF of normal skin (p < 0.05), and lower positive rates of autoantibodies against the 165-kDa LMa3 and the 145-kDa LMa3 (both p < 0.05) and higher positive rate of autoantibodies against LMg2 (p < 0.05) in IB of purified LM332 ( Table 4).
Compared with the DIF+/ssIIF+ group, the DIF+/ssIIF− group had a lower positive rate on IIF of normal skin for IgG reactivity to BMZ (p < 0.05) ( Table 5). Compared with the DIF+/ssIIF+ group, the DIF−/ssIIF+ group had lower positive rate of autoantibody against the 145-kDa LMa3 in IB of purified LM332 (p < 0.05) ( Table 5). Compared with the DIF+/ssIIF− group, the DIF−/ssIIF+ group showed no significant differences on clinical and immunological results.
Compared with the IIF− group, the IIF+ group showed significantly higher rate of ocular lesions (p < 0.05) ( Table 5).
Compared with the sole LM332 group, multiple-Ags group had similar statistical results to its comparison with the DIF+/LM332 group, particularly higher positive rate of autoantibodies against LMg2 (p < 0.001) ( Table 6). These results indicated that multiple autoantibodies might be related positively to the production of anti-LMg2 autoantibodies, but negatively to the productions of anti-LMa3 and anti-LMb3 autoantibodies. Compared with sole LM332 group, multiple-Ags group had higher rates of infiltration of eosinophils and neutrophils (both p < 0.05) ( Table 6), suggesting that multiple autoantibodies might enhance the infiltration of eosinophils and neutrophils.
In addition, oral score, mucosal score, and LM332 score were also used for statistical analyses but failed to find any correlated parameters in the present study.
DISCUSSION
In our previous article of the 55 LM332-MMP cases (12), we used very strict inclusion criteria of LM332-MMP: (i) IgG deposition to the BMZ in DIF, (ii) positivity to at least one of the three subunits (a3, b3, and g2) of LM332 in IB of purified human LM332 but negative for all other known skin autoantigens, and (iii) presence of mucosal lesions. These cases are designated as DIF+/LM332 group in the present study. Based on the results of these 55 cases, we concluded that ssIIF has a diagnostic value for LM332-MMP (12). Therefore, in the present study, we used newly revised inclusion criteria of LM332-MMP, in which the diagnosis can be made on the positive reactivity with dermal side of the split skin in ssIIF in cases without positive DIF result, and cases with additional autoantibodies against non-LM332 autoantigens were also included. The new inclusion criteria enable us to obtain as many as 133 patients with LM332-MMP, including the previous 55 DIF+/LM332 group patients, although the cohort included some cases of complicated LM332-MMP with multiple autoantibodies. In general, the 133 LM332-MMP cases in the present study showed similarly clinical and immunological features to those 55 cases in the DIF+/LM332 group, which were reported previously (12).
By comparing the DIF−/ssIIF+ group (12 cases) with DIF+/ LM332 group (55 cases) (12), most clinical and pathological features showed no statistical differences, excluding oral blister and nasal lesion (both p < 0.05). This finding indicated that these two groups belong to the same subgroup of LM332-MMP and further confirmed the diagnosis values of ssIIF for LM332-MMP.
Similarly, multiple-Ags group (45 cases) and DIF+/LM332 group (55 cases) showed no statistical differences in most clinical and pathological features, also indicating that these two groups belong to the same subgroup of LM332-MMP. Therefore, patients with mucosal lesion and anti-LM332 autoantibodies could be diagnosed as LM332-MMP, regardless of the concurrence of autoantibodies to non-LM332 antigens.
Based on the results mentioned above, we propose to use our new inclusion criteria for the diagnosis of LM332-MMP, rather than the very strict diagnosis criteria used in the previous study (12).
As for diagnostic sensitivity in the present study of 133 LM332-MMP cases, sensitivity of DIF for IgG deposition to BMZ and ssIIF for IgG reactivity with dermal side were the highest, followed by DIF for C3 deposition to BMZ and IIF of normal skin for IgG reactivity to BMZ. These results implied that ssIIF is a specific and sensitive method for the LM332-MMP diagnosis. Because the result of only one of the two methods (DIF and ssIIF) may be available for some cases, we suggest to use both methods for LM332-MMP diagnosis to avoid wrong diagnosis.
In the present study, autoantibodies against LMg2 subunit were most frequently found in LM332-MMP and may be correlated with multiple autoantibodies. These data implied that autoantibodies against LMg2 might be different from those against LMa3 and LMb3, in terms of pathogenic activity and possible close relation to antibodies to the other autoantigens.
Finally, in the present study, 17% of LM332-MMP patients had associated malignancies, and some patients had multiple tumors, further supporting the potential relationship between LM332-MMP and internal tumors suggested by previous reports (7,10,11).
In conclusion, the clinical, histopathological, and immunological features in our large cohort of LM332-MMP patients, including some complicated cases, overall confirmed the results of previous studies of LM332-MMP, including our own article (12). The diagnostic criteria for LM332-MMP, which we proposed in the present study, could benefit precise diagnosis and clinical studies on LM332-MMP in the future.
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 authors. | 2021-12-01T14:40:23.806Z | 2021-11-26T00:00:00.000 | {
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243860735 | pes2o/s2orc | v3-fos-license | String gravity in $D=4$
We revisit the four-dimensional theory of gravity that arises from string theory with higher-derivative corrections. By compactifying and truncating the ten-dimensional effective action of heterotic string theory at first order in $\alpha'$, and carefully dealing with field redefinitions, we show that the four-dimensional theory takes the form of an axidilaton model where the scalars couple to the Gauss-Bonnet and Pontryagin densities. Thus, the actual string gravity is a generalization of the well-studied Einstein-dilaton-Gauss-Bonnet and dynamical Chern-Simons models. Using this action we compute the stringy corrections to the Kerr geometry and we obtain, for the first time, the corrections to the entropy of the Kerr black hole at order $\alpha'^2$. We check that the first law of black hole mechanics is satisfied and discuss several properties of the solution. Our results suggest that there exist black hole solutions with $J>M^2$ and therefore the extremal ratio $J/M^2$ must be modified positively.
I. INTRODUCTION
String theory is thought to provide a quantum theory of gravity and a unified framework for all the forces of Nature. However, it possesses a huge landscape of low-energy effective theories that include all sorts of interactions, many of which we do not have any experimental evidence. One exception to this is gravity itself. Thus, it makes sense to wonder about the stringy prediction for the theory of gravity in our four-dimensional universe.
It has been known since long ago that the gravitational dynamics in string theories is not ruled by the Einstein field equations, but by higher-derivative extensions thereof. Indeed, the low-energy limit of the different types of string theories can be described by ten-dimensional supergravity actions with higherderivative corrections [1][2][3][4][5][6][7]. The compactification of these actions down to four dimensions leads to effective theories with many scalar and vector fields coupled to gravity, but it is interesting to ask what is the minimal truncation one could perform of these theories. Such minimal theory would correspond to the stringy description of pure gravity. Given that this theory will be different from general relativity (GR), it is simply natural to ask how are the vacuum GR solutions modified by string-theoretical corrections. In particular, one should wonder about the corrections to the arguably most important solution of Einstein's equations: the Kerr black hole.
In this paper we address these questions in the case of heterotic string theory (HST). As is well where α is a parameter with units of length square, and is the Gauss-Bonnet density. This theory has been the subject of very intensive research in the last years (see refs. [15][16][17][18][19][20][21][22][23] for for its black hole solutions), but we would like to revisit the claim about the stringy origin of this model. In fact, the EdGB theory cannot be the complete answer for the low-energy effective action of HST for an important reason: it lacks an axion field. In HST, the field strength of the Kalb-Ramond 2-form satisfies the Bianchi identity dH = α R ∧ R, and hence one cannot truncate this field, which must necessarily be a part of the gravitational sector together with the metric and the dilaton. In fact, this has inspired another family of well-studied models known as dynamical Chern-Simons theory [24,25], which can be written as arXiv:2111.04750v2 [hep-th] 26 Nov 2021 where in this casẽ is the Pontryagin density. Black hole solutions in this theory are also known [26][27][28][29][30][31]. Thus, it is clear that none of the two models above, EdGB and dCS gravities, can be, by themselves, the theory coming from the heterotic string effective action in four dimensions. On the other hand, it is not clear why one should not have other types of higher-derivative terms besides quadratic-curvature ones, such as (∂ϕ) 4 , (∂φ) 2 (∂ϕ) 2 , etc.
Therefore, the first question that we would like to clarify in this paper is what is the precise theory of gravity in four dimensions coming from HST. For that, we will make use of the ten-dimensional effective action at first order in α as given in [7]. As we show, the result is an almost (but not exactly) direct generalization of both EdGB and dCS theories. We cover this in section II.
The second question we want to address is that of the stringy corrections to the Kerr metric. It has been known for some time that, at first order in α , the Kerr black hole possesses an axidilatonic hair on account on the non-minimal coupling of these scalars to the curvature [32][33][34]. However, unlike the case of charged black holes, which receive first-order in α corrections [35][36][37][38], the geometry of neutral solutions such as the Kerr black hole is only modified at order α 2 . While these effects have been studied in the context of EdGB and dCS gravities (see the references above), the corrections to the Kerr metric in the actual string gravity model have been mostly ignored. As we show, the four-dimensional stringy action at first order in α can be consistently used to obtain the corrected Kerr metric at order α 2 . Thus, in section III we obtain the corrections to the Kerr geometry expressed analytically as a power series in the spin. We discuss in detail the thermodynamic properties of these rotating black holes, and we compute for the first time the α corrections to the entropy of the Kerr black hole by using Wald's formula. As an important test of our computations, we check that the first law of black hole mechanics is satisfied.
Finally, we provide some concluding remarks and discuss possible future directions in section IV.
Note on conventions: We follow the conventions of [39]: the metric has mostly minus signature and the Riemann tensor is defined by The Ricci tensor is defined in the usual way, R µν = R ρ µρν .
II. HETEROTIC SUPERSTRING EFFECTIVE ACTION IN FOUR DIMENSIONS
The first-order in α corrections to the heterotic string effective action are fully understood [3][4][5][6][7]. Here we will use the action given by ref. [7], which is obtained from the supersymmetrization of the Lorentz-Chern-Simons terms. The equivalence of this result with the ones obtained from string amplitudes was determined in [40].
Our starting point is the heterotic superstring effective action at first order in α , where we are already truncating away all of the gauge fields. In this action,R (−) is the curvature of the torsionful spin connection where a, b are Lorentz indices and ω a b is the usual spin connection. The curvatureR (−) can be written in terms of the Riemannian curvatureR and in terms ofĤ as follows, (8) On the other hand, the 3-form field strength is defined asĤ whereB is the Kalb-Ramond 2-form and ω L (−) is the Lorentz-Chern-Simons 3-form of the torsionful spin connection. Note that the relation (7) implies that H is defined in a recursive way that produces implicitly an infinite tower of α corrections according to (9) [41]. Also note that, due to the Chern-Simons term,Ĥ satisfies the Bianchi identity, Finally, the string coupling constant g s is related to the asymptotic vacuum expectation value of the dilaton according to g s = e φ ∞ , while the ten-dimensional Newton's constant reads G Our goal is then to find the simplest compactification and truncation of this theory down to four dimensions and to express it in the most compact or appealing way. We observe that the minimal consistent truncation we can perform consists in considering a direct product compactification on a six-torus, M 4 × T 6 . This is, the metric takes the form, where ds 2 is the four-dimensional metric in the string frame and the coordinates z i ∼ z i + 2π s parametrize the six-torus. Here we are taking all of the Kaluza-Klein vectors and scalars to be trivial. At the same time, the Kalb-Ramond 2-form and its 3-form field strength only have four-dimensional components, a fact that we express byB = B, H = H. That this is a consistent truncation follows from the fact that this compactification ansatz solves all of the ten-dimensional equations of motion once the lower-dimensional ones are satisfied, as one can check from direct inspection. 1 This trivial compactification gives rise to formally the same theory as (6) but in four dimensions and with a Newton's constant G N /(2π s ) 6 . In order to express this theory in a more appropriate form, we first dualize the Kalb-Ramond 2-form into a scalar field. Let us note that, in four dimensions, the Bianchi identity (10) can be expressed as Then, we can promote H to be the dynamical field instead of B by introducing the Bianchi identity in the action together with a Lagrange multiplier ϕ.
After integration by parts, we are left with the action.
1 Alternatively, one can be convinced of this by examining the full toroidal compactification of HST, whose action is provided in [42]. One can check that truncating all of the vectors and scalars, except for the dilaton, is consistent. where . (15) Now the variation of this action with respect to ϕ yields the Bianchi identity of H, while variation with respect to H yields a relation that allows one to remove H in terms of ϕ. However, in this case the dualization process is not so straightforward as the Lagrangian has a non-linear dependence on H through L R 2 . In fact, we get the following equation from the variation of H: In order to solve it, we expand H in a series in α , and we get the following result for the first two terms, We then have to plug H = H(ϕ) back into the action so that we eliminate the 3-form in terms of ϕ. Remarkably, when (17) is inserted in (14), we observe that no additional O(α ) terms are generated, and the action reads simplȳ We note, however, that the dualization introduces O(α 2 ) terms which were not present in the original action (6). These terms are given in Appendix A, where it is also argued that they become of order O(α 4 ) when the scalars are of order O(α ). This is relevant for computing corrections to vacuum solutions of GR, as we shall discuss later. Then, we have to evaluate the four-derivative term L R 2 by substituting the value of H (0) in (18), for which we also have to use (8). After a somewhat lengthy computation in which we make use of the properties of the Levi-Civita symbol, we find the following answer, where we have introduced A µ = e 2(φ−φ∞) ∂ µ ϕ. Herē R µνρσ is the standard Riemann tensor of the string frame metric, whileḠ µν is the Einstein tensor. Now we have to transform our theory to the (modified) Einstein frame. This is achieved by rescaling the metric asḡ The effect of this conformal rescaling on the twoderivative Lagrangian is well known: (23) On the other hand, it takes some computations to obtain the transformation of the four-derivative Lagrangian under this conformal rescaling. After making use of the transformation rules of the Riemann tensor and of the covariant derivative, and integrating by parts multiple times, we can express the result as follows, up to total derivatives that we omit: (24) This result is not very illuminating, but we can massage it a bit further. Let us first rewrite this expression in terms of the Gauss-Bonnet density, which is defined by Thus, we can simply replace the Riemann squared term by the Gauss-Bonnet density using R µνρσ R µνρσ = X 4 + 4R µν R µν − R 2 . Then, we can express the result as where we are simply collecting the rest of the terms in L , whose form, as we have seen, is quite complicated. However, this Lagrangian becomes much more illuminating if we write it in terms of the zeroth-order equations of motion, After some algebra, we obtain the following result Astoundingly, all of the terms in L are proportional to the zeroth-order equations of motion. This means that these terms can be removed via a redefinition of the fields of the form (31) These redefinitions introduce O(α ) terms proportional to the zeroth equations of motion, and hence we can use them to cancel all of the terms in L . We show the explicit redefinitions in the Appendix A. Of course, these redefinitions also modify the action at higher orders in α , and in particular they introduce O(α 2 ) corrections. Note that, for the terms in (30) that are quadratic in the zeroth order EOM, these O(α 2 ) corrections generated by the redefinitions are still proportional to the EOM and they can be further removed by an additional redefinition. These will then only contribute at order O(α 3 ). However, the two last terms in (30) are only linear in the equations of the scalar fields, and therefore we will introduce non-trivial O(α 2 ) corrections upon removing these terms at first order in α . We discuss these terms in more detail in Appendix A. 2 In sum, field redefinitions can be used to set L = 0. Finally, introducing the four-dimensional dilaton as φ = 2(φ −φ ∞ ) we have a very elegant form for the heterotic string effective action at first order in α in four dimensions: 3 4 This result deserves some comments. First, observe that it is very non-trivial that the only higherderivative corrections are given by the Gauss-Bonnet and Pontryagin densities. There are other four higher-derivative operators that could be present in the action and that could not be removed by field redefinitions, namely (∂φ) 4 , e 3φ (∂ϕ) 4 , e φ (∂ϕ) 2 (∂φ) 2 and e φ (∂ µ φ∂ µ ϕ) 2 . We remark that our result is completely general and we are not making any approximation, e.g., we are not neglecting those terms by assuming that the scalar fields are of order α . We simply find that these terms are not present. Second, precisely because we do not have those terms, this action is almost exactly equivalent to an axidilaton model where the curvature 2-form plays the rôle of the field strength of the gauge field. In fact, introducing τ = ϕ + ie −φ , and taking into account that the Gauss-Bonnet density can be written as X 4 = −R µνρσR µνρσ , whereR µνρσ is the double dual of the Riemann tensor, we can write the action in a suggestive way as Thus, except for the presence of RR instead of Riemann 2 (which would be the equivalent of F 2 ), this is essentially like an axidilaton model. 5 Third, note that the scalar fields cannot be truncated, and hence they form part of the gravitational sector. In particular, the axion must be non-trivial whenever R µνρσR µνρσ = 0. For the special case of spherically-symmetric solutions, the Pontryagin density vanishes, and hence one recovers the solu-Riemann tensor defined by [∇µ, ∇ν ]ξ ρ = R ρ σµν ξ σ , the only difference in the action would be a change of sign in the kinetic terms of the scalar fields, e.g., (∂φ) 2 → −(∂φ) 2 . 5 It is very tempting to wonder if this action possesses an SL(2, R) symmetry that involves the dualization of the curvature. We will not dwell in this in this paper but we think it would be worth to explore this idea elsewhere.
tions of EdGB gravity. However, whenever the Pontryagin density is non-trivial, an axion hair is generated, and the solutions will be different from those of EdGB and dCS theories. This happens of course for rotating black holes, but also, e.g., for linear perturbations around spherical black holes. Finally, observe that, for corrections over Ricci flat solutions, the scalar fields acquire non-trivial profiles of order α and these backreact into the geometry at order α 2 . The reason for this is that the two quadratic densities are topological, and thus they only contribute to the equations of motion as long as the scalars are non-trivial. Now, since the O(α 2 ) terms in the action (32) are proportional to derivatives of the scalars (see Appendix A), these terms actually contribute at higher orders when the scalars have profiles of order α . Therefore, the O(α ) action already captures all of the O(α 2 ) corrections to Ricci-flat solutions, such as the Kerr black hole, that we study in the next section.
III. THE KERR BLACK HOLE AT ORDER α 2
After having determined the form of the heterotic string effective action in four dimensions as given in (32), our goal now is to compute the α corrections to the Kerr metric in that theory. Throughout this section we will work in units of G (4) N = 1, so one has to bear in mind that every quantity is expressed in Planck units. Observe that the value of Newton's constant in our setup is given by G (4) N = g 2 s α /8, and therefore, in Planck units we have α = 8g −2 s . Hence, if we consider a weakly coupled string theory, g s << 1, we have α >> 1, and thus the corrections appear much below the Planck scale.
The first aspect that one notices about the solutions of the theory (32) is that they typically possess an axidilatonic hair generated by the coupling of the scalars to the quadratic invariants. The existence of this hair in the context of string theory was noted long ago [32][33][34], but let us quickly review it. The equations for the dilaton and the axion read and it is clear these scalars cannot be constant as long as the quadratic curvature invariants do not vanish. We can simplify these equations if we are interested in corrections to solutions that have trivial scalars at zeroth order in α -this is, corrections to GR solutions. Note that, since φ = 2(φ −φ ∞ ), the vacuum value of the four-dimensional dilaton must be zero according to string theory, while the axion can have an arbitrary vacuum value ϕ ∞ . Then, since the fluctuations of the scalars with respect to their vacuum values will be of order α , one can reduce their equations to the following, In the background of the Kerr black hole, one finds a unique stationary and axisymmetric solution to these equations by fixing the asymptotic values of the scalars to their vacuum values and demanding that they are regular at the horizon. This solution does not seem to admit a fully analytic expression but one can easily expand it in a series in the spin χ [18, 26-28, 34, 43]. In particular, let us remark that the axionic hair is vanishing for spherically symmetric solutions, but not for rotating ones.
While the profile of these scalars can only be obtained analytically through a power series in χ, it is possible to find the exact (in spin) value for the dilaton charge, Q φ , identified by the asymptotic expansion of the dilaton, φ = −Q φ /ρ when ρ → ∞. It turns out that this reads [43,44] where T is the Hawking temperature of the black hole. This result is known to hold in general for EsGB gravity with a linear coupling [44], but it holds for heterotic string theory only at first order in α . Likewise, one can find an exact expression for the dipole moment of the axion -see [43]. While the axion and the dilaton get corrections at first order in α , one can see that these in turn "backreact" in the geometry at order O(α 2 ). As we have shown, one can use the action (32) to consistently compute these corrections in heterotic string theory. Although the corrections to the Kerr metric associated to either the Gauss-Bonnet [15,[17][18][19][20][21][22][23] or the Chern-Simons [26][27][28][29][30][31] sectors have been separately studied in the literature, their joint action has been essentially missed.
In order to compute the corrections to the Kerr geometry, we will follow our previous work [43] that studies a more general theory motivated by EFT arguments and offers a systematic method to find the corrected Kerr metric in higher-derivative theories. As shown in that reference, one can capture the corrections to the Kerr metric by using the following ansatz, where Σ = ρ 2 + a 2 cos 2 θ and ∆ = ρ 2 − 2M ρ + a 2 , and the functions H i contain the corrections. These functions can be obtained analytically by performing a series expansion in the spin χ = a/M as where each term H (n) i (ρ, x) is simply a polynomial in 1/ρ and x = cos θ. When solving the equations of motion we get several integration constants, but we fix these by imposing asymptotic flatness and that the parameters M and J = χM 2 still represent the total mass and angular momentum. We show the few first terms of these functions in Appendix B, but one can solve the equations algorithmically and for the present work we managed to obtain the solution to order χ 40 . An analysis of convergence indicates that these series are convergent for χ < 1, and to order χ 40 they are accurate everywhere outside the horizon for χ < 0.9. It is to be expected that these series do not converge for χ ∼ 1, corresponding to near extremality. As a matter of fact, the metric above is not appropriate to study the corrections to extremal black holes, as the ansatz assumes that extremality happens for χ = 1 (when ∆ develops a double root), but this is no longer true in the presence of corrections [19,22,45]. One should use a different approach to study the corrections to extremal or near-extremal black holes. Nevertheless, we will try to say a few things about the extremal limit by extrapolating our results to higher values of χ.
One could study many properties of these corrected black hole geometries, but here we focus on the thermodynamic properties, which were not completely characterized in [43] -in particular, the entropy was not computed.
Let us start by characterizing the event horizon of (39). The main advantage of the coordinates used in the metric above is that the location of the horizon is determined by the largest root of ∆, and therefore its position in terms of ρ is not corrected. The Killing vector that generates the horizon must be of the form for certain constant Ω. Demanding that ξ becomes null at ρ = ρ + one finds that Ω is indeed the angular velocity of the horizon, This quantity does receive α corrections, and the few first terms in the χ expansion read (44) Notice that the fact that this quantity is actually constant means that the black hole horizon is a Killing horizon. This is a quite non-trivial check of the correctness of our solution. Also observe that the corrections to Ω are negative, so the stringy black holes spin more slowly than Kerr ones. In fig. 1 we show Ω as a function of the spin up to χ = 0.9 by using an expansion up to order χ 40 . The effect of the corrections becomes more relevant as we decrease the ratio M/ √ α , but they also increase for larger values of the spin.
Once we have determined the Killing vector ξ, one can compute its associated surface gravity at the horizon κ, and consequently obtain the Hawking's temperature of the black hole, which is given by We remind that the surface gravity is defined by the equation ξ ν ∇ ν ξ µ = κξ µ , which holds on the horizon. In practice, the computation of κ is a bit tricky, but it can be obtained with the methods of ref. [46]. The result can be expressed in terms of the H i functions (see [43]), and when evaluated on the stringy solution we get the following value for Hawking's temperature, In this case, we note that the corrections to the temperature are positive and they grow as we increase χ. This is even more evident in fig. 2, where we show the temperature up to χ ∼ 0.9 by using the O(χ 40 ) solution.
At this point, we may wonder about the extremal limit T = 0, but one has to be cautious with the α and χ expansions, which are both singular for χ ∼ 1. To understand this, let us consider the corrections to the temperature of a near-extremal black hole. Assuming the extremal limit T → 0 is regular, one can see that the temperature must be of the form where c is certain dimensionless constant. Essentially, this formula expresses the fact that the temperature will tend to zero as a square root, T ∝ (χ ext − χ) 1/2 . This is the type of behavior one naturally expects near extremality even in the presence of higher-derivative corrections. 6 If we now expand this expression in α we get Thus, when expressed in this way, the correction to the temperature is divergent for χ = 1, but this divergence appears because the series expansion in α is not valid anymore if 1 − χ 2 ∼ α 2 /M 4 , so it is just an artifact. Now we can ask if our correction to the temperature in eq. (46) behaves as ∼ 1/ 1 − χ 2 when χ → 1, but there are two caveats to this. In the first place, we do not know if the extremal limit is actually regular, and in fact studies of near-horizon geometries in EdGB and dCS theories suggest that it may not be [47]. 7 In that case, it is not guaranteed one can then use (47) for the near-extremal 6 This has been explicitly observed, e.g., in the case of charged black holes with α corrections [38]. 7 In particular, the near-horizon extremal geometries seem to be singular in EdGB gravity but they are regular for dCS theory [47]. However, the singularities in the EdGB case only appear at the poles of the horizon, and for instance the entropy is well-defined, so the thermodynamic properties probably behave regularly. It would be interesting to investigate what happens for the complete theory (32). temperature. On the other hand, as we have already remarked, our series expansions do not converge for χ ∼ 1. Thus, the best we can do is to examine the correction to the temperature up to a large enough value of χ (in our case we can reach χ ∼ 0.9) and try to extrapolate the result for χ ∼ 1. Let us define the dimensionless correction to the temperature by T = T Kerr + α 2 M 5 ∆T , so that ∆T is only a function of χ. Then, our idea is to fit this function to an expression of the form for values of χ as close to 1 as we can. The result of this fit in the interval χ ∈ [0.7, 0.9] -in which our series expansion in (46) is accurate -is shown in fig. 3. As we can observe, the fit with only three terms works very well, and it therefore suggests that the correction to the temperature really behaves as in (48). We also obtain a value for the constant c = 8πc 0 , namely c ≈ 8π × 0.0034 ≈ 0.086, although it is hard to say how accurate this result is -we would need to get closer to extremality to obtain a better estimation. On account of our previous discussion and equation (47), this would imply that the extremal limit is reached for Determining the precise value of the spin at the extremal limit would require other methods than the ones we have applied here, but all of the evidence points toward the existence of solutions with χ > 1. 8 Let us now turn our attention to the entropy of these black holes, which can be obtained by means of Wald formula [48,49]. This formula tells us that the entropy in a general diffeomorphism-invariant theory of gravity is given by where h and µν are, respectively, the induced metric and the binormal (normalized so that µν µν = −2) of any cross-section of the horizon Σ, 9 and is the variation of the four-dimensional action S with respect to the Riemann tensor. Let us remark that this formula can only be applied to theories in which all the fields are tensors, i.e., there should be no fields with internal gauge freedom. This is an issue for the original form of the heterotic string effective action (6), as the B-field contains Chern-Simons terms that are not (manifestly) invariant under diffeomorphisms. Historically, this has been a source of problems when dealing with the entropy of black holes in the heterotic theory (see for instance [51][52][53][54][55][56] and references therein), that only recently has been rigorously understood [57,58]. However, in the form in which we expressed (32), the Chern-Simons terms appear in a manifestly diff.-invariant form, and hence Wald's formula can be applied right away. The variation of the action with respect to the curvature reads wherẽ 8 As a matter of fact, this phenomenon has been observed for EdGB gravity [19,22], so it is not unexpected that the theory (32) shares this feature. 9 In the original formula by Iyer and Wald, Σ is assumed to be the bifurcation surface. However, it was later shown in [50] that the result is independent of which cross-section is chosen.
is the double dual of the Riemann tensor. However, it is known that the Gauss-Bonnet contribution to Wald's entropy can be simplified for stationary black holes, as the one at hand. In fact, by decomposing the Riemann tensor at the horizon using the Gauss-Codazzi equations it is possible to show that where R is the Ricci scalar of the induced metric on the horizon and O(K 2 ) are terms quadratic in the extrinsic curvatures of the horizon, that vanish for stationary black holes. Hence, the Wald formula reduces to the Jacobson-Myers result [59], which takes a simpler form as it only involves intrinsic quantities. On the other hand, a similar analysis does not seem to lead to anything particularly illuminating for the Chern-Simons contribution.
In sum, taking into account that µν µν = −2, we obtain the following general formula for the entropy of stationary black holes in the theory (32), where A Σ denotes the area of any cross-section of the horizon.
Let us compute the different contributions in this formula, for instance, for a t = constant slice of the horizon of our rotating black hole. The metric induced in this two-dimensional surface is the following: Therefore, the area is given by and evaluating it in our solution we get Let us remark at this point that the corrections to the area are strictly negative for every value of χ; these black holes are more compact than their GR counterparts. Now we have to evaluate the integral in (56). We note there is a first-order correction related to the Gauss-Bonnet term whose origin is topological. In fact, to first order in α we have e −φ = 1 − φ and hence we have the term where in the first equality we used the Gauss-Bonnet theorem and in the second one we took into account that the Euler characteristic of a (topologically) spherical horizon is χ(Σ) = 2. On the other hand, there is no analogous topological contribution from the Chern-Simons term, because its integral vanishes.
Finally, we have to compute the dynamical contribution to the entropy by integrating the combination −2φR + ϕR µνρσ µν ρσ . For that, we take into account that the scalar fields are already of order α and therefore we can evaluate the curvatures on the Kerr metric. In addition, the appropriately normalized binormal to the horizon reads The evaluation of the integral yields the following result, (62) Then, putting all of the pieces together, we find the following result for the entropy at order α 2 : Interestingly, we observe that all of the corrections to the entropy are positive, despite the area term being corrected negatively. Finally, we should check whether the first law of black-hole mechanics [60] is satisfied. For that, we must take into account that, in our solution, M represents indeed the physical mass of the black hole, while the angular momentum is J = χM 2 . Then, using (44), (46) and (63), we observe that the first law holds at order α 2 . Although here we are only showing the solution at order χ 6 , we have checked that the first law is satisfied for all the terms in the χexpansion that we are able to compute -up to ∼ χ 40 . This is a remarkably strong test on the validity of our computations, but also an interesting check on the validity of the first law of black hole mechanics for heterotic string theory. We offer two different visualizations of the entropy in fig. 4. In the top plot we show the entropy of rotating black holes relative to that of static ones, and we observe that, while the entropy always decreases with the spin, it does so more slowly when the α corrections are taken into account. In the second plot we graph the ratio between the entropy of the corrected black holes and the one of the Kerr black hole. We see that, not only the entropy of the stringy black holes is larger than the Kerr one, but the difference increases as we turn on the spin. All these effects seem to become more drastic as we approach χ ∼ 1, so it would be interesting to explore what happens in the near-extremal case, that is not accessible with our analysis.
IV. CONCLUSIONS
We have performed a dimensional reduction and truncation of the heterotic string effective action down to four dimensions. The resulting theory can be expressed in a very appealing form as given by eq. (32), which is a simple generalization of the well studied EdGB and dCS models. We have observed that the appearance of the Gauss-Bonnet and Pontryagin densities and no other higher-derivative terms is in fact a natural and non-trivial prediction of heterotic string theory. Interestingly, the 4dimensional action resembles that of a standard axidilaton model (see (33)) where the curvature plays the form of the field strength of a gauge field. It is therefore tantalizing to speculate whether such an action could contains a sort of SL(2, R) duality symmetry involving the curvature. If one could make sense of such a symmetry, this would be a very interesting way to constrain the additional α corrections in this minimal setup that only contains the metric and the axidilaton. Expressed as in eq. (32), the action contains O(α 2 ) corrections, which we show explicitly in the Appendix A. However, these terms become of higher order if one is interested in corrections to vacuum GR solutions (with trivial scalars at zeroth order). Therefore, the simple action (32) can be used to compute consistently all of the corrections to Ricci flat solutions to order α 2 . Then, using the methods of [43] we have obtained the perturbative α 2 corrections to the Kerr background expressed as a power series in the dimensionless spin parameter χ = J/M 2 . This approach allows one to study nonextremal black holes, but with enough terms in the series one can get close to extremality χ ∼ 1. In our case, we managed to obtain the solution to order χ 40 , which is accurate up to χ ∼ 0.9. We have focused on the thermodynamic properties of these stringy rotating black holes, and in particular we have obtained their entropy at order α 2 . We have found that, for a fixed angular momentum and mass, these black holes spin more slowly, are more compact, are hotter and are more entropic than their Kerr counterparts. In addition all these effects become more relevant as the spin increases. We have also checked that the first law of black hole mechanics holds at order α 2 , which is a very strong test of our calculations.
The positivity of the corrections to the entropy is line with general expectations, as it indicates that the underlying quantum theory contains more degrees of freedom that only become active at higher energies. On the other hand, the fact that the temperature is higher than for Kerr black holes suggests that the extremal value of the angular momentum will be corrected positively. We cannot obtain a precise value for the extremality bound with our current approach, but as discussed in section III, everything points towards the fact that extremality is modified as for a positive constant c. Interestingly, this is reminiscent of a form of the weak gravity conjecture according to which the corrections to the charge-tomass ratio of extremal charged black holes should be positive in string theory [61][62][63]. The positivity of the corrections to the entropy is also connected with this statement [64]. One of the reasons behind this conjecture is the requirement that extremal black holes should be able to discharge, which is possible if Q/M is modified positively by the higher-derivative corrections. In the case of rotating black holes, there does not seem to be an obvious reason why J/M 2 should be corrected positively, as the angular momentum can always be radiated away. 10 Our results suggest that J/M 2 > 1 anyway, in complete analogy with the charged case. It would certainly be interesting to better understand the properties of extremal and near-extremal black holes in the theory (32). One possibility to to simplify the problem would be to study the nearhorizon geometry of extremal black holes, which is itself a solution with enhanced symmetry to the equations of motion [47,67]. The near-horizon geometry allows one to obtain the entropy of extremal black holes, but the expression obtained is meaningless unless it can be written in terms of physical quantities. 11 One could compute the angular momentum by making use of generalized Komar integrals [68], in whose case one would obtain a physically meaningful relation S(J). However, one cannot identify the mass of the black hole from the near-horizon geometry. Thus, in order to compute the extremality bound one would need to study the global geometry of extremal black holes, which probably can only be accessed numerically -see [30] though. 12 Finally, it would be interesting to study the solutions to (32) even in a non-perturbative fashion. Although strictly speaking one would lose contact with string theory (because the stringy action contains more terms besides those in (32)), there are at least two reasons to do this. On the one hand, the theory (32) is, on its own, a well-motivated and interesting model. As it is known, the Gauss-Bonnet invariant leads to second order equations of motion, and the Pontryagin density has also reduced-order equations, namely, of third order (instead of fourth order, which is the case for typical higher-curvature terms). Thus, the theory might have a chance to lead to a well-posed dynamical evolution, which would be worth exploring [69]. On the other hand, non-perturbative solutions may offer new phenomena that are not seen when performing a perturbative expansion in α , hence their interest. Also, in that case the coupling between the axion and the dilaton and the presence of both Gauss-Bonnet and Pontryagin densities will make the non-perturbative solutions substantially different to their EdGB [19,22] and dCS [31] counterparts, leading perhaps to interesting new properties.
(A3)
One could now use (18) and (8) in order to find the expression of H (1) in terms of the Riemann tensor and the scalars, and then plug it back into (A1) to write down the α 2 corrections using these variables. This is however a long calculation that we are going to avoid since we do not need to know explicitly these terms. All we have to check, before ignoring them once for all, is that they will not induce α 2 corrections to vacuum solutions of GR. This is not difficult to see, as one can check by using the Bianchi identities of the Riemann tensor (namely,R µ[νρσ] = 0 and∇ σR σµνρ = 0) in the first term of (A2) that all the terms entering in the expression for H (1) contain derivatives of the scalars. This implies that the α 2 terms in the action actually become effectively of order O(α 4 ) when this action is used to compute corrections to vacuum solutions of GR. Hence, one can simply ignore them if we are just interested in the leading O(α 2 ) corrections.
Let us now give further details of the field redefinitions that one has to perform in order to cancel all the terms contained in L . Let us recall that, in terms of φ = 2(φ −φ ∞ ), the Lagrangian expressed in the modified Einstein frame reads where and where and we recall that A µ = e φ ∂ µ ϕ. We then perform field redefinitions of order α , It is not difficult to see that, up to boundary terms that we discard, these redefinitions have the following effect in the Lagrangian, Therefore, we can use these redefinitions to cancel all of the terms in L . This is for instance achieved by the choice being not unique. Notice that the redefinition not only introduces O(α ) terms canceling L , but also introduces an infinite tower of α n terms, that includes in particular α 2 corrections, besides those already present originally in (A4). These new terms are proportional to the ∆ shifts. In the case of ∆ µν , this quantity is also proportional to the zeroth-order equations of motion, and therefore the O(α 2 ) terms generated by the transformation ∆ µν can be removed by a new field redefinition of order α 2 . The same reasoning applies to the part of ∆ φ and ∆ ϕ that is proportional to the zeroth-order EOMs. Therefore, the only α 2 terms that we cannot a priori get rid of are those related to the transformations Since these are related to redefinitions of the scalars, it is easy to compute their effect, and we see that they generate the following contribution to the six-derivative Lagrangian (A15) We suspect that this Lagrangian, together with the (H (1) ) 2 contribution in (A1), can be simplified by integrating by parts and using additional O(α 2 ) field redefinitions. However, for the purposes of this paper we only need to note that the quantities∆ φ,ϕ are proportional to the square of ∂φ and ∂ϕ. Therefore, for corrections over vacuum GR solutions we have∆ φ,ϕ ∼ O(α 2 ) and in those cases the Lagrangian L (6) actually contributes to the equations of motion at order O(α 4 ). Hence, the first-order Lagrangian (32) already provides all of the α 2 corrections to the vacuum GR solutions. | 2021-11-10T02:15:47.039Z | 2021-11-08T00:00:00.000 | {
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244977642 | pes2o/s2orc | v3-fos-license | Computed Tomography Characterization of Emphysema in Combined Pulmonary Fibrosis and Emphysema (CPFE) and its Correlation with Lung Cancer with Comparison to Chronic Obstructive Pulmanory Disease (COPD)
Purpose: To investigate imaging features of emphysema on high-resolution computed tomography (HRCT) in combined pulmonary brosis and emphysema (CPFE) and its correlation with lung cancer, with comparison to chronic obstructive pulmonary disease (COPD). Methods: 160 consecutive patients (80 CPFE and 80 COPD ) were analyzed retrospectively. The HRCT imaging features of the two groups were compared, and the correlation between factors underlying CPFE and the occurrence of lung cancer was analyzed. Results: We found paraseptal emphysema to be the more common form of emphysema in CPFE (38.75%), with an incidence statistically higher than that of COPD (χ²=14.462, p=0.001). The extent of emphysema in each lung lobe in the CPFE group was statistically less than that in the COPD group (p<0.01). Homogeneous emphysema was common in both the CPFE (73.8%) and COPD groups (52.5%), and it showed a higher incidence in CPEF group (χ²=7.760, p=0.004). Homogeneous emphysema also showed signicantly high correlation to lung cancer (χ²=9.298, p=0.004). The incidence of lung cancer in the CPFE group (20%) was higher than that in the COPD group (8.8%) (χ²=4.113, p=0.035). There was a linear relationship between the incidence of lung cancer and the degree of pulmonary interstitial brosis and years of smoking in CPFE patients (χ²=6.679, p=0.01* ,b=0.148). Conclusion: Different emphysema features showed between CPFE and COPD, and CPFE showed higher incidence of lung cancer. Homogenous emphysema, severity of damage caused by interstitial brosis and the number of smoking years have high relationship with incidence of lung cancer in CPFE.
Introduction
Combined pulmonary brosis and emphysema (CPFE) is a clinical entity that has been increasingly diagnosed over the last two decades [1,2] and is broadly characterized by upper lobe emphysema and lower lobe brosis [3,4]. The upper lobe emphysema contributes to a static volume and late phase in the forced volume test. In contrast, the lower lobe brotic portion contributes to early phase ow in the ow volume curve, and the KL-6 value (a blood marker of pulmonary brosis) is usually highly elevated [5].
The co-existence of pulmonary brosis and emphysema is considered to be an independent syndrome having a speci c disease progression, and CPFE is frequently complicated by pulmonary hypertension, acute lung injury, and lung cancer, especially non-small cell lung cancer (NSCLC) [6][7][8]. Previous studies have shown the severity of brosis have correlation with prognosis in CPFE. Prognosis is generally poor, and studies have shown that forced vital capacity (FVC) and brosis scores (FS) can be used to predict survival in biopsy-con rmed CPFE [9,10]. However, few studies have explored the correlation between emphysema features and lung cancer in CPFE.
The two most prevalent forms of emphysema in CPFE are paraseptal emphysema, and bullae, whose prevalence rates among CPFE patients have been reported to be 93 %, and 54 %, respectively [11]. Clinically, emphysema was also commonly seen in chronic obstructive pulmanory disease (COPD), and centrilobular emphysema showed more common in COPD [9,12]. However, few studies [13] have explored the detailed characterisitics of emphysema between the two and compared the incidence of lung cancer.
Therefore, for better differentiation in CPFE and COPD and deeper understanding in prognosis in CPFE, in this retrospective cohort study, we investigated the emphysema features including types, volume and uniformity in CPFE, with COPD as comparison, and we also investigated the possible factor correlating to lung cancer including emphysema types and uniformity with multi-factor statistical analyses.
Patient population
This retrospective study was approved by the local ethics committee board (Peking University First Hospital Ethics commitment, and the number of ethics was 2017[1382]) with a waiver of written informed consent. The methods were carried out in accordance with the Declaration of Helsinki.
A total of 160 patients diagnosed with either CPFE or COPD by a multiple team discipline (MDT) of radiologists and pulmonologists from Jan 2013 to May 2019 were included in this study. The CPFE cohort (80 out of 160) was the observation group, and the COPD cohort (80 out of 160) was the control group. Exclusion criteria included any other respiratory diseases, missing information, poor image quality, or other factors resulting in insu cient diagnostic con dence. The smoking history was recored for every participant.
Diagnostic criteria
We used CT-based diagnostic criteria to diagnose emphysema and pulmonary brosis.
Emphysema is characterized as having cystic lung lesions with low-density shadows and a clear boundary. There is no wall structure and only a thin visible wall (< 1mm), and the diameter of the pulmonary bullae is greater than 1 cm. Pulmonary brosis is characterized as having grid and honeycombing shadows predominating in the peripheral pulmonary and lower pulmonary areas; stretch bronchiectasis with a small amount of local ground glass opacity and/or consolidation is also seen. All the diagnoses were made by two senior radiologists who were expertise in pulmonary eld in consensus.
Both groups of patients underwent HRCT examinations, using a full-body CT scanner (manufacturer GE). The following scanning parameters were used: voltage 120 KV, current automatic adjustment, pitch 1.375, slice thickness 1.25 mm, slice spacing 1 mm. Patients were in a supine position throughout the scan. Breath-holding training was routinely given to patients before scanning. The scan coverage was from the thoracic inlet to the top of the diaphragm.
All the patients were undergone pulmonary functional tests, and the nal diagnoses of CPFE and COPD were con rmed by the MDT of sophisticated radiologists and pulmonologists Emphysema volume CT post-processing technology was used in the determination of emphysema volume. Thoracic VCAR is a non-invasive CT image analysis software package, which may be used in conjunction with CT lung images to aid in the assessment of thoracic disease (emphysema, COPD) diagnosis and management. In this study, thoracic VCAR software was utilized to obtain the emphysema volume of the left and right lungs and each lung lobe. When the difference in the volume percentage of any one of the lung lobes was greater than 10%, it was de ned as heterogeneous emphysema (Figure 1a). If this was not the case, it was de ned as homogeneous emphysema (Figure 1b).
The degree of interstitial brosis was characterized in the three groups based on the imaging criteria, including ground-glass opacity, thickened lea et interval, grid shadow, honeycomb lung, and stretch bronchiectasis ( Figure 2).
Statistical analyses
Data were analyzed using SPSS program version 18.0. The patient demographic data were analyzed rst to identify any statistical differences between the CPFE and COPD groups. Emphysema volume and emphysema types with and without the presence of lung cancer were compared between these groups. In addition, the relationship between multiple factors (gender, age, smoking status, emphysema volume uniformity, emphysema type, pulmonary interstitial brosis, and years of smoking) and the presence of lung cancer was analyzed.
All measurements are presented as average ± standard deviation (X±S), based on the descriptive statistics. All data evaluate normal distributions. Counting data is expressed as percentages (%). Chi-square tests were performed, and a p<0.05 indicates that the differences were statistically signi cant. A Chi-square test of association was used to evaluate the linear correlation of pulmonary interstitial brosis and the presence of lung cancer or smoking status in the CPFE cohort.
Demographic data
For the CPFE observation cohort, there were 72 males and 8 females, and the average age were (70.5±9.6) years (ranging from 48 to 88 years). For the COPD control group, there were 74 males and 6 females, and the average age were (72.6 ±11.2) years (ranging from 45 to 101 years). There were no statistical signi cance in the demographic differences between the two groups (Supplementary 1).
Comparison of CPFE and COPD cohorts: Emphysema types characterized by HRCT
The most common type of emphysema in the CPFE cohort was the paraseptal type, followed by the mixed type, and the incidences for both types were statistically higher than for those in the COPD group. The COPD was dominated by the centrilobular type at a statistically higher level than for the CPFE group (p<0.05) (Table 1, Figure 3).
Comparison of CPFE and COPD cohorts: Emphysema volume and uniformity
The emphysema volumes of both the left and right lung were signi cantly lower in CPFE group (p<0.01), and also showed signi cantly lower in each lung lobe in CPFE group (p=0.001), compared with the COPD group.
In the CPFE group, 59 cases (incidence of 73.8%) were homogeneous emphysema, while 21 cases were heterogeneous. In the COPD group, 42 cases (incidence of 52.5%) were homogeneous emphysema, while 38 cases were heterogeneous. Thus, homogeneous emphysema had a higher incidence in CPEF group. The Chi-square test also con rmed that the incidence of homogeneous emphysema in the CPFE group was signi cantly higher than that of COPD group (χ²=7.760, p=0.004 < 0.01) ( Table 2). Correlation between multi-factors and lung cancer in the CPFE group: Lung cancer incidence and characteristics in CPFE patients The analysis of correlation between multi-factors and lung cancer incidence revealed a lack of gender difference was observed (p>0.05). Age, smoker status and emphysema types did not have signi cant impact on lung cancer incidence (p>0.05), but the uniformity of emphysema correlated with lung cancer incidence. Speci cally, homogeneous emphysema patients were more likely to develop lung cancer (42.6%) than were the patients with heterogeneous emphysema (11.9%). (Table 3) Correlation between multi-factors and lung cancer in the CPFE group: The correlation of lung cancer to pulmonary interstitial brosis and smoking history There was a linear relationship between the incidence of lung cancer and the degree of pulmonary interstitial brosis and years of smoking in CPFE patients. The incidence of lung cancer increased with the severity of interstitial brosis and the duration of smoking. For every severity degree increase of interstitial brosis or every 10 years of smoking history, the cancer incidence rose by 14.8% or 14.1%, respectively. (Table 4, Supplementary 2).
Discussion
In our study, we investigated the detailed imaging features of emphysema in CPFE, with comparsion to COPD, and our results showed paraseptal emphysema was the most common form in CPFE, with an incidence statistically higher than that of COPD (p=0.001). Homogeneous emphysema was common in both the CPFE (73.8%) and COPD groups (52.5%), and it showed a higher incidence in CPEF group (p=0.004). The extent of emphysema in each lung lobe in the CPFE group was statistically less than that in the COPD group (p<0.01).
Due to the histopathological heterogeneity of CPFE, researchers often rely on HRCT for the diagnosis of CPFE, rather than using a pathological diagnosis [14][15][16][17]. HRCT can be used to visualize early changes in emphysema, as well as the in-depth details of interstitial pneumonia. HRCT thus plays a fundamental role in diagnosis of CPFE syndrome. Choi et al. [17] suggested the following diagnostic criteria based on HRCT: an area of emphysema with a reduced translucency clearly demarcated from adjacent normal lung tissue with a very thin wall (<1 mm) or no wall, and/or multiple pulmonary bullae (>1 cm) in the upper lobe. Centrilobular emphysema is a long-term, progressive lung disease and is considered to be a form of COPD, and studies [9,12] have shown that centrilobular emphysema and/or bullous emphysema occurring predominantly in the upper lung is seen as a focal low-density area clearly demarcated from normal lung tissue, with visibly thin walls (<1 mm) or no wall.
Paraseptal emphysema (seen as low-attenuation areas in the subpleural zone) has been described in a majority of CPFE reports [1], and Araki [11] suggest that paraseptal emphysema is a typical feature of CPFE. However, determination of the emphysema type in CPFE is often very di cult because of alteration of its features due to coexisting brosis [18]. In spite of this di culty, the presence of thick-walled large cysts-2 cm or more in diameter and delimitated by a wall 1 mm or more in thickness-in an area of the lung where reticulation is present, is considered to be one of the characteristic features of CPFE [19]. Based on the analysis of emphysema types in CPFE patients, this current study revealed that paraseptal type emphysema is the most common, a nding consistent with other studies [11,13]. And we also used COPD as control to further pove the emphysema differences between the two enteties.
In our study, by using the thoracic VCAR post-processing software, we found that homogeneous emphysema was the main type in CPFE patients. This research has not been conducted before between CPFE and COPD. In addition, the emphysema volume was lower than that of COPD patients. This may be due to the different pathological mechanisms of emphysema formation by the two diseases. Although both can lead to poor alveolar elasticity, valve action caused by chronic bronchiolitis might also contribute to the pathogenesis of COPD. A larger sample study is required to con rm this hypothesis.
This study also explored the incidence of lung cancer in CPFE patients. Our results show that CPFE patients are more likely to develop lung cancer, compared with COPD patients, with squamous cell carcinoma being the most commonly seen. These results are consistent with previous studies published by Kwak et al. [20]. Upon analyzing the relationship between multiple factors of CPFE and lung cancer, we found that lung cancer incidence is higher in homogeneous emphysema patients and increases both with the severity of pulmonary interstitial brosis and the number of years of smoking. We speculated that homogeneous emphysema, severe brosis, and length of smoking history are the principal risk factors for lung cancer in CPFE patients. The diffuse pulmonary interstitial brosis seen in CPFE often presents as reticular blurry shadows, honeycomb patterns, distortion of alveolar structures in the subpleural and lower lobe, and/or traction bronchial or bronchiolar dilation [13,19]. Focal ground glass opacity and/or alveolar consolidation may also occur but is not the primary manifestation. Occasionally, ground-glass opacity can be the only evidence of interstitial lung disease, and in this situation, lung biopsy becomes necessary [21].
In a few cases, pulmonary nodules and consolidation have also been found in CPFE, ndings which are correlated with a higher incidence of lung cancer [20].
Previous studies have shown that smoking is closely related to the occurrence of CPFE and lung cancer.
However, further research is needed to describe the correlation of homogeneous emphysema, degree of brosis, and lung cancer incidence. This information would contribute to both earlier detection of lung cancer and earlier treatment of patients, making increased survival of CPFE patients possible.
The limitations of this study include small sample size, and lack of in-depth characterization of interstitial brosis. In future study, we should conduct further investigation.
Conclusions
Pulmonary interstitial brosis with emphysema (CPFE) features unique imaging characteristics. Different emphysema features showed between CPFE and COPD, and CPFE showed higher incidence of lung cancer.
Homogenous emphysema, severity of damage caused by interstitial brosis and the number of smoking years have high relationship with incidence of lung cancer in CPFE. Radiologists treating patients with CPFE should be alert to the high incidence of lung cancer in this population. Clinical manifestations and imaging should also be included when managing CPFE patients. ). The need for written informed consent was waived by the institutional review board due to the retrospective design of the study.
The methods were carried out in accordance with the Declaration of Helsinki.
Consent for publication
All the authors consent for the publication.
Availability of data and material The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
Competing interests
There is no competing interests among authors. Yan Xiong: Visualization, Investigation. | 2021-10-17T15:13:21.319Z | 2021-10-15T00:00:00.000 | {
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236480288 | pes2o/s2orc | v3-fos-license | Real-time Sampling and Estimation on Random Access Channels: Age of Information and Beyond
Efficient sampling and remote estimation are critical for a plethora of wireless-empowered applications in the Internet of Things and cyber-physical systems. Motivated by such applications, this work proposes decentralized policies for the real-time monitoring and estimation of autoregressive processes over random access channels. Two classes of policies are investigated: (i) oblivious schemes in which sampling and transmission policies are independent of the processes that are monitored, and (ii) non-oblivious schemes in which transmitters causally observe their corresponding processes for decision making. In the class of oblivious policies, we show that minimizing the expected time-average estimation error is equivalent to minimizing the expected age of information. Consequently, we prove lower and upper bounds on the minimum achievable estimation error in this class. Next, we consider non-oblivious policies and design a threshold policy, called error-based thinning, in which each source node becomes active if its instantaneous error has crossed a fixed threshold (which we optimize). Active nodes then transmit stochastically following a slotted ALOHA policy. A closed-form, approximately optimal, solution is found for the threshold as well as the resulting estimation error. It is shown that non-oblivious policies offer a multiplicative gain close to $3$ compared to oblivious policies. Moreover, it is shown that oblivious policies that use the age of information for decision making improve the state-of-the-art at least by the multiplicative factor $2$. The performance of all discussed policies is compared using simulations. The numerical comparison shows that the performance of the proposed decentralized policy is very close to that of centralized greedy scheduling.
shown to be optimal for point-to-point channels with a random delay and closed form solutions are found for the optimal threshold value. It is further shown that the oblivious policies can be far from optimal. We build on our prior work in [31] that concerned AoI minimization and propose decentralized threshold policies for minimizing estimation error in random access channels with many users.
Distributed decision making: In random access networks, a large number of sensors communicate with a single fusion center over a wireless channel. To avoid collision, most works in this direction have considered centralized oblivious policies that do not observe the process realizations for decision making (see, e.g., [43]- [49] and the references therein). In IoT applications, however, it is not realistic to assume a central scheduler that monitors all the sensors for decision making. We seek decentralized solutions in which each sensor decides when to sample and transmit information based only on its local observations. In decentralized setups (and in the context of control, rather than estimation) [50], [51] consider wireless control architectures with multiple control loops over a random access channel and optimize the access rate of the sensors who randomly communicate. Policies that adapt to the state of the systems are proposed in [52].
The work [53] (which was carried out concurrently and independently) designs decentralized policies for the remote estimation of i.i.d processes over a collision channel. Decision making in both [52] and [53] is thresholding and based on the realization of the process (or a function of that). But since neither of the two works exploits channel collision feedback, adaptations of them (or other policies that impose fixed transmission probabilities on the channel) are far from optimal in our setup.
C. Contributions
We study sampling and remote estimation of M independent random walk processes over a wireless collision channel. As opposed to all prior works, we seek decentralized solutions in which decision at each node is based solely on its local observations and channel collision feedback. Our goal is to minimize the estimation error, and specifically a normalized metric that we call the normalized average estimation errors (NAEE). This metric looks at the expected time-average estimation error, normalized by the number of source nodes M . We are interested in the asymptotic regime where M → ∞.
Two general classes of policies are considered, namely oblivious policies and non-oblivious policies. In the former class, decision making is independent of the processes that are monitored and we prove that minimizing the expected time-average estimation error, in the class of oblivious policies, is equivalent to minimizing the age of information. This leads to lower and upper bounds on the minimum achievable estimation error in this class along with efficient oblivious policies that are age-based. In particular, the NAEE under age-based policies is lower bounded by .88σ 2 and upper bounded by e 2 σ 2 . We next ask if non-oblivious policies can provide a significant gain by observing the processes as they progress. Since all source nodes are provided with the channel collision feedback, they can compute their age-function and reproduce their respective estimated processes (at the destination) in each time slot. Furthermore, using the collision feedback, the nodes can implicitly coordinate for communication. We define the notion of error process at each node which is a function of the sample values and age. We then propose a threshold policy, called error-based thinning, in which source nodes become active only when their corresponding error process is beyond a given threshold. Once a node becomes active, it transmits stochastically following a slotted ALOHA policy.
To find an optimal threshold and find a closed-form solution for the resulting NAEE, we first provide a closed-form expression for the NAEE that is a function of the peak age, the transmission delay, a term which we call the silence delay, as well as the process realization. We approximately find the NAEE under an optimal threshold policy by considering the underlying autoregressive Markov process as a discretized Wiener process. An optimal threshold is then shown to be approximately σ √ eM and the resulting NAEE to be e 6 σ 2 . The approximation error increases linearly as a function of the variance of the innovation process and decreases as M gets large.
Next, we extend our framework to unreliable random access channels. The closed-form of NAEE under oblivious and non-oblivious policies are provided. The multiplicative gain is the same as that in reliable random access channels, which equals to 3, and is independent of the channel erasure probability . Under non-oblivious policies, an optimal threshold is approximately σ eM/(1 − ) and the corresponding NAEE is e 6(1− ) σ 2 . Simulation results show that the proposed decentralized threshold policy outperforms oblivious policies. Moreover, oblivious policies are shown to outperform all state-of-the-art policies (both oblivious and non-oblivious) that impose a fixed rate (without using the collision feedback).
Finally, it is numerically shown that the performance of the optimal threshold policy is very close to that of centralized greedy policies that schedule transmissions according to the instantaneous 6 error reduction or age reduction.
The paper is organized as follows. In Section II, we introduce the system model. Oblivious policies are studied in Section III and non-oblivious policies are discussed in Section IV. The framework is extended to unreliable random access channels in Section V. Simulation results are presented for various policies in Section VI and our assumptions and derivations are verified numerically. Finally, we conclude in Section VII.
D. Notation
We use the notations E[·] and Pr(·) for expectation and probability, respectively. Scalars are denoted by lower case letters, e.g. s, and random variables are denoted by capital letters, e.g.
S.
The notation A ∼ B implies that A has the same distribution as B and N (0, σ 2 ) stands for the Gaussian distribution with mean 0 and variance σ 2 . The notations O(·) and o(·) represent the Big O and little o notations according to Bachmann-Landau notation, respectively.
II. SYSTEM MODEL Consider a system with M statistically identical sensors and a fusion center. We often refer to the sensor nodes as nodes or transmitters and the fusion center as the receiver/destination. For analysis tractability, we focus on symmetric cases where all nodes have similar configuration.
Let time be slotted. Each node i, i = 1, 2, · · · , M , observes a process {X i (k)} k≥0 which is a random walk process as follows where W i (k) ∼ N (0, σ 2 ). The processes {X i (k)} ∞ k=0 are assumed to be mutually independent across i and for simplicity we let X i (0) = 0.
At the beginning of each time slot, the nodes have the capability to sample the underlying process and decide whether or not to communicate the sample with the receiver. The communication medium is modeled by a collision channel: If two or more nodes transmit in the same time slot, then the packets interfere with each other (collide) and do not get delivered at the receiver. We use the binary variable d i (k) to indicate whether a packet is transmitted from node i and delivered at the receiver in time slot k. Specifically, d i (k) = 1 if node i delivers a packet successfully; d i (k) = 0 otherwise. We assume a delay of one time unit in delivery for packets. At the end of time slot k, all transmitters are informed (through a low-rate feedback link) whether or not collision occurred, which is indicated by an indicator c(k). If collisions happen in time slot k, then c(k) = 1; if a packet is delivered successfully at the receiver or no packet is transmitted, then c(k) = 0. Note that if c(k) = 1, then d i (k) = 0; if c(k) = 0, then d i (k) = 0 when node i does not transmit, and d i (k) = 1 when node i transmits a packet.
We assume that the buffer size of every transmitter is one packet and that new packets replace older undelivered packets at the transmitter. This assumption relies on the fact that the underlying processes that are monitored are Markovian.
The receiver estimates the process in every time slot based on the collection of the received samples. Denote byX i (k) the estimate of X i (k) in time slot k. We define the following normalized average sum of estimation errors (NAEE) as our performance metric: where M is the number of sources, π ∈ Π refers to the sampling and transmission policy in place, and Π is the set of all decentralized sampling and transmission policies. Note that the metric (2) is normalized by M . This allows us to study the asymptotic performance in the regime of large M . The minimum attainable NAEE is then denoted by L(M ): Our objective is to design decentralized sampling and transmission mechanisms to attain L(M ).
Consider the i th node. Let {k (i) } ≥0 be the sequence of time slots at the end of which packets are received at the destination from node i. In any time slot k, k , the latest sample from node i is received at the end of k (i) −1 and since collisions may happen, then it is time stamped at the beginning of time k with k ≤ k (i) −1 . So the age of information (AoI) [31] with respect to node i, denoted by h i (k), is Without loss of generality, assume k (i) 0 = 0. At the beginning of time slot k, the receiver knows the information of all packets delivered before time k, i.e., {X i (k (i) t )} −1 t=0 and reconstructsX i (k) by the minimum mean square error (MMSE) estimator: For the class of policies that we consider in this paper (oblivious policies and symmetric thresholding policies), the MMSE estimator reduces to a Kalman-like estimator: One of the major challenges in this problem arises from the decentralized nature of decision making. A decentralized policy is one in which the action of each node is only a function of its own local observations and actions. In this setup, the action of node i at time k depends on the history of feedback and actions as well as casual observations of the process {X i (j)} j≤k .
We also consider a simpler class of policies Π , called oblivious policies, in which the action of each node depends only on the history of feedback and actions at that node. In particular, oblivious policies do not take into account the realization (value) of the samples, but only the time they were sampled, transmitted, and received (if successfully received). We denote the minimum attainable NAEE in the class of oblivious policies bȳ We argue in section III that this simplification equivalently transforms the estimation problem into the problem of timely communication of packets for age minimization. By additionally exploiting the value of the samples, in Section IV, we design and analyze decentralized mechanisms that outperform oblivious schemes in minimizing the expected average estimation error.
III. OBLIVIOUS POLICIES AND AGE OF INFORMATION
Oblivious policies are independent of the processes they observe and they are therefore less costly to implement. Moreover, they can still benefit from the channel collision feedback to (i) quantify how stale the information at the receiver has become (in order to decide when to sample and communicate) and (ii) adapt to the channel state (for communication purposes). In this section, we show that minimizing NAEE in the class of oblivious policies is equivalent to minimizing the normalized average sum of AoI (NAAoI) as we have previously defined in [31].
First, we establish the following relationship between the expected estimation error and the expected age.
Lemma 1. In oblivious policies, the expected estimation error associated with process i has the following relationship with the expected age function: Proof. At the beginning of time slot k, the estimation error is By the stationarity of {W i (k)} ∞ k=1 and using (4), we conclude under oblivious policies. Therefore, using Wald's equality, we find Remark 1. Lemma 1 does not hold for non-oblivious policies. As a matter of fact, finding in closed-form is non-trivial and its numerical computation can be intractable when M is large. The reason is that even though the estimation error is the sum of h i (k) Gaussian noise variables, once we condition on h i (k), their distributions change because h i (k) can be dependent on the process that is being monitored.
Lemma 1 is reminiscent of [54,Lemma 4]. Using Lemma 1, the metric NAEE in (2) can be re-written as follows: where Note that J π (M ) is only a function of the age function h π i (k). The metric in (9) is the NAAoI defined in [31] and, therefore, the decentralized threshold policies of [31] apply directly. Note that the generation rate of packets for every sensor is θ ∈ (0, 1] in [31], while θ should be set to 1 in the model defined in Section II. This is because we assume that sensor i can observe the process {X i (k)} k for every k. In particular, [31, Algorithm 2] outlines a stationary agebased thinning (SAT) policy in which a source transmits only when the corresponding AoI is larger than a pre-determined threshold. Using this algorithm, it was shown that the following age performance can be achieved in the limit of large M : Results from [31, Proposition 1] also lead to the following lower bound on NAAoI J π (M ) for any decentralized policy π: Using (11) and (12), we arrive at the following proposition.
Proposition 1. The minimum attainable NAEE in the class of oblivious policies is characterized by the following bounds
A. Comparison with Oblivious Centralized Policies
In this section, we compare the SAT policy in [31,Algorithm 2] with an oblivious centralized policy -the Max-Weight (MW) policy [24], [26], [30], [31], [55]. Denote We devise the MW policy using techniques from Lyapunov Optimization. Define the Lyapunov function and the one-slot Lyapunov Drift We devise the MW policy such that it minimizes the one-slot Lyapunov Drift.
Definition 1. At the beginning of each slot k, the MW policy chooses the action i * such that Note that this policy is exactly the MW policy derived in [55] Proof. The proof of Proposition 2 is given in Appendix A.
Comparing (11) with (17), we have The NAEE of the decentralized SAT policy is e times that of the optimal centralized policy in the limit of large M . The conclusion coincides with one's intuition: the throughput of the decentralized SAT policy in [31] is e −1 , while the throughput of the centralized MW policy is 1, which implies the amount of delivered fresh packets in the centralized MW policy is e times that of the decentralized SAT policy. We illustrate their performances through simulations in Section VI.
IV. NON-OBLIVIOUS POLICIES
We now consider a more general class of policies in which the nodes can observe their corresponding Markov processes for decision making. In other words, we seek to benefit from not only the AoI, but also the process realization (in a casual manner). Clearly, if all nodes try to transmit their samples at every time slot, no packet will go through due to collisions.
The nodes, therefore, need to transmit packets with a lower rate. This means that they have to decide, in a decentralized manner, when to transmit. Motivated by the optimality of threshold policies in various point-to-point setups [32], [35], [36], [39], as well as their applications in age minimization over many-to-one random access channels [31], we propose threshold policies for decision making.
A. Error-based Thinning
Define the error process ψ i (k) at node i as follows: Since the transmitters have access to collision feedback, they can calculateX i (k), and hence ψ i (k), in each time slot and use this information for decision making. One way to understand ψ i (k) is as follows. At time k, if the sample of node i is successfully delivered, the estimation error will reduce by ψ i (k). So ψ i (k) quantifies the amount of instantaneous estimation error reduction upon successful delivery from transmitter i. With this viewpoint, we devise a threshold policy in which transmitters prioritize packets that have large ψ i (k). In particular, we design a fixed threshold β to distinguish and prioritize nodes that offer a high instantaneous gain.
The action of each node is thus as follows: node i becomes "active" if the error process ψ i (k) has crossed a pre-determined threshold β. Once a transmitter is active, it remains active until a packet is successfully delivered from that node. Active nodes transmit stochastically following In particular, each active node transmits its sample with probability p b (k) which is calculated adaptively as follows based on an estimate of the number of active nodes 1 : Here,λ(k) is an estimate of λ(k), and λ(k) is the sum arrival rate in time slot k. It is well-known that the maximum sum throughput of the slotted ALOHA is e −1 [56, Chapter 4.2.3] and the regime of interest is λ(k) < e −1 when k is sufficient large. In our setup, λ(k) corresponds to the expected number of nodes that become active in time slot k (see Definition 2 ahead). We refer to λ(k) as the activation rate or the effective arrival rate in time slot k.
So far, we have outlined a threshold policy in which a node decides to become active if its local error process is larger than a pre-determined threshold value β. We call this procedure Error-based Thinning (EbT). The main underlying challenge is, however, in the design of the optimal β. In the rest of this section, we will find an approximately optimal choice for β and analyze the corresponding NAEE. We start by some preliminaries.
B. Preliminaries
Consider node i and an inter-delivery interval (k Figure 1). The inter-delivery time I (i) is given by , we can write the 1 Since the sensors have unit buffer sizes, the number of "backlogged" nodes N (k) in Rivest's algorithm is at most M . One notes that this has been incorporated in (19).
error process ψ(k) as follows: Note that from (4), −1 , the term on the right hand side of (20) is the sum of h i (k) independent Gaussian noise variables. Indeed, (20) demonstrates that ψ i (k) contains both the information of sample values as well as the age with respect to source i. We next define "active" nodes as follows.
Definition 3 (Silence Delay and Transmission Delay). Let k 0 be as defined in Definition 2. We −1 as the silence delay, and U (i) = k (i) − k 0 + 1 as the transmission delay (see Figure 1).
An active source becomes inactive immediately after a successful delivery. By the above two definitions, the inter-delivery time I (i) consists of two components -the silence delay J (i) and the transmission delay U (i) : In this equation, J (i) is the first time slot after k (i) −1 at which ψ i (k) ≥ β (as defined in Definition 3). So J (i) −1 represents the number of time slots in which node i is not active, and U (i) represents the number of time slots in which node i is in active state. Recall that active nodes transmit with probability p b (k). So U (i) may be larger than 1 either because the node is active and it does not transmit or because the node transmits and experiences collision. By the stationarity of the transmission scheme, the processes across i and . We define I β , J β , and U β to have the same distributions as Using the definition of h i (k) in (4), and by the stationarity of {W j } j , we conclude that Recall that J β has the same distribution as J (i) . Then, J β is the smallest time index at which Finding an optimal β is non-trivial because β impacts both J β and U β . In the remainder of this subsection, we establish some useful expressions for the expectations of I β and U β in an optimal design.
Let a(k) denote the number of newly active nodes at time k, i.e., the number of nodes that become active from inactive states. We have E[a(k)] = λ(k), where λ(k) is the expected sum arrival rate in time slot k (imposed by our sampling and transmission policy). Now recall that in a traditional slotted Aloha-based random access channel, the maximum sum throughput is asymptotically e −1 . This is true also for the case with buffer size 1 where only the latest packets are stored, as discussed in [31, Appendix E]) and which applies to our setting here. Define c(M ) as the sum rate/throughput when the system contains M sources.
We provide our analysis under the following two assumptions: 2 Here, contrary to traditional slotted ALOHA schemes, the term "stabilized" does not refer to "stability of queues" in our problem setup. However, the term "stabilized" implies that the system is stationary, when the sum arrival rate is less than e −1 .
Assumption 2. Under an optimal β, when M is sufficiently large, the random access system is Assumptions 1, 2 are given for analysis tractability, and we will verify them for our proposed β later. In the rest of the paper, let M be sufficiently large. We seek to find an optimal β under assumptions 1, 2. To transmit as many fresh samples as possible, β is designed such that λ(k) is as large as possible. Thus, we focus on the regime where λ(k) is close to e −1 when k is large, and from Assumption 2, λ m ≈ e −1 . For tractability in analysis, we let the estimateλ(k) = e −1 for all k. Specifically, we replaceλ(k) with e −1 in (19). [58].
Recall that N (k) is the number of active nodes at the beginning of time slot k. The fraction of active nodes at the beginning of time slot k is hence N (k)/M .
Definition 5.
Define α β (k) as the expected fraction of active nodes: If β = 0, then all nodes are active and α 0 (k) = 1; if β = +∞, then all nodes are inactive and α +∞ (k) = 0. In the limit of k → ∞, we denote the expected fraction of active nodes by α β : The limit in (25) exists because the transmission policy is stationary and hence the sequence in the expectation above is stationary in the steady state. Continuing from (25), we have where step (a) holds by the dominated convergence theorem because the sequence in the expectation (26) is a fraction and bounded by 1. Utilizing the symmetry and stationarity with respect to various nodes (the system), we prove the following lemma in Appendix C, signifying that α β represents the fraction of time that each node is active in the limit of K → ∞, hence represents the probability of each node being active when the system is steady.
Lemma 2. When the system is stabilized, α β exists, and α β = Since α β exists, then, when k → ∞, the expected number of nodes that become active in From Assumption 2, λ m ≈ e −1 < 1. Using (27), one sees that M α β is an infinitesimal of higher order than M . Now using Lemma 2, we can show that E[U β ] is an infinitesimal of higher order than M , as discussed in the following lemma.
Lemma 3. When the system is stabilized, where α β is the expected fraction of active nodes in the steady state as defined in (25). Proof. The proof of Lemma 3 is given in Appendix D.
C. The closed form of NAEE
We next derive a closed form expression for the attained NAEE, L EbT (M ). Using (22), we re-write (2) as follows. Define Since h i (k) has the same distribution in the interval [k Proof. The proof of Lemma 4 is given in Appendix E.
Similar to (31), ∆ β can be expressed as From (33), the NAEE in (32) can now be re-written as follows where L EbT L EbT The equality in (36) is proved in Appendix F. Note that L EbT is a function of the peak age I β , the silience delay J β , the transmission delay U β , and the process realization through W j .
D. Optimizing β Approximately
Finally, we find approximate closed form expressions for L EbT (36) in the limit of large M : The following lemma comes in handy in our approximations. Proof. The proof of Lemma 5 is given in Appendix G.
For any j, S j σ is Gaussian with mean zero and variance j. We propose to use B j as an approximation of S j σ . Letting a = β/σ in Lemma 5, we obtain The approximation error analysis is provided in Section IV-E.
Substituting (39) into (34), we find the following approximation for L EbT : Theorem 1. Let M be sufficiently large. The optimal β * is approximately given by Proof. The detailed proof of Theorem 1 is given in Appendix H. Here, we only provide a roadmap of the proof. using (11) and (41), we obtain The NAEE of oblivious SAT policy is around three times that of the EbT policy. From (13), the NAEE of the oblivious MW policy of Section III is asymptotically σ 2 2 and comparing with e 6 σ 2 = 0.455σ 2 one concludes that the NAEE of the EbT policy is close to that of the oblivious MW policy. We remark that sinceL EbT (M ) is an estimate of L EbT (M ), these comparisons are not exact. We will also compare the numerical performance of Algorithm 1 with oblivious policies as well as other state-of-the-art algorithms in Section VI. Algorithm 1 below summarizes the proposed decentralized error-based transmission policy.
E. Approximation Error Analysis
Note that approximations are used in (38) and (39), now we analyze the approximation error in terms of σ 2 . The approximation error of L EbT consists of (i) the approximation error in (38) and (ii) the approximation error in (39), both of which are incurred when approximating an autoregressive Markov process with a Wiener process. In other words, the approximation error is due to the discretization of the Wiener process. This discretization is analyzed by the Langevin dynamics in [60]. In particular, Sn σ = n i=1 W i ≈ B n can be regarded as an overdamped Langevin dynamics with step size 1 to approximate the Brownian motion. The approximation error in each step remains constant due to the unit step size. Set β * = σ √ eM . repeat Step 1: For each node i, observe the collision feedback c(k − 1) and d i (k − 1) at the end of time slot k − 1, and update k (i) 's andX i (k), respectively.
Step 2: For each node i, observe X i (k) which evolves according to (1) and compute ψ i (k) by (18).
Step 3: If ψ i (k) < β * , then node i does not transmit packets; otherwise it transmits a packet with probability p b (k).
Then, we consider (39). J β is an approximation of J, and The distribution of J does not change with σ, nor does the distribution of J β . The terms S j σ ∼ N (0, j) inside the sum in (43) are independent of σ. The distribution of J β j=1 S 2 j /σ 2 does not change with σ. Thus, the approximation error in (39) increases linearly with σ 2 .
Recall that the approximation error in (39) increases linearly with σ 2 , thus the approximation error in E[J 2 ] also increases linearly with σ 2 . From (40), the approximation error in L EbT (M ) increases linearly with σ 2 .
V. UNRELIABLE RANDOM ACCESS CHANNELS
In this section, we generalize our model to account for unreliability in random access channels, i.e., erasure channels. Related works such as [61], [62] investigated Age of Information in unreliable channels, while optimal power allocation strategies in unreliable channel with respect to remote estimation has been considered in [63]. However, in this section, we aim to minimize NAEE defined in (2) under oblivious and non-oblivious policies in unreliable channels.
In the model defined in Section II, sensors can deliver packets successfully if no collisions happen. Now, we assume that packets are erased with some probability even if no collisions happen in the channel. In particular, suppose that if the channel is not in collision, the packet can be delivered with probability 1 − , where is the channel erasure probability. We do not introduce another feedback, i.e., we assume that only collision feedback (not the full feedback) can be transmitted to sensors. Same as Section II, (active) sensors transmit packets through the slotted ALOHA algorithm (19). For clarity of exposition, we assume that packets erasure happens in the end of every time slot (after channel collisions).
From Section IV, in the limit of M , the channel throughput/rate is around e −1 when = 0. Now, note that the channel erasure probability is , which implies when no collisions occur, every packet chosen by slotted ALOHA (19) is delivered with probability 1 − . Thus, the throughput/rate is around e −1 (1 − ). In this section, we let c(M ) = e −1 (1 − ).
We first consider oblivious schemes. By a proof similar to that of Lemma 1, (7) and (8) still hold in our unreliable random access setting. Since the channel throughput/rate is around e −1 (1 − ), we use [31,Theorem 5] to obtain the following NAAoI: Note that (7) and (8) Now, we consider non-oblivious schemes. The analysis in Section IV can be generalized to yield the following theorem.
Theorem 2. Let M be sufficient large. An optimal β * is approximately given by Remark 3. The new threshold in Theorem 2 is larger than that in Theorem 1, i.e., β * ≥ β * for 0 ≤ < 1. The expected number of newly active nodes is reduced. This is because (i) if a packet is erased, then the corresponding sensor is still active in the next time slot; (ii) the channel throughput/rate is decreases to around e −1 (1 − ), not e −1 . (45) and (46), we still have L SAT /L EbT ≈ 3 in erasure channels.
Remark 4. Comparing
Proof. The proof of Theorem 2 is the similar to that of Theorem 1. The only difference is
VI. NUMERICAL RESULTS
In this section, we verify our findings through simulations. Figure 2a compares the NAEE of our proposed policy with the state of the art for M = 500 under different σ 2 . In this plot, the green (plus) curve corresponds to an optimal stationary randomized policy in which each node transmits with an optimal pre-determined probability. The performance of threshold policies like [52], [53] that impose an optimal (fixed) transmission rate for each sensor also coincides with this curve, i.e, the green (plus) one. These policies do not exploit the available feedback for decision
NAEE
Optimal Stationary Randomized Policy Stationary Age-based Thinning in [31] Adaptive Age-based Thinning in [31] Error-based Thinning Oblivious MW Policy Non-oblivious Greedy Policy Estimated L EbT in (41) (a) NAEE as a function of σ 2 for various stateof-the-art schemes with M = 500. (Figure 3b). This coincides with the analysis in Section IV-E. In the simulation, we numerically find the expected fraction of active nodes to be α β = 0.0173. Substituting This is because more packets are erased when channel erasure probability is larger, hence a larger estimation error is occurred. In addition, the proposed algorithm, i.e., the EbT policy outperforms all other policies.
VII. CONCLUSION AND FUTURE WORK
We considered the problem of real-time sampling and timely estimation over wireless collision channels with M independent and statistically identical Gauss-Markov processes (sources).
We studied a normalized metric of estimation error which we termed the normalized average estimation error (NAEE), and focused on the regime of large M . We proposed two general classes of policies: oblivious policies and non-oblivious policies. We showed in the former class that minimizing the expected estimation error is equivalent to minimizing the expected age and consequently provided lower and upper bounds on the optimal estimation error. We then proposed and analyzed a (non-oblivious) threshold policy in which (1) nodes become active if their estimation error has crossed a threshold and (2) active nodes transmit stochastically with probabilities that adapt to the state of the channel (exploiting the collision feedback). We showed that the NAEE performance of oblivious (age-based) policies is at least twice better than the stateof-the-art schemes (which impose a fixed rate of transmission at the nodes) such as standard slotted ALOHA and optimal stationary randomized policy. Moreover, our proposed threshold policy offers a multiplicative gain close to 3 compared to oblivious policies. Finally, we extended our framework to incorporate unreliabile random access channels with erasure probability . The proposed optimal threshold and the corresponding NAEE increase with . Numerical results show that the multiplicative gain is 3 and independent of (which is consistent with Remark 4), and the additive gain (offered by non-oblivious policies compared to oblivious policies) increases with . Our findings suggest that designing optimal multiple access systems for the future IoT and CPS applications requires going beyond traditional metrics of rate, reliability, latency, and age. Recall that the proposed policy is oblivious to the monitored process. So Wi(j)'s are independent of hi(k). Using (7), (14), and (15), we write Moreover, the age functions have the following recursion: where di(k) ∈ {0, 1} indicates a successful delivery from source i at time k. Note M i=1 di(k) = 1. Under the MW policy, no collisions occur in every time slot, so hi(k) is a scalar (not a random variable) for all i, k. Substituting hi(k + 1) from (48) into (47), we obtain Thus, minimizing LD(k) is equivalent to choosing i * such that hi * (k) = maxi hi(k).
Since we assumed hi(0) = 1 for all nodes, from Lemma 2 in [26, Section III], the above MW policy is equivalent to a Round-Robin policy. Consequently, for all i = 1, . . . , M , and k ≥ i, we get hi(k) = 1, 2, · · · , M successively and periodically, and Therefore, which implies the correlation between I and U (i) m . We first claim that the Markov process S(k) = N (k),N (k) is geometrically ergodic [64]. In fact, by Assumption 2, the system is stabilized. Note that we setλ(k) = e −1 in (19) and λ(k) < e −1 for all k. Since λm = lim sup k λ(k) < e −1 =λ(k). From [64, Theorem 3.1 and Section IV], Markov process S(k) is geometrically ergodic. Define the state space of S(k) as P. For any i, j ∈ P, define pij(k) = [P (k)]ij and Π = [πi]i as the transition probability in time slot k and the stationary distribution, respectively. A Markov chain is geometrically ergodic [65] if there are ρ < 1 and C < ∞ such that for all i, j, k From (49), in the limit of k, the transition probability equals to the stationary distribution, i.e., lim k→∞ pij(k) = πj for any i, j ∈ P. as s1 and s2. Define the state of S(k) just before U (i) m as sm. In the following steps, we use πs i and Pr(si) interchangeably. Then, due to the Markovity of S(k), From (49), where | ij (k)| ≤ Cρ k for all i, j ∈ P. Note that the number of time slot between U Therefore, Note that (m ) ≤ Cρ m , so The last equality holds because m ≥ m and ρ < 1.
where p (i) a is the fraction of time that node i is active in the limit of K → ∞, Furthermore, (2) Using Lemma 2, we then obtain E[U (27), noting that α β ≤ 1, we find Therefore, we have From Assumption 2, under an optimal β, if M is sufficient large, then c(M ) ≈ e −1 , so there exists > 0, such that c(M ) > e −1 − . Therefore, from (29), we find where (a) holds because U β and J β are independent given β, (b) holds because E[U β ] = o(M ) (see (29)) and J β < I β , and (c) holds by (21) and the independence of U β and J β which leads to V ar(U β ) + V ar(J β ) = V ar(I β ).
Substituting (28) and (38) into (55), we obtain Note that J β , as defined before, is a stopping time of the discretization of the considered Wiener process B(t), and therefore J β > J, almost everywhere. We thus conclude that Delay per transmission is 1 time slot, so U β ≥ 1. Using (28) and (57), we can write Substituting E[J] = β 2 σ 2 (see (38)) into (58), we find .
For sufficiently large M , c(M ) ≈ e −1 and Assumption 2 is (approximately) satisfied.
Recall that E[a(k)] = λm when k → ∞, i.e., the system is stationary. Note that a(k) is non-negative, so by Markov's Inequality, when M is sufficiently large. Assumption 1 is thus (approximately) satisfied. | 2020-07-08T01:01:41.474Z | 2020-07-07T00:00:00.000 | {
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246840596 | pes2o/s2orc | v3-fos-license | Design and Development of Self-Made Cost-Effective Microsoft Excel Visual Basic Application for Livestock Ration Formulation
One of the most important aspects of the livestock sector is ration cost optimization, which results in profit and ideal animal health. Manually preparing rations is time consuming and unsafe. Whereas computers can quickly formulate a ration that meets all of the nutritional requirements, after giving standard data on feeds. However, the existence of the ideal computer programme is questionable; if it exists, it is more expensive, less user-friendly, exclude local feeds, be limited to a particular region/country, feed composition may differ. As a result, in this chapter, the user will learn how to create and develop a self-made least-cost ration formulation using the locally available feeds, so that user may easily build their computer Programme using Visual Basic application of Microsoft Excel. There are three phases to ration formulation for any animal (ruminant or non-ruminant). The first phase requires the user to know the available feeds and their nutrient composition. The second part involves determining which nutrients are important for animals and creating nutrient equations. The third phase involves the creation of a linear programming model. Finally, the interface is being designed. Each phase is thoroughly explained in excel, with suitable data and reference coding.
Introduction
The primary goal of ration formulation in animal production is to offer a balanced diet that supports physiological functions such as growth, maintenance, reproduction, and lactation while also providing energy for physical and metabolic activity [1]. A standard and efficient feed formulation must include all the classes of feedstuffs (Animal Nutrition Group, India) [2] as provided in Figure 1.
A concentrate is a feed or feed mixture that provides increased levels of primary nutrients (protein, carbohydrates, and fat) while containing less than 18 percent crude fiber (CF) and low moisture. These are high in nitrogen-free extract (NFE) and Total Digestible Nutrients (TDN) and low in crude fiber [3]. There are classified into two categories: energy-rich concentrates and protein-rich concentrates, based on the content of crude protein (CP). When the crude protein content of dry concentrates is less than 18 percent, they are categorized as energy-rich concentrates, and when the CP value is greater than 18 percent, they are defined as protein-rich concentrates [4,5].
Roughages are heavy foods that contain substantially less digestible material, such as crude fiber greater than 18% and a low TDN content (about 60%) on a dry basis. Roughages differ in the level of protein, mineral, and vitamin composition. Some roughages, particularly legumes, are good suppliers of calcium and magnesium [6]. The phosphorus concentration is likely to be moderate to low, whereas the potassium content is likely to be high, these concentrations are affected by the plant species, soil, and fertilization strategies all have an impact on trace minerals. Roughages are categorized into two classes based on moisture content: dry and green or succulent roughages [7]. Green roughages often have 60-90 percent moisture, while dry roughages have just 10-15 percent moisture. Green roughages are divided into several types for ease of use, including pasture, produced fodder crops, tree leaves, and roots. Based on the nutritional content and preparation methods, dry roughages have been further categorized as hay and straw [8]. Minerals available in the feeds are of different types i.e., Micro minerals, macro minerals and chelated minerals. Microminerals, also known as trace minerals, are needed in milligrams (mg) or microgram (g) quantities [9]. They're found in trace amounts in animal tissues and feeds. They're frequently found in enzyme cofactors and hormones. Cobalt, iodine, zinc, copper, manganese, and selenium are examples of micro or trace minerals. Macro-minerals play a specific role in the formation and function of the animal's body. Animals require the following seven macro-minerals: calcium (Ca), phosphorus (P), sodium (Na), magnesium (Mg), potassium (K), sulfur (S), chlorine (Cl) [10]. Chelated minerals are a class of organic minerals that are divided into proteinates, chelates, and other complexes based on their molecular structure [11]. A chelated mineral is one that has two or more chemical interactions with peptides or amino acids, such as copper or zinc. Each one has a different level of absorption and effectiveness.
The National Research Council has studied nutrient requirements based on several criteria such as dry matter intake (DMI), total digestible nutrients (TDN), crude protein (CP), metabolic energy (ME), calcium (Ca), phosphorus (P), and other elements that affect intake and techniques of prediction [12]. The entire weight of feed minus the weight of water in the feed is expressed as a percentage and is known as dry matter. In feeding studies, dry matter intake is determined by weighing the whole ration supplied and the amount of feed left by the animal [13]. The term "total digestible nutrients" stems from an old system of calculating available energy in feeds and animal energy requirements using a complex calculation of measured nutrients. The whole amount of protein present, determined from the total nitrogen available, is referred to as a crude protein. The percent nitrogen is multiplied by 6.25 to get the percent protein. The digestible energy intake minus the energy in the urine minus the energy in the gaseous result of digestion equals the metabolizable energy. Calcium is required for bone formation, neuron function, and the production of milk and eggs in animals. Phosphorus is also included in a wide range of co-enzymes, nucleic acids, proteins and amino acids [14].
It is essential to know the significance of these nutrients in animal feed. Animal disease control, as well as feed and fodder shortages, are the most pressing issues in animal husbandry. Farmers frequently encounter the following issues.
1. Nutrient requirement for the livestock animal, 2. The amount of feed that must be supplied every day to boost productivity, 3. Feed ingredient costs must be kept under control.
Animal nutrition is necessary for livestock production to be effective. Animal feed efficiency and growth rate can both benefit from good nutrition. Diets that meet the demands of animals must be provided.
This work used goats to fully understand nutritional needs and feed composition through the use of a visual basic application. The three phases of the study are explained in this chapter, the first phase is the selection of feeds and understanding their nutrient composition. The second part involves determining which nutrients are important for animals and creating nutrient equations. The third phase entails the establishment of a linear programming model, followed by the design of the interface.
Data collection
Goats can grow well and produce maximum milk if balanced and nutritious food is fed [15]. A balanced ration should contain digestible nutrients, vitamins, and minerals, including concentrate feeds and green and dry roughages. The feed list was created based on the most commonly used feeds, fodders and its nutrient composition was used as per ICAR (2013) given in Table 1.
Nutrient requirements and estimation of nutrient requirements
Nutrient requirements are estimated based on the Indian Council of Agricultural Research (ICAR) (2013) and it was programmed in Excel VBA. A balanced ration should meet the nutrient requirement. If the growing goat does not get the nutrients, it will affect milk yield and weight at the time of slaughter [16,17]. The nutrient requirements for the growing goats are given in Table 2. There are three major factors of balanced ration: DMI, CP and TDN.
Dry Matter Intake (DMI)(kg/d): Dry matter intake is dependent upon many factors like fodder quality and quantity, climate condition, and nutrient requirement of goat. The DM requirements of goats for different body weights and growth rate functions are different [18]. The dry matter requirement is calculated based on body weight and average daily gain as per the Indian standard [19]. The total DMI intake calculated is in terms of 'kg' and the formula used in VBA code is given below:
Dim a As Integer
Dim z As Integer Dim y As Integer Energy: Energy is expressed as "Total Digestible Nutrients" (TDN). Energy allows doing physical reproductivity. It also provides for the development, lactation, reproduction, and other physiological functions such as feed digestion [20]. The TDN is calculated based on metabolic body weight and average daily gain as per the Indian standard (ICAR 2013).
From the Table 1. Will find CF1 (common factor 1) for 5, 10, 15, 20, 25, 30 kg with respective metabolic body weight (BW^0.75). the formula of CF1 is given below in eq (1) 15 kg are taken as 30.04 (average), for more than 15 kg can be taken as 30.13 (average of 3). Then we find CF2 (common factor 2) with respective average daily gain (ADG) by the following formula (eq (2)) CF2 values are found to be 1.6. Therefore (see eq (3)) ME (Mcal/d) can be found by using the following formula (eq (4)) Where 0.28 is the common factor. Protein: Protein is expressed as crude protein (CP). It is one of the major nutrients in terms of nutrition and cost. CP represents the percentage of protein present in feedstuff. CP is essential for maintenance, increasing the forage intake [21,22]. It varies for every stage of goat life. Therefore, setting CP level is very important. If the balanced ration does not meet the CP requirement, protein supplies are to be used at a greater cost [23]. At the same time, they were producing a balanced ration for these four requirements to be met compulsorily with the least cost [24]. Then the production will be more and farmers will be benefitted. The CP is calculated based on metabolic body weight and average daily gain as per the Indian standard (ICAR 2013).
CF1
CF2 values are 0.46 for less than 10 kg and 0.44 for greater than 10 kg. Therefore (see eq (7)
Research design
The developed tool (RBT) uses VBA (Visual Basic Application) as front end and back end as excel. It is a simple excel file that is saved as .xlsm form and integrated with VBA code [25]. The user form or first page asks for input data, mainly body weight (BW) in kg and expected average daily gain (ADG) in g, depending upon which, it will calculate the minimum nutrient requirements, i.e., total DMI in 'Kg', CP in 'g', TDN in 'g'. Then should be selected from the list on the second page from roughages, concentrate, and minerals. Once this information is fed, tool RBT will solve for minimization of cost with DMI, TDN, CP as constraints. The tool RBT followed the following linear programming model [26]. (see eq (8)) Objective Function: Where c i is the cost of each feed ingredient, x j represents the number of feeds, a i represents the composition of DM, TDN and CP in all feeds, b min and b max are the minimum and maximum requirement of DMI, TDN and CP, c max is the maximum limit for each feed.
Results and discussion
Ration balancing tool for growing goata Microsoft Excel VBA based software can calculate the nutritional requirements for animals, such as dry matter intake (DMI), crude protein (CP), total digestible nutrient (TDN), and metabolizable energy (ME), for which equations are derived by using common factor method based on the data of ICAR ( Once the nutrients requirements are found, the application asks the user to select the available feeds which are listed in the application. Then it will provide a balanced ration using LP model [27]. The expert nutritionist has examined the created application in NIANP, and the results of some specific animal categories (Goat 1 and Goat 2) are given in Table 3. The developed RBT will find the least-cost ration with the consumer selected feed without breaching the nutrient requirement shown in the sixth column of Table 3. Table 3, It is observed that two different categories of goat listed for validation, DMI, CP, TDN and ME criteria, are determined depending on both the weight and average daily gain as in the second column of Table 3. The developed RBT will find the least-cost ration with the consumer selected feed without breaching the nutrient requirement shown in the fifth column. To find the optimal solution, the RBT uses the Excel solver. The Excel Solver is efficient in obtaining feasible solutions nonlinear model for goats and increased daily gain and milk yield [28]. The nutrients TDN and CP required for growing goat according to Mandal, 2005 [29] for goat 1 with the body weight of 20 kg and an ADG of 75 g are 351 g and 79 g, respectively, for goat 2 with the body weight of 20 kg and 100 g of ADG, the requirements are 446 g and 100 g respectively. The nutrients TDN and CP calculated by developed application for Goat 1 are 349.5 g and 77.74, and for Goat 2 are 444.7 g and 99.51 g. There is a very small difference 1.5 g (0.4%) and 1.26 g (0.3%) in TDN and CP for goat 1, for goat 2 TDN and CP difference is 1.3 g (1.6%) and 0.49 g (0.49%) between RBT and Mandal et al. (2017). The required nutrients for two different goat categories and calculated by balanced ration are shown in Figures 5 and 6 for goat1 and 2, respectively. From the above studies and evaluation, it can be confirmed that the calculated values for DMI, TDN and CP are almost equal to the values of ICAR (2013). A Ration Balancing Tool (RBT)" is developed using Excel VBA, which gives a balanced ration for the goats with the available ingredients that satisfy all the nutrient requirements. Many software programs are available to customize ration for the lowest cost [30]. However, scanty applications are available for goat least-cost ration formulation. This study explains how the application is exceptional and more efficient and convenient compared to all other software programs, most of which are not user-friendly, and farmers must rely on expert assistance to implement it. For the commercial business reason, many software programs are developed for the client, wherein small dairy farmers still have to rely on specialists for optimized rationing. This tool is very simple to execute and user friendly. It is designed to determine the nutritional requirement of goats, depending on their weight and daily gain and to optimize goat ration at least cost.
Steps followed for formulation
Step1: once the excel sheet is open, page 1 of the user form will appear as shown in Figure 7. This page contains three tabs 'Introduction', 'Methodology' and 'Help'. Detailed information on goat feed formulation is given in the 'Introduction' tab. The procedure to use the application is given step by step in the 'Methodology' tab. How to add excel options to the applications is mentioned in the 'help' tab.
Step2: Clicking the NEXT button, page 2 will be displayed as shown in (Figure 8) where users are required to enter input data such as body weight (kg), expected average daily gain (g) of goat. Depending on the input data, the tool RBT calculates the DMI, TDN and CP required for a particular goat. Step3: Users must select feeds from roughages, concentrate and minerals on page 3 as shown in (Figure 9). The option is given to fix the minimum and maximum feed selected. If not, it will take standard values set by the tool. Users can also add new feeds depending on the availability in the subpage shown in (Figure 10a).
Step4: After the selection of feed has been completed, the tool RBT will provide an optimized ration cost to satisfy all nutrients at the minimum cost. If any nutrient requirement has not been met, the app will ask for feed refining where the user has to reselect the feed, this case is shown in (Figure 10b).
Step5: For the final output, the user must click on the 'Go to Result'. It opens excel sheet where the user can find.
2. Cost of 100 kg ration on dry matter basis.
Ration cost for the number of goats available.
Printouts can be taken for all the results. The features of the developed application are as follows, firstly Data Maintenance; if no feed is listed, the feed with nutrient composition can be uploaded by the consumer while selecting the feed. It allows the consumer to reasonably apply the feed available locally and reduce the cost. Consumers can get optimized ration in maximum effective steps by selecting the animal data and picking the feeds and then tapping on the "Solve" icon to get the result as it is user-friendly. For Display and printing, after the solution is found, there is an option for the result to be printed on a fed basis with feed quantity and total DM intake per kg. System requirements are also minimal as All MS Office versions can be used, and no special hardware or RAM is needed. The macros and solver options in VBA reference need to be enabled. The application provides the result in three ways: cost of single goat ration: Here, the total DMI is provided for one goat. This will help the consumer get an optimized ration and provide the right amount of roughage, concentrate, and minerals to be included in the ration. Finally, the price of each feed is given in the result. Cost of 100 kg ration on dry matter basis: Here, it estimates 100 kg at a time, which can be fed to goats at periodic intervals. The amount of roughage, concentrates, and minerals to be added to make 100 kg and its cost will be shown. Ration cost for the number of goats available: Here, the ration will be estimated on a dry matter basis for the number of available goats. The output is given in Figure 11.
Conclusions
This study showed that how the excel VBA can be used to analyzing the nutrient requirement and producing a balanced ration for livestock (goat) are fundamental aspects of reducing goats' feeding cost. Hence Excel Visual Basic Application (VBA) has been developed. Developed 'RBT' for beneficial for dairy farmers, which is based on a linear programming model. The ICAR (2013) table values for nutrients and the software-calculated values have high R2 values (DMI-0.989; TDN-1; CP-0.999). It can be confirmed from the aforementioned studies and evaluations that the computed values for DMI, TDN, and CP are nearly identical to the ICAR (2013) values. Just by giving the goat's minimum input, the application will calculate the nutrient requirement and the balanced ration at the lowest cost. Adding extra feed allows the user to add the feed available, which can lower the cost. The answer produced by the application is verified by a nutritionist at the National Institute of Animal Nutrition and Physiology (NIANP), Bangalore. Hence, it is concluded that even this application could be used quite effectively by dairy farmers. By understanding the nutrient requirements, the same can be developed for other livestock animals such as cattle, buffalo, and pigs. | 2022-02-16T16:13:01.672Z | 2022-02-04T00:00:00.000 | {
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118605035 | pes2o/s2orc | v3-fos-license | Marder's Two-Fluid Dark Energy Cosmological Models In Saez-Ballester Theory of Gravitation
The present paper deals with cylindrically symmetric metric in the form of Marder (1958) with Saez-Ballester theory of gravitation in the presence of perfect fluid and dark energy. In order to obtain the deterministic solution of the field equations we have assumed that the expansion scalar in the model is proportional to the Eigen value of the shear tensor. We have also assumed that the two sources, here the perfect fluid and dark energy interact minimally with separate conservative parts of their energy momentum tensors together with the constant EoS parameter of the perfect fluid. The role of the dark energy in the present model with variable equation of state parameter is studied more in detail. Some physical properties of model are also discussed.
Introduction
The most remarkable astrophysical observations in the modern cosmology have revolutionized our understanding about cosmology. According to the cosmologists our current universe is not only expanding but also accelerating. The direct evidence comes from distance measurements and analysis of type Ia supernovae (SN Ia), measurements of cosmic microwave background as well as large scale structure strongly suggest that present universe is dominated by the standard candle known as dark energy and it is due to because of cosmic accelerated expansion of the universe [1]- [9]. According to Einsteins general theory of relativity in order to have such type of accelerated expansion of the universe, it is required to introduce new component to matter or the perfect fluid distribution of the universe with a large negative pressure. This new component most commonly known as dark energy. Thus from the recent observations obtained by the cosmologists it has been realized that without dark energy we can not explain the universe. The exact nature of the dark energy is known to be very homogeneous and not interact with any other fundamental forces except gravity. The form of the dark energy is not very dense so it is difficult to detect in the laboratory for the cosmologists. As the nature of dark energy and dark matter is unknown many radically different models have been proposed such as quintessence, tachyon, chaplygin gas, as well as generalized chaplygin gas etc [10]- [15]. Having some limitations in Einstein theory of general relativity since it does not seems to resolve some of the important problems in the cosmology such as dark matter or the missing matter problems many researchers attracted towards the alternative theories of gravitation. Brans Dicke theory, Barbers self creation theory, Saez-Ballester theory of gravitation are some of the alternative theories of gravitations [16]- [25]. Also,different two fluid models discussed by some researchers [26]- [29] The present paper deals with Saez-Ballester theory of gravitation. In Saez-Ballester theory of gravitation metric is coupled with a dimensionless scalar field φ in a simple manner. This φ-coupling gives a satisfactory description of the weak fields. Inspite of the dimensionless character of the scalar field, an antigravity regime appears in this theory [30]- [35]. In order to obtain the deterministic solution we have assumed that two sources of the perfect fluid and dark energy interact minimally with independent conservation of their energy momentum tensors with constant EoS parameter of the perfect fluid.. We have investigated cylindrically symmetric cosmological model in the form of Marder (1958) . We have obtained some physical parameters and also discussed their physical behaviors [36]- [39].
Model and Field Equations
We consider cylindrically symmetric metric in the form of Marder (1958) given by Where the metric potentials A 1 , A 2 , A 3 are the functions of cosmic time.
Here it is important to note that by using the co-ordinate transformations t → A 1 (t)dt metric given by equation (1) can turned into Bianchi type I. But for the sake of simplicity in the present paper we retain the metric given by equation (1). The field equations of the Saez-Ballester scalar tensor theory are Where the scalar field φ satisfying the equation is the overall energy momentum tensors with T (m) ij as the energy momentum tensor of the ordinary matter or the perfect fluid and T (de) ij as the energy momentum tensor of the dark energy component. These are respectively given by Where ρ (m) , p (m) are the energy density and pressure of the perfect fluid component respt. Whileρ (de) , p (de) are the energy density and pressure of the DE component respectively where as . Also in equation (2)̟ and n are constants. Now, the Saez-Ballester field equations (2) and (3) for the metric (1) with the help of equations (5) and (6) yield the following system of equations Also the energy conservation equationT ij ;j = 0 yieldṡ
Solution of the field equations
The field equations (7) to (11) is a system of five independent equations in eight unknowns A 1 , A 2 , A 3 , φ, ω (m) , ρ (m) , ω (de) and ρ (de) . Therefore in order to obtain an explicit solution of the system we require three more suitable assumptions relating these three unknowns. Let us first assume the condition that the expansion scalar in the model is proportional to the shear scalar which leads to Differentiating equation (13) and with some little manipulation we havė Now comparing equation (8) and (9) we geẗ Substracting equation (9) from equation (7) we geẗ Equation (17) with the equations (14) and (15) gives For the sake of simplicity by setting the relation we have used the substitutions So that Then the equations (16) and (18) respectively takes the form and d dt λ λ +γ γ λ = 0 Integrating equations (21) , (22) we get Where c 2 and c 3 are the constants of integrations. Thus from equations (13) and (23) we get a metric potential Equation (21) using equation (23) we get Where b and c 4 are the constants. From equation (20) with the equations (23) and (25) we get the remaining metric potentials Where b, c, D are the constants. Thus our required cosmological model for the metric (1) is given by Scalar field φ from equation (11) with the help of the equations (24), and (26) for the model (27) is given by Where k 0 is constant. To determine the energy density of the perfect fluid and DE components as well EoS parameters of the perfect fluid and DE components we have to assume following two more additional constraints. As per the proposed assumption according to Akarsu and Kininc [11] let us suppose that the two sources of perfect fluid and dark energy interact minimally. Therefore energy conservation equation given by (12) can be split up into two separately additive conserved components which are given byρ Finally we have assumed that the EoS parameter of the perfect fluid to be constant, i.e. ω (m) = p (m) ρ (m) = constant. While ω (de) is allowed to be a function of cosmic time since the current observational cosmological data from SN Ia, CMB and large scale structures mildly favor dynamically evolving dark energy crossing the phantom divide line (PDL).
Some physical parameter
The directional Hubble parameters of the model (27) are defined as Therefore mean Hubble parameter for the model is found to be The mean anisotropy parameter ∆ of the expansion for the model is obtained as By the definition shear scalar and expansion scalar for the model (27) are respectively found to be Integraing equation (29) by using assumption of EoS parameter ω (m) of the perfect fluid to be constant we get Where k 2 being constant of integration. Equation (10) with the help of the equations (24), (26) and (35) gives the energy density of the DE component as Equation (8) with the help of the equations (24), (26), (35) and (36) gives EoS parameter of the DE component as 4 Discussion and conclusion: The metric potentials A 1 , A 2 , A 3 all are finite at the initial moment but vanish when t = − c 3 c 2 and increases with increase in cosmic time. Thus model have point type singularity at the initial epoch. Similarly the directional Hubbles parameters as well as mean Hubble parameter are the functions of cosmic time t. All these parameters are finite at the early time of universe and diverge at initial singularity t = − c 3 c 2 but vanish when cosmic time is infinite. The mean anisotropy parameter of the model is constant throughout the evolution of the universe. The shear scalar as well as expansion scalar having the same behavior as that of the Hubble parameters. The energy density of the perfect fluid is constant when cosmic time is zero and decreases with the expansion of the universe. In the present model EoS parameter of dark energy is a function of cosmic time. The nature of the dark energy depends on constants involved in the expression of ω (de) . Also lim σ 2 θ 2 = 0 when t → ∞. In the present paper we have investigated cylindrically symmetric metric in the form of Marder by assuming that the two sources of the perfect fluid and dark energy interact minimally with EoS parameter of the perfect fluid to be constant. Also we have assumed the present paper is that current universe is dominated by the dark energy which can describe that the current universe is accelerating and consistent with observations. | 2016-02-10T12:26:35.000Z | 2016-02-10T00:00:00.000 | {
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52938507 | pes2o/s2orc | v3-fos-license | The mycoparasitic yeast Saccharomycopsis schoenii predates and kills multi-drug resistant Candida auris
Candida auris has recently emerged as a multi-drug resistant fungal pathogen that poses a serious global health threat, especially for patients in hospital intensive care units (ICUs). C. auris can colonize human skin and can spread by physical contact or contaminated surfaces and equipment. Here, we show that the mycoparasitic yeast Saccharomycopsis schoenii efficiently kills both sensitive and multi-drug resistant isolates of C. auris belonging to the same clade, as well as clinical isolates of other pathogenic species of the Candida genus suggesting novel approaches for biocontrol.
SCIenTIfIC REPORTS | (2018) 8:14959 | DOI: 10.1038/s41598-018-33199-z microorganisms, rare feature of being unable to assimilate sulfate as their sole source of sulfur. Recently, we reported the lack of eight genes in the sulfate assimilation pathway in draft genomes of Saccharomycopsis fodiens and Saccharomycopsis fermentans 15,16 .
Here we show that S. schoenii efficiently attacks and kills a range of pathogenic Candida species, including the newly emerged human pathogenic fungus C. auris. We follow the predation process using time lapse microscopy in combination with fluorescent dyes. Efficient predation as shown here could be useful for biocontrol purposes in either clinical settings for skin clearance or in agricultural settings for combatting plant pathogens.
Results and Discussion
In this study, we prospected the use of a predatory yeast, Saccharomycopsis schoenii, as a potential biocontrol agent against human fungal pathogens of the Candida clade with a focus on C. auris. To this end we confronted multiple drug resistant strains including C. auris NCPF8985#20, a multi-drug resistant isolate from the South Asian clade (India), with S. schoenii (Supplementary Table 1). Equal numbers of dimorphic S. schoenii and ovoid C. auris NCPF8985#20 cells were seeded on minimal media agar on microscopy slides to offer solid support for a potential S. schoenii interaction. This minimal media lacked methionine and thus did not support proliferation of S. schoenii in pure culture. We found that S. schoenii attacked C. auris cells upon contact and killed prey cells using specialized penetration pegs ( Fig. 1; Supplementary Movies 1-3). The chitin-staining fluorescent dye Calcofluor White, revealed septa at the bases of penetration pegs indicating the sites of entry ( Fig. 1a; Supplementary Movies 1 and 2). Within minutes after S. schoenii cells generated penetration pegs, C. auris cells started to vacuolarize, take up dyes such as propidium iodide that are not permeable to living cells and then collapse, presumably because of feeding and material transfer to the predating S. schoenii cell ( Fig. 1a; Supplementary Movies 1-3). We prepared Transmission Electron Microscopy (TEM) images of interactions between S. schoenii and C. auris after 1 h of co-culture on minimal media (Fig. 1b-e) and found that C. auris cells attacked by S. schoenii cells were necrotic (Fig. 1b). Penetration pegs were directed at prey cells (Fig. 1c) and cell wall interactions led to the formation of penetration peg start sites (# Fig. 1d). Ultimately this led to degradation of the prey cell wall (Fig. 1e). After killing of prey cells, penetration pegs did not grow further or develop into buds or daughter cells We determined rates of killing over a 6 h time-course using morphology and/or propidium iodide staining (PI) stain ( Supplementary Figs 1 and 2). Several prey cells were found to accumulate PI staining upon predation, however, many prey cells were apparently killed without being stained by PI. In these cases, killed prey cells were "flattened" and or shrunken in size. This resulted in the death of around 34% of C. auris cells within a period of 6 h of co-culture with S. schoenii (Fig. 2, middle panel; Supplementary Table 2). As a control, almost none of the C. auris cells (0.6%) had died after 6 h when cultured alone under identical experimental conditions. To examine if predator-prey interactions differ with different C. auris isolates that exhibit variable drug resistance phenotypes, we analysed predator-prey interactions in three additional C. auris isolates (Supplementary Table 1). Furthermore, to elucidate host range of predator-prey interactions within Candida species we included clinical isolates of Candida albicans, Candida glabrata, Candida lusitaniae, Candida parapsilosis and Candida tropicalis in this analysis. For reference, we used Saccharomyces cerevisiae and Schizosaccharomyces pombe, two previously known prey species of S. schoenii 14 . All isolates of Candida species tested, including several drug resistant C. auris strains, were susceptible to predation by S. schoenii (Fig. 2 and Supplementary Table 2).
Collectively, these results demonstrate that the predator yeast S. schoenii provides a novel opportunity to develop biocontrol methods for skin disinfection. Saccharomycopsis predator are unique within the Saccharomycetes in displaying predatory behaviour. Thus these yeasts may harbor potential as biocontrol agents of other fungi including human and plant pathogens. Based on genome survey sequencing, Saccharomycopsis yeasts, like the distantly related filamentous ascomycete Trichoderma, harbor multi-gene families of proteases and chitinases 15,16 (our unpublished data). These multi-gene families probably represent a resource for the identification of lytic enzymes that have the potential to generate novel antifungal compounds.
Microscopy. Imaging was performed using the PerkinElmer UltraVIEW VoX Spinning Disk Confocal
Microscope controlled by Volocity software. Images for movies were captured 2-4 times/min for up to 2 h, using the Nikon Perfect Focus System to autofocus. For kill curve analyses, three frames were captured every hour per species and time point. FIJI/ImageJ 17 was used for image processing and analysis. Drift in movies frames was corrected using the macro NMS fixTranslation v1 and the plugin Image Stabiliser. For kill curve analyses, individual cells were counted using the Cell counter plugin.
For TEM images, S. schoenii and C. auris cells were separately pre-cultured to log phase in YPD media, then washed and mixed together at equal ratios. A total 1*10 8 cells were seeded on SD media solidified with 2% agarose. After 1 h of co-culture, cells were scraped off, washed and pelleted. High Pressure Freezing was carried out using a Leica EM PACT 2 (Leica Microsystems, Milton Keynes, UK) and samples were freeze substituted in a Leica AFS 2. Freeze substitution was carried out using the following program: −95 °C to −90 °C for 30 h with 2% OsO 4 in acetone, −90 °C for 10 h with 2% OsO 4 in acetone, −90 °C to −30 °C for 8 h with 2% OsO 4 in acetone, −30 °C to −10 °C for 1 h with acetone, −10 °C to 4 °C for 1 h in acetone, 4 °C to 20 °C for 1 h in acetone. Samples were then removed and placed in 10% Spurr's (TAAB, UK): acetone for 72 h, followed by 30% Spurr's overnight, 50% Spurr's for 8 h, 70% Spurr's overnight, 90% Spurr's for 8 h and embedded in Spurr's resin at 60 °C for at least 24 h. Ultrathin sections were cut to 90 µm using a diamond knife (Diatome Ltd, Switzerland) onto copper grids (TAAB, UK) using a Leica UC6 and were contrast stained with uranyl acetate and lead citrate in a Leica AC20. Samples were imaged on a JEM 1400 plus (JEOL UK) Transmission Electron Microscope and captured using an AMT UltraVUE camera (AMT, USA). All relevant data are available from the authors. | 2018-10-22T17:25:40.381Z | 2018-10-08T00:00:00.000 | {
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3870254 | pes2o/s2orc | v3-fos-license | AHCYL1 Is Mediated by Estrogen-Induced ERK1/2 MAPK Cell Signaling and MicroRNA Regulation to Effect Functional Aspects of the Avian Oviduct
S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as IP3 receptor-binding protein released with IP3 (IRBIT), regulates IP3-induced Ca2+ release into the cytoplasm of cells. AHCYL1 is a critical regulator of early developmental stages in zebrafish, but little is known about the function of AHCYL1 or hormonal regulation of expression of the AHCYL1 gene in avian species. Therefore, we investigated differential expression profiles of the AHCYL1 gene in various adult organs and in oviducts from estrogen-treated chickens. Chicken AHCYL1 encodes for a protein of 540 amino acids that is highly conserved and has considerable homology to mammalian AHCYL1 proteins (>94% identity). AHCYL1 mRNA was expressed abundantly in various organs of chickens. Further, the synthetic estrogen agonist induced AHCYL1 mRNA and protein predominantly in luminal and glandular epithelial cells of the chick oviduct. In addition, estrogen activated AHCYL1 through the ERK1/2 signal transduction cascade and that activated expression of AHCYL1 regulated genes affecting oviduct development in chicks as well as calcium release in epithelial cells of the oviduct. Also, microRNAs, miR-124a, miR-1669, miR-1710 and miR-1782 influenced AHCYL1 expression in vitro via its 3′-UTR which suggests that post-transcriptional events are involved in the regulation of AHCYL1 expression in the chick oviduct. In conclusion, these results indicate that AHCYL1 is a novel estrogen-stimulated gene expressed in epithelial cells of the chicken oviduct that likely affects growth, development and calcium metabolism of the mature oviduct of hens via an estrogen-mediated ERK1/2 MAPK cell signaling pathway.
Introduction
S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a member of AHCY family of proteins involved in metabolism of S-adenosyl-L-homocysteine [1]. AHCYL1 consists of 540 amino acid residues and has a domain homologous to AHCY in the Cterminal region and multiple potential phosphorylation sites in the N-terminal region [2]. Unlike AHCY that catalyzes a reversible reaction for S-adenosylhomocysteine hydrolysis [3,4], AHCYL1 does not have hydrolase activity for adenosylhomocysteine due to the substitution of important amino acids in the critical enzymatically active site [5,6] although it has an AHCY-like domain in the C-terminal domain [1]. However, AHCYL1 plays important role in the inositol phospholipid (IP) signaling pathway by interacting with the inositol 1,4,5-trisphosphate (IP 3 ) receptor, which is an intracellular Ca 2+ release channel located on the endoplasmic reticulum [2,5,7,8,9,10]. Therefore, AHCYL1 influences the IP 3 -induced Ca 2+ signaling cascade essential for numerous cellular and physiological processes such as organ development, fertilization, and cell death [8,11,12]. Recently, Cooper and co-workers identified two zebrafish AHCYL1 orthologs and found that the function of AHCYL1 is different from AHCY and that it plays a key role in zebrafish embryogenesis in response to IP 3 receptor function for release of intracellular calcium [1]. Although AHCYL1 is highly conserved among various species, little is known about its expression and functional roles in chickens or any other avian species.
In mammals, the oviduct undergoes diverse cellular and molecular changes in response to sex steroids during the estrous/menstrual cycle and peri-implantation period as these actions are pivotal to establishing an optimal microenvironment from gamete transport and embryonic development [13]. Of these steroid hormones, estrogen is the primary female sex hormone that controls a number of biological events including cell proliferation and differentiation, protection against apoptosis, and diabetes [14,15]. To investigate mechanisms of action of sex steroids, their biological effects and signal transduction cascades, the chicken oviduct is an established model due to its responsiveness to steroid hormones [16]. The chicken oviduct is a highly differentiated linear organ with compartments that undergo structural, cellular and biochemical changes in response to sex hormones during egg formation and oviposition [17]. The oviduct of egg-laying hens consists of the infundibulum, magnum, isthmus, and shell gland essential for fertilization, production of egg-white proteins, formation of the soft shell membrane, and formation of the outer egg shell, respectively. In the chicken oviduct, estrogen induces both cell proliferation and differentiation, as well as anti-apoptotic effects on cells [18,19]. In particular, estrogen stimulates formation of oviductal tubular glands and differentiation of epithelial cells into goblet and ciliated cells [20]. In addition, estrogen regulates the mechanism for Ca 2+ release necessary for formation of the egg shell in the shell gland of the chicken oviduct [21,22].
We used differential gene profiling data from the chicken oviduct to identify the avian homolog of the human AHCYL1 transcript and found it to be highly expressed in chicks treated with the synthetic estrogen agonist diethylstilbestrol (DES) [23].
There is little known about the expression or function of AHCYL1 in most species, except for humans and zebrafish [1,2,6]. Therefore, the objectives of this study were to: 1) compare the primary sequences of chicken AHCYL1 with those of selected mammalian species; 2) determine tissue-and cell-specific expression of AHCYL1 gene in various organs of the mature chicken; 3) determine whether estrogen regulates expression of AHCYL1 mRNA and protein during development of the chick oviduct; 4) determine whether AHCYL1 regulates calcium release and expression of genes related to development of the chicken oviduct through an estrogen-induced MAPK signaling pathway(s); and 5) investigate post-transcriptional regulation of AHCYL1 expression in the chicken oviduct. Results of this study provide novel insights into the AHCYL1 gene with respect to its sequence, tissue specific expression and hormonal regulation of its expression during development of the chicken oviduct.
Sequence Comparison, Pair-wise Alignment and Phylogenetic Tree Analysis of AHCYL1
The chicken AHCYL1 gene spanned 10.3 kb on chromosome 26. The gene consists of 16 exons and the mRNA has 3,445 bp encoding a protein with 540 amino acid residues. The primary sequence of chicken AHCYL1 was compared to those of six mammals and the zebrafish. Chicken AHCYL1 protein contained an NAD(P)-binding motif required for catalysis of S-adenosyl-L-homocysteine into adenosine and homocysteine as for mammalian AHCYL1s ( Figure S1). This verified that AHCYL1 has a different function from AHCY because AHCYL1 lacks several binding sites for S-adenosyl-L-homocysteine, irrespective of the conserved cysteines required for a tight globular structure of AHCY and NAD + binding motifs [1]. In pair-wise comparisons of chicken AHCYL1 proteins with seven other vertebrates, chicken AHCYL1 protein has high homology to mammalian AHCYL1 proteins (91-98%, Table 1). The phylogenetic tree was constructed using the neighbor-joining method ( Figure S2). The human and orangutan AHCYL1 genes clustered together and formed a larger cluster with mice and cattle, and an even larger cluster with sister groups was detected for dog and zebrafish. However, chicken AHCYL1 is in a separate branch, but closer to rat than to other mammalian species. These results indicate that chicken AHCYL1 diverged from mammalian AHCYL1 at very early stage in its evolution.
AHCYL1 mRNA Expression in Various Organs from Chickens
Tissue specific expression of AHCYL1 mRNA in brain, heart, liver, kidney, muscle, gizzard, small intestine, ovary, oviduct and testis of 1-to 2-year-old males and females was determined by RT-PCR. High levels of expression of AHCYL1 mRNA were detected in kidney and testis from male, and liver and oviduct from female chickens ( Figures 1A and 1B), and lower expression in liver, gizzard and small intestine from males and kidney and gizzard from females. However, expression of AHCYL1 mRNA was not detected in other organs analyzed regardless of sex. Based on differential gene profiling data of the chicken oviduct, the avian homolog of the human AHCYL1 transcript is highly expressed in the oviduct of chicks treated with DES [23]. Since little is known about expression and function of AHCYL1 in the oviduct of any species [1,2,6], this study focused on its expression in the chicken oviduct.
Localization of Chicken AHCYL1 mRNA and Protein in Chicken Oviduct
In situ hybridization analysis was used to determine cell-specific localization of AHCYL1 mRNA in the chicken oviduct. The oviduct of egg-laying hens includes the infundibulum (site of fertilization), magnum (production of components of egg-white), isthmus (formation of the shell membrane), and shell gland (formation of the egg shell). As illustrated in Figure 1C, AHCYL1 mRNA was most abundant in the luminal epithelium (LE) of the infundibulum and shell gland, and it was also expressed at lower abundance in glandular epithelium (GE) and LE of the magnum and isthmus, and GE of the infundibulum and shell gland. Little or no mRNA was detected in stromal cells, blood vessels, immune cells or myometrium of the oviduct. Results of immunohistochemial analysis ( Figure 1D) were consistent with results from in situ hybridization analyses. The AHCYL1 protein was abundant in LE of all segments of the oviduct, but less abundant in GE of magnum, isthmus and shell gland. The nonspecific mouse IgG, used as a negative control, did not detect AHCYL1 protein in any segment of the oviduct.
Effects of DES on AHCYL1 mRNA and Protein Expression in the Chicken Oviduct
Cell-specific expression of AHCYL1 in the oviductal segments of mature hens suggested regulation by estrogen during development of the chick oviduct. We reported that exogenous DES affects growth, development and differentiation of the chick oviduct and discovered candidate genes and pathways regulating oviduct development [35]. Therefore, we examined the effects of DES on AHCYL1 expression in the chick oviduct. As shown in Figures 2A and 2B, semi-quantitative RT-PCR analysis indicated that DES increased AHCYL1 mRNA in all segments of the 37-dayold chick oviduct. Further results from quantitative PCR revealed that DES induced a 3.3-fold increase (P,0.01) in oviductal AHCYL1 mRNA as compared to 37-day-old chicks that were not treated with DES ( Figure 2C). In addition, DES stimulated 2.3-, 3.4-, 2.6-and 4.3-fold increases (P,0.01) in AHCYL1 mRNA in the infundibulum, magnum, isthmus, and shell gland, respectively ( Figure 2D). In situ hybridization analyses revealed that AHCYL1 mRNA is expressed specifically in superficial GE in close proximity to LE in all segments of the oviduct of chicks treated with DES ( Figure 2E). AHCYL1 mRNA is also expressed at lower abundance in LE of oviducts from untreated chicks. Consistent with results from in situ hybridization analyses, immunoreactive AHCYL1 protein was detected predominantly in superficial GE of magnum and isthmus, and to a lesser extent in GE of shell glands of oviducts treated with DES and LE of oviducts from untreated chicks ( Figure 2F).
DES Activates ERK1/2 Signal Transduction in Chicken Oviduct Cells
Epithelial cells from the chicken oviduct were isolated and cultured in the presence or absence of DES and subjected to protein extraction. Based on results from our preliminary experiments, we focused on MAPK signaling cascades, especially, the extracellular-signal regulated kinase1/2 (ERK1/2) signaling pathway. Based on dose-response experiments ( Figure 3A), DES was used at 2 mg/ml in all experiments to determine cell signaling pathways mediating effects of DES on activation of ERK1/2 proteins. Western blot analyses of whole oviduct cell extracts with antibody to phosphorylated target proteins indicated that DES increased phospho-ERK1/2 (p-ERK1/2) 15.7-fold (P,0.01) over basal levels within 5 min and this effect was maintained to 30 min ( Figure 3B). In addition, the same dose of DES increased AHCYL1 protein approximately 3-fold within 2 h and further stimulated it 6.3-fold over 24 h. Next, we examined effects of an ERK1/2 inhibitor (U0126) on the ability of DES to increase synthesis of AHCYL1 protein ( Figure 3C). As illustrated in Figure 3D, 1 to 10 mM U0126 decreased the abundance of AHCYL1 protein in response to DES treatment. To verify these results, we performed immunofluorescence analyses and compared Figure 3E). Immunoreactive AHCYL1 protein was most abundant in the cytoplasm of chicken oviduct epithelial cells treated with DES, but detectable at lower abundance in the cytoplasm of cells receiving no treatment or DES treatment with U0126. Furthermore, DES stimulated calcium release from epithelia cell of the magnum of the chicken oviduct in a dosedependent manner ( Figure 3F). However, calcium release was reduced in these cells when cultured in the presence of both DES and U0126 as compared to DES alone ( Figure 3G).
AHCYL1 Knockdown and Expression of Genes Related to Oviduct Development in Response to Estrogen
The constitutive expression of AHCYL1 after transfection was not significantly different in chicken oviduct epithelial cells at 0.5, 1, 10, 25 and 50 nM of AHCYL1-specific siRNA. However, AHCYL1 protein expression was inhibited 25.3% at 48 h post- Figure 2. Effect of DES on tissue specific expression of chicken AHCYL1. Both RT-PCR [A and B] and q-PCR [C and D] analyses were performed using cDNA templates from DES-treated and untreated oviducts. These experiments were conducted in triplicate and normalized to control GAPDH expression.
[E] In situ hybridization analyses revealed cell-specific expression of AHCYL1 mRNA in oviducts of DES-treated and untreated chicks. Crosssections of the four segments of chicken oviduct (infundibulum, magnum, isthmus, and shell gland) treated with DES or vehicle were hybridized with antisense or sense chicken AHCYL1 cRNA probes.
[F] Immunoreactive AHCYL1 protein in oviducts of DES-treated and untreated chicks. For the IgG control, normal goat IgG was substituted for the primary antibody. Sections were not counterstained. See Materials and Methods for complete description. Legend: Untreated oviduct, non-treated whole oviduct; DES Treatment, DES treated whole oviduct; LE, luminal epithelium; GE, glandular epithelium; Scale bar represents 100 mm. The asterisks denote statistically significant differences (***P,0.001 and **P,0.01). doi:10.1371/journal.pone.0049204.g002 In the DES-treated (2 mg/ml) and non-treated chicken oviduct cells, AHCYL1 protein levels were investigated to determine time-dependent effects of DES.
[D] In chicken oviduct cells treated with DES (2 mg/ml) or both DES and an ERK1/2 inhibitor (U0126) for 24 h, according to results of a preliminary study to optimize time-dependent treatment effects, AHCYL1 protein decreased due to effects of U0126. In [C and D], blots were imaged to calculate the normalized values presented in graphs (bottom) for relative abundance of AHCYL1 protein and alpha-tubulin (TUBA) protein.
[E] Immunofluorescence microscopy detected AHCYL1 protein in chicken oviduct epithelial cells treated with DES or both DES and an ERK1/2 inhibitor. AHCYL1 protein was barely detectable in untreated, as well as DES-and ERK1/2 inhibitor-treated cells, but abundant in cytoplasm of DEStreated oviduct epithelial cells. Cell nuclei were stained with DAPI (blue). All images were captured at 40X objective magnification. [F and G] Cells were grown in media with various concentration of DES for 24 h or both DES and an ERK1/2 inhibitor. Then, calcium concentration from the cells was measured. The asterisk denotes a significant effect (***P,0.001, **P,0.01 and *P,0.05). See Materials and Methods for complete description. doi:10.1371/journal.pone.0049204.g003 transfection with AHCYL1 siRNA at 100 nM ( Figures 4A and 4B). Therefore, we investigated whether DES affects levels of AHCYL1 expression in chick oviduct cells transfected with AHCYL1 siRNA for 48 h and then treated with 2 ug/ml DES for 0, 6 or 24 h. Cell transfected with AHCYL1-specific siRNA had less AHCYL1 compared to sham-treated cells (p,0.001) and cells transfected with control siRNA (p,0.001) at each time point ( Figure 4C). Sham and control siRNA cells treated with DES for 6 and 24 h had a greater increase in AHCYL1 protein compared to sham and control siRNA cells not treated (0 h) with DES (p,0.05). To examine expression of genes related to chicken oviduct development and major egg white proteins in response to estrogen, we examined expression of cathepsin B (CTSB), CTSC, CTSS, ERBB receptor feedback inhibitor 1 (ERRFI1), pleiotrophin (PTN), gallinacin 11 (GAL11), ovalbumin, lysozyme (LYZ) and LYZ2 using quantitative PCR analyses. As illustrated in Figure 4D to 4F, the expression levels for CTSB, CTSC, CTSS, ERRFI1, PTN, ovalbumin and LYZ2 mRNAs were decreased significantly by AHCYL1 knockdown as compared to naïve, sham and control siRNA treatments: CTSB to 0.49-(p,0.001), CTSC to 0.51-(p,0.001), CTSS to 0.46-(p,0.001), ERRFI1 to 0.22-(p,0.001), PTN to 0.41-(p,0.001), ovalbumin to 0.61-(p,0.05) and LYZ2 to 0.31-fold (p,0.01). However, the expression of GAL11 and LYZ mRNAs increased significantly in response to AHCYL1 knockdown: GAL11 to 1.64-(p,0.05) and LYZ to 2.88-fold (p,0.001). In addition, calcium release was reduced significantly in response to AHCYLI knockdown as compared to control or control siRNA treated cells ( Figure 4G). Moreover, immunoreactive AHCYL1 protein was less abundant in the cytoplasm of the chicken oviduct cells receiving DES treatment and AHCYL1 siRNA transfection as compared to treatment with DES and the control siRNA ( Figure 4H).
Post-transcriptional Action of miRNAs on AHCYL1
Expression of AHCYL1 may be regulated at the post-transcriptional level by microRNAs (miRNAs); therefore, we performed a miRNA target validation assay. Analysis of potential miRNA binding sites within the 39-UTR of the AHCYL1 gene using the miRNA target prediction database (miRDB; http://mirdb.org/ miRDB/) revealed six putative binding sites for miR-124a, miR-1602, miR-1612, miR-1669, miR-1710 and miR-1782 ( Figure 5A). Therefore, we determined whether these miRNAs influenced AHCYL1 expression via its 39-UTR. A fragment of the AHCYL1 39-UTR harboring binding sites for the miRNAs were cloned in downstream of the green fluorescent protein (GFP) reading frame, thereby creating a fluorescent reporter for function of the 39-UTR region ( Figure 5B). After co-transfection of eGFP-AHCYL1 39-UTR and DsRed-miRNA, the intensity of GFP expression and percentage of GFP-expressing cells were analyzed by fluorescence microscopy and FACS. As illustrated in Figures 5C and 5D, in the presence of miR-124a, miR-1669, miR-1710, miR-1782, the intensity and percentage of GFP-expressing cells (27.7% in control vs. 14.2% in miR-124a, 17.1% in miR-1669, 19.4% in the miR-1782 and 23.2% in miR-1710) were decreased. However, in the presence of miR-1602 and miR-1612, there was no significant decrease in green fluorescence compared to the control (data not shown). Further results from quantitative PCR revealed that DES induced a 3.38-and 3.18-fold increase (P,0.01) in expression of miR-1710 and -1782, respectively, in the oviduct as compared to control chicks ( Figure 5E). However, expression of miR-124a and miR-1669 was decreased in DES-treated oviducts (P,0.05). These results indicate that miR-124a, miR-1669, miR-1710 and miR-1782, influence AHCYL1 expression in vitro via its 39-UTR which suggests that post-transcriptional events regulate or influence AHCYL1 expression in the chick oviduct.
Discussion
Results of the present study are novel as they provide the first comparisons among chicken and mammalian AHCYL1 genes with respect to structure, phylogenetic evolution, tissue specific expression of AHCYL1 mRNA and protein, and regulation of expression by estrogen in an ERK1/2-dependent cell signaling cascade. Our results also indicate that AHCYL1 is posttranscriptionally regulated by several miRNAs and knockdown of AHCYL1 results in down-regulation of genes critical to development of the chick oviduct in response to estrogen in chicks. These results support our hypothesis that AHCYL1 is required for growth, development and functional aspects of the mature oviduct of hens in response to estrogen during their reproductive cycle.
We reported that differential gene profiling data of the chick oviduct showed that the avian homolog of human S-adeonosylhomocysteine hydrolase like protein 1 (AHCYL1) transcript is highly expressed in chicks treated with DES [35]. AHCYL1 regulates numerous important cellular processes, especially Ca 2+ -dependent processes, by modulating concentrations of Ca 2+ in cytoplasm of cells [11,12]. However, little is known about expression and function of AHCYL1 in the oviduct of any species [1,2,6] although AHCYL1 has potential role(s) in many important biological events such as development, fertilization, gene expression, secretion and cell death [8,11,12].
In the present study, we found that the chicken AHCYL1 gene consists of 16 exons encoding 540 amino acid residues and that it has high homology (greater than 90%) to mammalian AHCYL1 proteins (Table 1). In addition, expression of AHCYL1 mRNA in kidney, liver, testis, oviduct, and to a lesser extent, in gizzard and small intestine of chickens was found. However, expression was not detected in other organs analyzed in either sex. Furthermore, as illustrated in Figures 1C and 1D, AHCYL1 mRNA and protein were most abundant in the LE of the infundibulum and shell gland, and at lower abundance in GE and LE of the magnum and isthmus, and GE of the infundibulum and shell gland of the oviduct. These results indicate that cell-and tissue-specific expression of AHCYL1 may be associated with functional mechanism(s) of chicken oviduct functions including calcium metabolism for formation of the egg shell and oviposition (egg laying).
Generally, the biological actions of estrogen are mediated by its cognate nuclear receptors, estrogen receptors alpha (ESR1) and beta (ESR2) which activate and recruit a variety of transcription factors with estrogen response elements to the 59 upstream region of target genes [15,16]. Indeed, several steroid hormones, including estrogen, are involved in many physiological and developmental events requiring modification of cell-type and tissue-specific gene expression [16,36]. Although various animal models have been used to investigate developmental and hormonal mechanism of oviduct growth, development and differentiation, the most well studied and informative model is the chick oviduct [16]. During development of the chicken oviduct, estrogen stimulates proliferation and cytodifferentiation of epithelial cells to tubular gland cells and expression of oviductspecific genes [37,38]. In particular, the differentiated tubular gland cells of the magnum synthesize and secrete the egg-white proteins including ovalbumin, lysozyme, ovotransferrin, ovomucoid and avidin during egg formation [39]. Indeed, the magnum is the most estrogen-responsive segment of the chicken oviduct. The administration of exogenous estrogen to neonatal chicks stimulates an 8-fold increase in wet weight of the magnum within three days [40]. Consistent with these results, we reported that exogenous DES affects growth, development and differentiation of the chicken oviduct [26] and discovered candidate genes and pathways regulating oviduct development in chickens [35]. In the present study, as illustrated in Figure 2, DES treatment increased AHCYL1 mRNA and protein in LE of the infundibulum and magnum, and GE of the isthmus and shell gland of the chick oviduct. These results strongly support our hypothesis that estrogen-mediated AHCYL1 gene expression plays a crucial role(s) in growth, differentiation and function of the chicken oviduct.
Results of the current study revealed that estrogen stimulates activation of ERK1/2 phosphorylation, expression of AHCYL1, and calcium release by oviduct cells of chicks. Mitogen-activated protein kinases (MAPKs) are highly conserved in most organisms and respond to various extracellular stimuli such as mitogens, heat shock, stress and cytokines [41]. Among the three well-characterized subfamilies of MAPKs, the ERK1/2 MAPK pathway plays important roles in growth and differentiation processes of female reproductive organs during early pregnancy, including embryonic and placental development [42,43,44,45]. However, little is known about the ERK1/2 MAPK signal cascade in growth, development and differentiation of female reproductive tract such as oviduct and uterus. In the present study, DES induced a rapid increase in phosphorylation of ERK1/2 MAPK by 5 min and this effect was maintained to 30 min before declining by 60 min (Figure 3). In addition, the same dose of DES increased AHCYL1 protein 3-fold within 2 h and 6.3-fold by 24 h and induced calcium release in a dose-dependent manner. Meanwhile, treatment of chicken oviduct epithelial cells with both an ERK1/2 inhibitor (U0126) and DES decreased AHCYL1 protein in the cytoplasm of those cells and inhibited calcium release despite DES treatment. These results strongly suggest that estrogen influences development and differentiation of the chick oviduct by activating AHCYL1 and calcium release in an ERK1/2 MAPK-dependent manner.
RNA interference methods such as the siRNA-mediated recognition of homologous target mRNA molecule have been used successfully in biological research to examine effects of silencing target genes [46]. In this study, we determined that AHCYL1 knockdown decreases expression of several genes associated with oviduct development and differentiation including several members of the cathepsin (CTS) family of lysosomal proteases. CTSs degrade extracellular matrix (ECM) molecules including collagens, laminin, fibronectin and proteoglycans and they are also involved in catabolism of intracellular proteins and processing of pro-hormones. In addition, the CTSs regulate intracellular protein metabolism [47], bone resorption [48] and antigen presentation [49], as well as cell transformation, differentiation, motility, and adhesion [50]. In the present study, the expression of cathepin B (CTSB), CTSC, and CTSS mRNAs was significantly decreased by AHCYL1 knockdown compared to naïve, sham and control siRNA treatments ( Figure 4D). Furthermore, the expression of ERBB receptor feedback inhibitor 1 (ERRFI1), pleiotrophin (PTN), gallinacin 11 (GAL11), ovalbumin, lysozyme (LYZ) and LYZ2 mRNAs, which are estrogen-induced genes or genes for egg white proteins expressed in the oviduct epithelial cells of the chicken were also significantly affected by AHCYL1 knockdown. These results suggest that estrogen-induced AHCYL1 regulates downstream genes for oviduct growth/remodeling and maintenance of oviduct function during the reproductive cycle of chickens.
MicroRNAs (miRNAs), as post-transcriptional regulators, play essential roles in a wide variety of biological processes including vertebrate growth, development and differentiation [51]. In the current study, we performed a miRNA target validation assay based on the hypothesis that AHCYL1 expression is regulated at the post-transcriptional level by miRNAs. As illustrated in Figure 5, co-transfection of eGFP-AHCYL1 39-UTR and DsRed-miRNA decreased the percentage of GFP-positive cells and GFP fluorescence density in miR-124a, miR-1669, miR-1710 and miR-1782 transfected cells, but not in cell transfected with miR-1602 and miR-1612 when compared to untreated control cells. However, as illustrated in Figure 5E, the in vivo DES-mediated decrease in miR-124a and miR-1669 supports this hypothesis, whereas the DESmediated increase in miR-1710 and miR-1782 is inconsistent with the in vitro data. These results indicate that miR-1710 and miR-1782 may act indirectly or regulate expression of other DESregulated genes in vivo. Collectively, these four miRNAs influence AHCYL1 expression in vitro via its 39-UTR which suggests that post-transcriptional regulation influences AHCYL1 expression in the chick oviduct. In addition, we propose that, of these four miRNAs, miR-124a and miR-1669 are closely related to the regulatory pathways of oviduct development and differentiation in chickens; however, this requires further investigation.
Based on the collective results from the present studies, we propose a model ( Figure 6) in which estrogen activates receptor tyrosine kinase (RTK) and phosphorylated RTK activates RAS-RAF-MEK to stimulate the ERK1/2 signal transduction cascade to effect expression of genes affecting growth-and/or development of the chick oviduct and to stimulate oviduct-specific genes for the production of egg white proteins and calcium release during egg formation. In conclusion, results of the present study provide important insights into the mechanism by which AHCYL1 regulates growth, development and functional aspects of the mature oviduct of hens in response to estrogen-mediated ERK1/2 MAPK cell signaling during their reproductive cycle.
Experimental Animals and Animal Care
The experimental use of chickens for this study was approved by the Institute of Laboratory Animal Resources, Seoul National University (SNU-070823-5). White Leghorn (WL) hens, roosters, and chicks were subjected to standard management practices at the University Animal Farm, Seoul National University, Korea. All chickens were exposed to a light regimen of 15 h light and 9 h dark with ad libitum access to feed and water.
Tissue Samples
Following euthanasia of the WL hens and roosters, tissue samples were collected from brain, heart, liver, kidney, muscle, small intestine, gizzard, ovary, oviduct and testis of 1-to 2-yearold males (n = 5) and females (n = 5). The collected samples were frozen or fixed in 4% paraformaldehyde for further analyses. Frozen tissue samples were cut into 5-to 7-mm pieces, frozen in liquid nitrogen vapor, and stored at 280uC. The other samples were cut into 10 mm pieces and fixed in fresh 4% paraformaldehyde in PBS (pH 7.4). After 24 h, fixed tissues were changed to 70% ethanol for 24 h and then dehydrated and embedded in Paraplast-Plus (Leica Microsystems, Wetzlar, Germany). Paraffinembedded tissues were sectioned at 5 mm.
Diethylstilbestrol (DES) Treatment and Oviduct Retrieval
Female chicks were identified by PCR analysis using W chromosome-specific primer sets [24]. Treatment with DES and recovery of the oviduct were performed as reported previously [25,26]. Briefly, a 15 mg DES pellet was implanted subcutaneously in the abdominal region of 1-week-old female chicks for 10 days. The DES pellet was removed for 10 days, and a 30 mg dose was administered for 10 additional days. Five 37-day-old chicks in each group were euthanized using 60%-70% carbon dioxide. Subsets of these samples were frozen or fixed in 4% paraformaldehyde for further analyses. Paraffin-embedded tissues were sectioned at 5 mm.RNA Isolation.
Total cellular RNA was isolated from frozen tissues using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations. The quantity and quality of total RNA was determined by spectrometry and denaturing agarose gel electrophoresis, respectively.
Sequence Analysis
For pair-wise comparisons and multiple sequence alignment, the amino acid sequences encoded by AHCYL1 genes from each species were aligned using Geneious Pro Version 5.04 [27] with default penalties for gap and the protein weight matrix of BLOSUM (Blocks Substitution Matrix). A phylogenetic tree was constructed using the neighbor-joining method [28] of the Geneious Pro Version 5.04 [27]. To determine the confidence level for each internal node on the phylogenetic tree, 1,000 nonparametric bootstrap replications were used [29].
Semiquantitative RT-PCR Analysis
The expression level of AHCYL1 mRNA in various organs from chickens, including the oviduct, was assessed using semi-quantitative RT-PCR as described previously [30]. The cDNA was synthesized from total cellular RNA (2 ug) using random hexamer (Invitrogen, Carlsbad, CA) and oligo (dT) primers and AccuPo-werH RT PreMix (Bioneer, Daejeon, Korea). The cDNA was diluted (1:10) in sterile water before use in PCR. For AHCYL1, the sense primer (59-TTT GGA GGG AAG CAA GTG GC-39) and antisense primer (59-GCT CAA TCA GAG CCA GAG CC-39) amplified a 481-bp product. For GAPDH (housekeeping gene; glyceraldehyde 3-phosphate dehydrogenase), the sense primer (59-TGC CAA CCC CCA ATG TCT CTG TTG-39) and antisense primer (59-TCC TTG GAT GCC ATG TGG ACC AT-39) amplified a 301-bp product. The primers, PCR amplification and verification of their sequences were conducted as described previously [30]. PCR amplification was conducted using approximately 60 ng cDNA as follows: 1) 95uC for 3 min; 2) 95uC for 20 sec, 60uC for 40 sec, and 72uC for 1 min for 35 cycles (AHCYL1) and 30 cycles (GAPDH); and 3) 72uC for 5 min. The amount of DNA present was quantified by measuring the intensity of light emitted from correctly sized bands under UV light using a Gel Doc TM XR+ system with Image Lab TM software (Bio-Rad).
Quantitative PCR Analysis
Total RNA was extracted from each oviduct of untreated and DES-treated chicks using TRIzol (Invitrogen) and purified using an RNeasy Mini Kit (Qiagen). Complementary DNA was synthesized using AccuPowerH RT PreMix (Bioneer, Daejeon, Korea). Gene expression levels were measured using SYBRH Green (Sigma, St. Louis, MO, USA) and a StepOnePlus TM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The PCR conditions were 95uC for 3 min, followed by 40 cycles at 95uC for 20 sec, 64uC for 40 sec, and 72uC for 1 min using a melting curve program (increasing the temperature from 55uC to 95uC at 0.5uC per 10 sec) and continuous fluorescence measurements. Sequence-specific products were identified by generating a melting curve in which the C T value represented the cycle number at which a fluorescent signal was significantly greater than background, and relative gene expression was quantified using the 2 -DDCT method [31]. For the control, the relative quantification of gene expression was normalized to the C T value of the untreated oviduct.
In Situ Hybridization Analysis
For hybridization probes, PCR products were generated from cDNA with the primers used for RT-PCR analysis. The products were gel-extracted and cloned into pGEM-T vector (Promega). After verification of the sequences, plasmids containing gene sequences were amplified with T7-and SP6-specific primers
Immunohistochemistry
Immunocytochemical localization of AHCYL1 protein in the chicken oviduct was performed as described previously [32] using an anti-human AHCYL1 monoclonal antibody (catalog number: ab56761; abcam, Cambridge, UK) at a final dilution of 1:500 (1 mg/ml). Antigen retrieval was performed using the boiling citrate method as described previously [32]. Negative controls included substitution of the primary antibody with purified nonimmune mouse IgG at the same final concentration.
Western Blot Analyses
Chicken oviduct cells were isolated and cultured with minor modifications as we described previously [33]. Cells were grown in DMEM-F12 containing 10% charcoal-stripped FBS until they were 80% confluent and starved in serum free medium for 24 h before DES treatment. The protein content was determined using the Bradford protein assay (Bio-Rad, Hercules,CA) with bovine serum albumin (BSA) as the standard. Proteins were denatured, separated using 10% SDS-PAGE and transferred to nitrocellulose. Blots were developed using enhanced chemiluminescence detection (SuperSignal West Pico, Pierce, Rockford, IL) and quantified by measuring the intensity of light emitted from correctly sized bands under ultraviolet light using a ChemiDoc EQ system and Quantity One software (Bio-Rad, Hercules, CA). Immunoreactive AHCYL1 and phosphorylated ERK1/2 protein was detected using an anti-human AHCYL1 monoclonal antibody (catalog number: ab56761; abcam, Cambridge, UK) at a final dilution 1:500 and anti-mouse phospho-ERK1/2 monoclonal IgG (catalog number: sc-7383; Santa Cruz, CA) at a final dilution 1:1000, respectively. As a loading control, western blotting with mouse anti-beta actin IgG (catalog number: sc-47778; Santa Cruz, CA) or anti-rabbit total ERK1/2 polyclonal IgG (catalog number: 9102; Cell signaling Technology) was performed. Figure 6. Schematic illustrating the current working hypothesis on estrogen-induced ERK1/2 MAPK signaling cascades in chicken oviduct cells. Evidence from the present study indicates that estrogen stimulates the classical estrogen-and alternative ERK1/2 MAPK signaling pathways. Legend: RTK, receptor tyrosine kinase; RAS, synaptic Ras-GTPase-activating protein; RAF (also known as MAPK3), mitogen-activated protein kinase (MAPK) kinase kinase; MEK (also known as MAPK2), MAPK kinase; ERK1/2, extracellular signal-regulated kinase; ERE, estrogen response element; ER, endoplasmic reticulum. doi:10.1371/journal.pone.0049204.g006
Immunofluorescence Microscopy
Oviduct cells obtained from laying hens were examined for AHCYL1 protein expression patterns by immunofluorescence microscopy as described previously [34]. Briefly, oviduct cells were cultured to 80% confluency in charcoal-stripped FBS to remove sex steroids, starved and then treated with 2 mg/ml DES and 10 mM U0126 (ERK1/2 inhibitor) for 24 h. Each type of cell was seeded onto Lab-Tek chamber slide (Nalge Nunc International, Rochester, NY). After 24 h, cells were fixed with 220uC methanol and immunofluorescence staining was performed using an antihuman AHCYL1 monoclonal antibody (catalog number: ab56761; abcam, Cambridge, UK). Cells were then incubated with Alexa Fluor 488 rabbit anti-mouse IgG secondary antibody (A21204, Invitrogen). Slides were overlayed with DAPI before images were captured using a Zeiss confocal microscope LSM710 (Carl Zeiss) fitted with a digital microscope camera AxioCam using Zen 2009 software.
Target-specific siRNAs for AHCYL1 Knockdown
For messenger RNA sequences of chicken AHCYL1 (NM_001030913.1), three potential small interfering RNA target sites were determined using the Invitrogen design program. The most effective target sequence (GTG AGA AGC AGC AAA CCA A) was screened out and synthesized. Silencer Negative Control 1 siRNA (Ambion, Austin, TX), with no homology to any known gene, was used as a negative control. Down-regulation of AHCYL1 expression was confirmed by quantitative PCR and Western blotting analyses. The information on primers used for q-PCR is described in Table 2.
Transfection
Chicken oviduct cells were treated with specific AHCYL1 siRNA or controls that included naïve treatment (no siRNA or Lipofectamine 2000) and sham treatment (Lipofectamine 2000 only). Transfection of siRNA was according to the manufacturer's procedure. To analyze the effect of DES on chicken oviduct cells, DES was added to the culture medium 48 h post-transfection and the incubation continued for either 6 or 24 h. Using red fluorescein-labeled control siRNA duplexes (Invitrogen), we estimated that more than 95% of the cells were transfected.
MicroRNA Target Validation Assay
The 39-UTR of AHCYL1 was cloned and confirmed by sequencing. The 39-UTR was subcloned between the eGFP gene and the bovine growth hormone (bGH) poly-A tail in pcDNA3eGFP (Clontech, Mountain View, CA) to generate the eGFP-miRNA target 39-UTR (pcDNA-eGFP-39UTR) fusion constructs. For the dual fluorescence reporter assay, the fusion constructs containing the DsRed gene and either miR-124a, miR-1602, miR-1612, miR-1669, miR-1710 or miR-1782 were designed to be co-expressed under control of the CMV promoter (pcDNA-DsRed-miRNA). The pcDNA-eGFP-39UTR and pcDNA-DsRed-miRNA (4 mg) were co-transfected into 293FT cells using the calcium phosphate method. When the DsRed-miRNA is expressed and binds to the target site of the 39-UTR downstream of the GFP transcript, green fluorescence intensity decreases due to degradation of the GFP transcript. At 48 h post-transfection, dual fluorescence was detected by fluorescence microscopy and calculated by FACSCalibur flow cytometry (BD Biosciences). For flow cytometry, the cells were fixed in 4% paraformaldehyde and analyzed using FlowJo software (Tree Star Inc., Ashland, OR).
Calcium Release Assay
Chicken intact or AHCYL1 siRNA knockdowned oviduct cells were treated with various concentrations of DES for 24 h and the supernatant was used to evaluate the release of calcium using Calcium Assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. The 100 ml working detection reagent was added to 10 ml supernatant and gently mixed, incubated at room temperature for 5 min. Calcium concentration was quantified using a microplate reader with a 595 nm absorbance and compared to a calcium standard curve.
Statistical Analyses
Differences in the variance between untreated and DES-treated oviducts were analyzed using the F test, and differences between means were detected using the Student's t test. The probability value of P,0.05 was considered statistically significant. Excel (Microsoft, Redmond, WA, USA) was used for statistical analyses. Figure S1 Multiple sequence alignment of chicken, fish and mammalian AHCYL1 proteins. (A) The amino acid sequences of AHCYL1 proteins from chicken (Gallus gallus), human (Homo sapiens), orangutan (Pongo abelii), mouse (Mus musculus), rat (Rattus norvegicus), cattle (Bos taurus), dog (Canis lupus familiaris) and zebrafish (Danio rerio) were aligned using Geneious Pro Version 5.04 [27] with default penalties for gap and the protein weight matrix of BLOSUM (Blocks Substitution Matrix). Shaded amino acid sequences are identical among all species examined. Dashes represent gaps among the sequences. The conserved functional domains in AHCYL1 proteins were identified using the Pfam-A family matrix and NCBI conserved domain database. (TIF) Figure S2 The phylogenetic tree generated from alignments of primary sequences of chicken, fish and mammalian AHCYL1 proteins. The amino acid sequences were obtained from each GenBank ( Table 1). The phylogenetic tree was constructed by the neighbor-joining method using the Geneious program. The | 2017-04-15T08:20:43.367Z | 2012-11-07T00:00:00.000 | {
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259270298 | pes2o/s2orc | v3-fos-license | In the Pursuit of Metabolic Markers of Systemic Sclerosis—Plasma Adiponectin and Omentin-1 in Monitoring the Course of the Disease
Systemic sclerosis (SSc) is a connective tissue disease leading to cutaneous and visceral fibrosis. Pathological features of SSc include immune dysregulation, vasculopathy, and impaired angiogenesis. Adipokines act as cytokines and hormones and are involved in various pathological processes, including metabolic disorders, inflammation, vasculopathy, and fibrosis. This study aimed to determine the level of omentin-1 and adiponectin to evaluate their potential role in the pathogenesis of SSc. We assessed serum omentin-1 and adiponectin as well as metabolic parameters in 58 patients with SSc and 30 healthy controls. The follow-up was performed in SSc individuals. Omentin-1 levels were significantly higher in SSc individuals as compared to the controls. In post-hoc analysis, omentin-1 was higher in the group with disease duration ≥7 years than in the control group. A positive correlation was noted between disease duration and both adipokines and increased with longer disease duration. However, there were no correlations between selected adipokines and metabolic parameters. Enhanced omentin-1 levels and higher levels of omentin-1 in patients with longer disease duration may suggest that omentin-1 is involved in the pathomechanisms of SSc as its concentrations are not directly related to BMI, age, and insulin resistance.
Introduction
Systemic sclerosis (SSc) is a chronic and progressive connective tissue disease leading to skin, blood vessels, and visceral organ fibrosis. The characteristic clinical features include skin sclerosis, lung fibrosis, and gastrointestinal, cardiovascular, renal, and osteoarticular disorders. Depending on the clinical features, diffuse cutaneous (dc) and limited cutaneous (lc) SSc are distinguished [1][2][3][4]. The etiopathogenesis of SSc has not been fully understood, but autoimmune processes, vascular endothelium dysfunction, and excessive synthesis of collagen leading to tissue fibrosis play a crucial role in the development of the disease [5][6][7].
Adipose tissue is a source of biologically active compounds. Cells (primarily macrophages) located in adipose tissue synthesize and secrete increased amounts of pro-inflammatory factors (e.g., IL-1, IL-6, tumor necrosis factor α-TNFα). In addition, adiposetissue has immunomodulatory effects [8,9]. There are reports that obesity worsens the course of rheumatoid arthritis, systemic lupus erythematosus, and psoriatic arthritis [8]. Although the majority of patients with systemic sclerosis have a normal Body Mass Index (BMI), studies have indicated unfavorable changes in the concentrations of some adipokines in this group of patients [10][11][12].
Insulin resistance (IR) is a pathological condition of impaired biological response of tissues to insulin, despite normal or elevated insulin concentration in the blood. As a result of metabolic disorders associated with IR, chronic low-grade inflammation is enhanced [13]. Studies have shown a correlation between IR and pro-inflammatory cytokines, in particular TNF-α [14]. Interestingly, IR incidence has been higher in patients with systemic connective tissue diseases, such as rheumatoid arthritis and systemic lupus erythematosus [15]. Furthermore, despite BMI being within a normal range in the majority of patients with systemic sclerosis, IR is observed in this group, primarily in those with ulceration of the fingers [16].
Adipokines act as cytokines and hormones. They are involved in the regulation of energy metabolism, food intake, and numerous physiological and pathophysiological processes [17,18]. Adipokines are capable of activating immune cells leading to the accumulation of inflammatory cells and affecting T-cell differentiation, and surprisingly showing both pro-inflammatory and anti-inflammatory effects [19][20][21]. Studies have shown that a disturbed adipokine profile could be potentially involved in various pathological processes in cardiovascular, metabolic, rheumatic, and autoimmune disorders, including SSc [20,[22][23][24][25][26].
Omentin-1 is a 313 amino-acids adipokine. The biological role of omentin-1 in the physiological and pathophysiological processes is still not fully understood [27,28]. It is synthesized predominantly in visceral adipose tissue (VAT). Two isoforms of this protein are distinguished: Omentin-1 (circulating isoform) and omentin-2 secreted into the intestine lumen [28]. It has been presumed that omentin-1 shows vasoprotective and vasodilatory effects by inhibiting cyclooxygenase-2 (COX-2) and reactive oxygen species (ROS) in endothelial cells and stimulating nitric oxide (NO) synthesis. Moreover, omentin-1 suppresses the expression of adhesions molecules, including intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) by the impact on the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway and inhibition of monocytes adhesion. Omentin-1 also affects the 5 AMP-activated protein kinase (AMPK) pathway. Therefore, it may show anti-inflammatory properties [28][29][30]. Recently, the potential role of omentin-1 in autoimmune disorders, including psoriasis, has been suggested [31,32]. The role of omentin-1 in the pathogenesis of systemic sclerosis is still under study.
However, it is suggested that the loss of the protective effect of omentin-1 on blood vessels may be involved in pathomechanisms leading to vascular damage in the course of systemic sclerosis [33]. Ometin-1 activates the endothelial nitric oxide synthase (eNOS) signaling pathway and inhibits oxidative stress, leading to increased survival and proliferation of endothelial cells and the promotion of revascularization [34]. In the early stages of systemic sclerosis, characterized by excessive activation of the vascular endothelium, decreased omentin-1 levels may contribute to the loss of neovascularization and abnormal vascular remodeling leading to vascular damage and vasculopathy [33].
Adiponectin is mainly synthesized by adipocytes. Other cells have also been shown to secrete adiponectin, including macrophages, leukocytes, fibroblasts, and endothelial cells [35,36]. This adipokine is a polypeptide of 244 amino acids and its molecular weight is 30 kDa [17,36]. Three forms of adiponectin are formed: Low molecular weight (LMW) fraction, medium molecular weight (MMW) fraction, and high molecular weight (HMW) fraction. Adiponectin is also found in the globular form [35,37]. The biological activity of adiponectin is mainly due to the high-molecular fraction, HMW [36,38], that has a stronger metabolic, vascularprotective, and cardioprotective effect compared to total adiponectin [39,40].
The concentration of adiponectin is reduced in obesity and shows a negative correlation with the amount of adipose tissue, BMI, and indicators of insulin resistance. Adiponectin synthesis increases with weight loss and is inhibited by pro-inflammatory molecules (TNF-α, IL-6) [17]. Adiponectin enhances insulin sensitivity and has a beneficial effect on the lipid profile [36], as well as shows anti-inflammatory and anti-atherosclerotic effects. It directly affects the vascular endothelium showing a vasoprotective effect. It reduces the accumulation of lipids in the vessels, inhibits the transformation of macrophages into foam cells and the proliferation of vascular smooth muscle cells, and reduces the pro-duction of pro-inflammatory cytokines (including TNF-α), as well as adhesion molecules. Adiponectin also stimulates the production of NO, promotes repair mechanisms of endothelial cells, and stimulates angiogenesis [35,[41][42][43].
Data on the role of adiponectin in systemic sclerosis are limited. Reduced levels of adiponectin in patients with SSc have been shown [44,45]. Significantly lower concentrations of adiponectin were also observed in patients with diffuse cutaneous systemic sclerosis compared to those with the limited form [44,46]. In addition, it has been found that the concentration of adiponectin changes with the duration of the disease, as a lower concentration of adiponectin occurs in patients in the early stage of the disease (≤5 years) when the process of fibrogenesis is the most intense. Most of the published data suggest that decreased adiponectin levels may be associated with skin fibrosis [25,44,[46][47][48]. The results also indicate a negative correlation between the level of adiponectin and the severity of skin fibrosis according to the modified Rodnan skin score in patients with diffuse cutaneous SSc [46,47].
The results obtained so far do not fully explain the role of omentin-1 and adiponectin in systemic sclerosis. Therefore, this study aimed to determine the level of selected adipokines, omentin-1, and adiponectin, concerning the metabolic status and to evaluate their potential role in the pathological processes in SSc.
Results
In the group of patients with systemic sclerosis, the BMI index had a median of 23.35, and in the control group the median was 24.71. There were no significant differences between the two groups for BMI as well as for other anthropometric parameters, body composition, and HOMA-IR. Biochemical parameters between the SSc and control groups markedly differed for the triglyceride level and C-reactive protein (CRP). Triglycerides were the highest in the SSc group (p = 0.024). The CRP level was also higher in patients with SSc compared with the control group (p = 0.029) ( Table 1). The comparison of adipokine levels revealed a significant difference in the concentration of omentin-1 between the SSc group and the control group, with the higher level in the SSc group (p = 0.030). For total adiponectin and HMW adiponectin, we did not reveal statistical differences between both analyzed groups ( Table 2). In addition, we assessed correlations between adipokines and age and adipokines and CRP. No correlations were observed (Table 3). In the whole SSc group, the diffuse form of the disease was present in 40 cases (69% of all SSc subjects), while the limited form was diagnosed in 18 individuals (31%). When anthropometric and biochemical parameters and adipokines were analyzed between the subgroups of SSc and the controls (diffuse SSc vs. limited SSc, diffuse SSc vs. controls and limited SSc vs. controls) the significant differences were observed between limited SSc and the controls in terms of age and triglycerides (p = 0.04 and p = 0.034, respectively (Table 4). When the SSc subjects were divided according to the disease duration with a cutting point of 7 years in the group of shorter disease duration (n = 26) the diffuse form was found in 19 individuals, while the limited form was observed in 7 cases. When the group of patients with longer disease duration (n = 32) was analyzed, it was revealed that the diffuse form was found in 21 subjects, and the limited form was seen in 11 cases.
Furthermore, amongst the SSc group, pulmonary involvement confirmed with computed tomography was present in 33 patients (56.9%), arthritis was observed in 26 (44.8%) individuals and gastrointestinal tract complications were seen in 32 patients (55.2%). In addition, 1 patient had a scleroderma renal crisis in anamnesis.
The assessment of the correlation of pulmonary involvement with adipokines revealed no correlation in all studied models (SSc with pulmonary involvement vs. SSc without pulmonary involvement, SSc with pulmonary involvement vs. controls, and SSc without pulmonary involvement vs. controls).
The antibodies pattern in the SSc patients showed that Anti-Scl70 was found in 36 (62.1%) subjects. The measured antibodies pattern is presented in Table 5. The SSc group was divided according to the duration of the disease using the median value (7 years). A group of patients with a disease duration of fewer than 7 years (26 individuals) and a group of individuals with a disease duration of 7 years or more (32 individuals) were created. Then, two groups were compared with the control group in terms of individual anthropometric, biochemical, and adipokine parameters. There was no significant difference in age and BMI values between the three analyzed subgroups. Only omentin-1 significantly differed between the analyzed groups: Omentin-1 (p = 0.019). Post-hoc analysis showed that omentin-1 was higher in the group with disease duration ≥7 years than in the control group. For total adiponectin and HMW adiponectin, no significant differences were found between subgroups (Table 6). The correlation between the modified Rodnan skin score and adipokines was also assessed, and the marked correlation and HMW-adiponectin were revealed (rho −0.27, p = 0.048) ( Table 7). A significant correlation was confirmed between the duration of the disease and the level of omentin-1 (rho = 0.32) and adiponectin (rho = 0.38) (Figures 1 and 2, respectively). The correlation between the modified Rodnan skin score and adipokines was also assessed, and the marked correlation and HMW-adiponectin were revealed (rho −0.27, p = 0.048) ( Table 7). A significant correlation was confirmed between the duration of the disease and the level of omentin-1 (rho = 0.32) and adiponectin (rho = 0.38) (Figure 1 and Figure 2, respectively). However, omentin-1, adiponectin, and HMW adiponectin did not change between measurements during the 9-month follow-up (Table 8). Table 8. Comparative analysis of selected anthropometric parameters, the modified Rodnan skin However, omentin-1, adiponectin, and HMW adiponectin did not change between measurements during the 9-month follow-up (Table 8). Table 8. Comparative analysis of selected anthropometric parameters, the modified Rodnan skin score, and adipokine concentrations in patients with systemic sclerosis in a 6-month and 9-month follow-up. The frequency of comorbidities between the groups was also analyzed. Hypertension was significantly more frequent in the SSc group (38%) than in the control group (13%), RR = 2.84, CI95 [1.08; 7.50], p = 0.025. The incidence of other diseases (type 2 diabetes, ischemic heart disease, myocardial infarction) did not differ significantly between the two groups ( Table 6). An additional statistical analysis showed no influence of the presence of arterial hypertension on the obtained results (Table 9).
Discussion
Studies on the occurrence of metabolic disorders, insulin resistance, and body composition analysis in patients with systemic sclerosis are scarce. Interestingly, despite BMI within the normal range, which is present in the majority of patients with systemic sclerosis, results of some studies indicated disturbed body composition in this group of patients [49]. In addition, the research by Park and co-workers demonstrated a statistically higher incidence of insulin resistance in patients with SSc as compared to the control group, even though significantly lower BMI in the group of patients suffering from SSc was seen [16]. It has been suggested that factors contributing to weight loss in patients with systemic sclerosis include malnutrition and muscle atrophy [50]. In addition, malfunction of internal organs, in particular the gastrointestinal tract, in the course of systemic sclerosis may lead to appetite loss and, consequently, reduced food intake and changes in body composition and structure. It is worth noticing that gastrointestinal symptoms in SSc patients are a risk factor for malabsorption and malnutrition, which lead not only to weight loss but also to a worse prognosis [10].
There is also a suggestion that IR is related to systemic connective tissue diseases. A higher incidence of IR has been demonstrated in patients with rheumatoid arthritis as well as those with systemic lupus erythematosus when compared to the controls [15,51]. Moreover, the study of Park et al. revealed a higher HOMA-IR index in patients with systemic sclerosis in comparison with the control group. In addition, this study also showed a positive correlation between HOMA-IR values and the presence of finger erosions. The authors suggested a potential role of insulin resistance in the pathogenesis of vascular abnormalities leading to erosion of the fingertips in SSc [16].
In contrast to the above-presented findings, in the present study, we did not find any significant differences in insulin and HOMA-IR in subjects with systemic sclerosis compared to their healthy counterparts. Furthermore, these parameters did not vary between patients with limited and diffuse cutaneous SSc. However, it should be highlighted that the median HOMA-IR showed a non-significant trend towards higher values in individuals with scleroderma. Finally, our results revealed that there was no relationship between tested adipokines and both insulin and the HOMA -IR index.
Studies on SSc pathogenesis have also indicated the potential involvement of adipokines in the pathogenesis of systemic sclerosis. However, the results of scientific research published so far are often inconclusive, and the role of these molecules in SSc is not fully understood [25].
The studies published so far have shown a reduced concentration of adiponectin in patients with diffuse cutaneous SSc compared to the control group and, additionally, in patients at an early stage of the disease when the fibrosis process is most intense [44][45][46]48]. A negative correlation between the level of adiponectin and the severity of skin fibrosis according to the modified Rodnan skin score in patients with dcSSc was also observed [46,48]. In a study by the team of Neumann, reduced expression of adiponectin in the lung tissue of individuals with pulmonary fibrosis in the course of SSc was demonstrated. Moreover, decreased expression of adiponectin was observed in the gastric tissue of these patients [52].
Contrary to published studies, we found no significant difference in adiponectin and HMW adiponectin levels in patients with systemic sclerosis compared to the control group. During the 9-month observation, no changes in the concentration of this adipokine were noticed. However, a negative correlation between the concentration of adiponectin and the long-time duration of the disease was found in our group under the study. Moreover, our observation of the negative correlation of the modified Rodnan skin score with the level of HMW adiponectin is noteworthy. This finding is in concordance with data from the literature suggesting that reduced adiponectin levels may be associated with skin fibrosis [25,[46][47][48]. It is also speculated that lower levels of adiponectin in the blood serum in conjunction with the reduced activity of peroxisome proliferator-activated receptor γ (PPARγ) may cause the activation and progression of skin fibrosis, especially in the early stages of SSc [47]. Furthermore, the study by Arakawa et al. showed reduced serum adiponectin concentration as well as decreased adiponectin mRNA levels in the skin of individuals with diffuse cutaneous systemic sclerosis. Decreased levels of adiponectin were associated with more advanced skin sclerosis and a higher incidence of pulmonary fibrosis, suggesting that adiponectin concentration may be a useful marker for assessing fibrosis in the course of SSc [48]. However, in our study, there were no correlations between pulmonary involvement and adipokine concentration. Nevertheless, it has been assumed that the antifibrotic effect of adiponectin may be associated with the induction of 5 AMP-activated protein kinase (AMPK) and the effect on PPARγ activity [53]. It has also been observed that the treatment of systemic sclerosis may affect the concentration of adiponectin. Noticeably, a significant increase in the concentration of adiponectin during the treatment of systemic sclerosis with epoprostenol was shown, and this phenomenon may have a beneficial effect by inhibiting the progression of fibrosis in the course of the disease [54]. Unfortunately, due to the complex regiment of the rheological therapy of our patients, there was no possibility of evaluating the correlation between this kind of treatment and adipokines.
So far, to our knowledge, only one study evaluated the concentration and influence of omentin-1 on the course of SSc. The authors showed no differences in the concentration of omentin-1 in the sera of patients with systemic sclerosis compared to the control group. However, lower concentrations were observed in individuals with diffuse cutaneous systemic sclerosis compared to its limited form and in SSC patients with shorter disease duration (≤5 years) compared to controls [33].
Our study revealed a significantly higher concentration of omentin-1 in patients with systemic sclerosis compared to the control group. Higher levels of this adipokine in patients with SSc were not directly related to BMI, patients' age, and insulin resistance indices (HOMA-IR, fasting insulin levels), which were comparable in the SSc and control groups. In addition, a significant correlation was confirmed between the duration of the disease and the level of omentin-1 (rho = 0.32). Moreover, post-hoc analysis showed that omentin-1 was higher in the group with disease duration ≥7 years than in the control group. However, we are aware of the influence of small study groups on our findings. Nevertheless, our results are in concordance with a study by Miura et al., who also showed a similar correlation in patients with dSSc [33].
Omentin-1 has vasoprotective, vasodilating, and anti-inflammatory properties. It could be speculated that the enhanced omentin-1 serum concentration observed in our study might serve as a compensatory mechanism for vascular dysfunction associated with pathological processes in the course of SSc. The study by Miuro et al. showed elevated omentin-1 levels in SSc patients with increased right ventricle systolic pressure RVSP, which may be associated with a loss of response to the vasodilating effect of omentin-1 caused by eNOS suppression in epigenetic mechanisms or a compensatory increase in omentin-1 levels. These authors suggest that the level of omentin-1 may be a useful marker reflecting the advancement of pulmonary vascular involvement leading to the development of pulmonary hypertension as a result of a compensatory enhancement in the concentration of this adipokine caused by an increase in pulmonary artery pressure [33]. Taking into account the results obtained in our study as well as in the study by Miura et al., it could be hypothesized that omentin-1 may be involved in the process of vasculopathy in systemic sclerosis, especially in the early stages of the disease and additionally, in the development of pulmonary hypertension. However, further intensive research should be performed on a larger cohort of patients to evaluate the exact role of omentin-1.
The Patients with Systemic Sclerosis
The study group comprised 58 patients, including 46 women (79.3%) and 12 men (20.7%). The mean age was 54.38 ± 13.42 years (between 33 to 75 years). The study included individuals with systemic sclerosis that was diagnosed based on the criteria of the European League Against Rheumatism (EULAR) (2013) and the American College of Rheumatology (ACR) [55]. The disease duration from the diagnosis of SSc ranged from 3 months to 46 years (median 7 years). Forty (69%) patients were diagnosed with diffuse cutaneous sclerosis, and 18 (31%) with limited systemic sclerosis.
Patients were recruited from the Department of Dermatology, Centre of Postgraduate Medical Education located in The National Institute of Medicine of The Ministry of Interior and Administration in Warsaw, Poland.
The Control Group
The control group consisted of 30 individuals, 25 women (83.3%) and 5 men (16.7%). The mean age in the control group was 49.57 ± 10.68 years. The control group comprised patients from the Department of Dermatology, Centre of Postgraduate Medical Education, and The National Institute of Medicine of The Ministry of Interior and Administration in Warsaw, Poland who were without any signs of systemic sclerosis and in whom autoimmune skin diseases were excluded. No significant differences were found for BMI, anthropometric parameters, and body composition in patients with systemic sclerosis and the control group ( Table 1). The comorbidities comparison of both groups is presented in Table 9.
Criteria for Exclusion from the Study and Informed Consent
Criteria for exclusion from the study. Each participant was informed about the purpose of the study, and written consent was obtained at the beginning. The research project protocol was approved by the Bioethical Committee of the Central Clinical Hospital of the Ministry of Interior and Administration in Warsaw (resolution 2/2016 of 13 January 2016). The study was in accordance with the Declaration of Helsinki.
Clinical Evaluation
A detailed medical history was collected from all the study participants. In the study group, the severity of skin lesions was estimated according to the modified Rodnan skin score, in which the degree of skin hardening is assessed with a scale of 0-3 points (0-no sclerosis, 1-slight sclerosis, 2-medium degree of induration, 3-high degree of induration) in 17 body areas (fingers, hands, forearms, arms, thighs, face, chest, feet). The total scale range is 0-51 [56].
Physical examination was performed on all study participants and included measurement of blood pressure and anthropometric parameters (BMI, waist, and hip circumference). In addition, the amount of adipose tissue using the bioimpedance method (BIA) (Tanita) was also assessed.
Blood Collection
Blood for laboratory tests was collected from all participants in standardized conditions after at least 6 hours of fastening. For the determination of adipokines, blood samples were collected in tubes containing EDTA, then centrifuged (3000 rpm) at 4 • C. The obtained plasma was stored at −80 • C.
Blood samples of systemic sclerosis individuals were obtained on the baseline and then after 6-and 9-month follow-ups.
The concentration of omentin-1 in the blood plasma samples was determined using a commercial enzyme immunoassay ELISA kit (BioVendor, Brno, Czech Republic). The sensitivity of the test was 0.5 ng/mL. The inter-and intracoefficients of variations were <5%.
Total adiponectin and its high molecular weight fraction (HMW) in blood plasma samples were determined using a commercial enzyme immunoassay kit ELISA (ALPCO, Salem, NH). The sensitivity of the test was 0.034 ng/mL. The inter-and intracoefficients of variations were 4% and 5%, respectively.
The concentration of insulin in blood plasma samples was measured with a commercial RIA radioimmunoassay kit (DIAsource ImmunoAssays S.A., Louvain-la-Neuve, Belgium). The sensitivity of the kit was ≤1 µIU/mL. The inter-and intracoefficients of variations were 2% and 7%, respectively.
The insulin resistance was calculated based on the HOMA-IR index (calculated using the formula fasting glucose x fasting insulin/405, a value more than 2.5 was recognized as insulin resistance).
Statistical Analysis
The statistical analysis was performed in the R statistical package, version 4.0.5 (http://cran.r-project.org (accessed on 2 June 2021)). The significance level α = 0.05 was accepted as significant. The number of occurrences (n) and frequency (%) describe the nominal data. The normal distribution in individual subgroups was checked using the Shapiro-Wilk test. A comparison of the level of variance between subgroups was performed using Levene's test. To determine whether there was a significant difference between the expected and observed frequencies in subgroups, the chi-square test was used.
The comparative analysis of the two subgroups was performed using the chi-square test or Fisher's exact test for qualitative variables and for quantitative variables using the Student's t-test for independent measurements or the Mann-Whitney U-test, as appropriate. When comparing the 3 subgroups, ANOVA was used with Tukey's post-hoc test or Kruskal-Wallis test with Dunn's post-hoc test with Bonferroni's correction, depending on whether the assumptions were met. The analysis between individual measurements within the research group was performed. ANOVA for dependent measures or the Wilcoxon test for pairs was used in cases where the assumptions of ANOVA were not met. The relationship between the selected quantitative variables was verified using the Spearman correlation coefficient. In the research group, the correlation analysis was performed for each measurement separately due to the necessity to meet the assumption of the independence of observation.
Conclusions
Enhanced omentin levels were observed in patients with SSc as well higher levels of omentin-1 in patients with longer than 7 years disease duration may suggest that this adipokine is involved in the pathomechanisms of SSc as its concentrations are not directly related to BMI, age, and insulin resistance indices (HOMA-IR, fasting insulin levels). However, this hypothesis needs further evaluation in the larger patient cohort.
Our findings of the negative correlation between the concentration of adiponectin and the long-time duration of the disease, as well as the negative relation between the modified Rodnan skin score with the level of HMW adiponectin, strongly suggest that adiponectin is an important player in systemic sclerosis. However, intensive research is also needed here to clarify the exact role of adiponectin and its fraction in SSc. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions. | 2023-06-29T05:10:00.785Z | 2023-06-01T00:00:00.000 | {
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244928721 | pes2o/s2orc | v3-fos-license | Observable player behaviours and playing performance following helmet strikes in elite cricket
Objectives Investigate the observable player behaviours and features of both concussive (HS-C) and non-concussive (HS-NC) helmet strikes and describe their impact on playing performance. Methods Elite male cricketers sustaining helmet strikes between the 2016 and 2018 seasons were identified by the England and Wales Cricket Board. Medical records identified players sustaining a concussion and those in whom concussion was excluded. Retrospective cohort analysis was performed on batting and bowling performance data available for these players in the 2 years prior to and 3 months post helmet strike. Video analysis of available incidents was conducted to describe the characteristics of the helmet strike and subsequent observable player behaviours. The HS-C and HS-NC cohorts were compared. Results Data were available for 194 helmet strikes. 56 (29%) resulted in concussion. No significant differences were seen in playing performance in the 3 months post concussive helmet strike. However, a significant decline in batting performance was seen in this period in the HS-NC group (p<0.001). Video features signifying motor incoordination were most useful in identifying concussion post helmet strike, however, typical features suggesting transient loss of consciousness were not seen. Features such as a longer duration pause prior to the batsman resuming play and the level of concern shown by other players were also useful features. Conclusion HS-NC may be more significant for player performance than previously thought. Guidance for using video replay to identify concussion in cricket may need to be modified when compared with other field sports.
Methods Elite male cricketers sustaining helmet strikes between the 2016 and 2018 seasons were identified by the England and Wales Cricket Board. Medical records identified players sustaining a concussion and those in whom concussion was excluded. Retrospective cohort analysis was performed on batting and bowling performance data available for these players in the 2 years prior to and 3 months post helmet strike. Video analysis of available incidents was conducted to describe the characteristics of the helmet strike and subsequent observable player behaviours. The HS-C and HS-NC cohorts were compared.
Results Data were available for 194 helmet strikes. 56 (29%) resulted in concussion. No significant differences were seen in playing performance in the 3 months post concussive helmet strike. However, a significant decline in batting performance was seen in this period in the HS-NC group (p<0.001). Video features signifying motor incoordination were most useful in identifying concussion post helmet strike, however, typical features suggesting transient loss of consciousness were not seen. Features such as a longer duration pause prior to the batsman resuming play and the level of concern shown by other players were also useful features.
INTRODUCTION
Head impacts occur in elite cricket at a rate of 7.2 per 1000 player days with concussions occurring at 2.3 per 1000 player days. Although the risk of both is not uniformly spread through the team, this equates to one player receiving a head impact every 12 days of play and a concussion every 36 days of play, per team. 1 These injuries are a source of concern. Partly, this relates to research linking concussive head injuries to long-term psychiatric and neurodegenerative disease, although Jones et al 2 reported no increased risk of dementia in retired professional cricketers. However, there is also the potential for impaired playing performance, increased musculoskeletal injury risk and, in elite sport, limitation of career length and earning potential in the short-and medium-term following concussion. [3][4][5][6][7] There are also concerns around repetitive 'sub-concussive' impacts and the potential for these accumulated impacts to be as important in the short-and long-term brain health of athletes. [8][9][10] Improved understanding of head impact injuries in cricket is important, particularly given approximately 70% of concussions occurring after such injuries are not diagnosed at the time of the strike. Possibly, this may be because the 'traditional' observable features signifying transient loss of consciousness seen
Key messages
What is already known ► Helmet strikes are common in cricket and are a source of concern in both the short and long term. ► Approximately, 70% of concussions occurring after such impacts are not diagnosed immediately demonstrating the need to understand their recognition better. ► Sporting performance can be impaired following sporting head injury in other codes.
What are the new findings ► Helmet strikes that result in concussion do not appear to significantly impact batting or bowling performance post return to play in elite cricket. ► However, helmet strikes which do not result in concussion do appear to result in significant batting performance impairment for up to 3 months post impact. ► Observable features and player behaviours of concussed players following helmet strike appear to differ from those seen in other sports and this should be widely disseminated to aid concussion detection in cricket.
Open access after concussion in other sporting codes are not common in the elite cricket setting. 1 11 Thus, our objective was to investigate helmet strikes occurring in elite English cricket from two perspectives, to inform the detection of significant strikes and the aftercare of players suffering them. First, to describe the observable player behaviours seen on video review following helmet strike and analyse any differences seen between players subsequently diagnosed with concussion versus those in whom concussion is excluded. Second, to describe effects on playing performance following helmet strike and, again, differences seen based on concussion diagnosis.
Study design
Helmet strikes were defined as the ball hitting the batsman's helmet, via any mechanism, be that directly from the bowler, from the throw of a fielding player, etc. Players sustaining helmet strikes between 2016 and 2018 seasons while playing in matches, where video recording and documentation are mandated (First Class County and International), were identified through video analysis supplied by England and Wales Cricket Board (ECB) Performance Analysts.
Concussion injuries were recorded within the ongoing ECB injury surveillance programme, which is centrally coordinated by an 'Injury Surveillance Officer' (ISO) who monitors compliance and injury definitions. The diagnosis of concussion was made by the doctor and physiotherapist at each team according to the ECB and International Cricket Council Concussion Guidelines, written with reference to the most recent Concussion in Sport Group Consensus Statements at the time. The data collection spans two Consensus Statements. 4 12 13 All injuries were recorded on purpose built central online medical records systems: Profiler (The Profiler Corporation, New Zealand (2016)), Cricket Squad (The Sports Office, UK (2017-2018 inclusive)), supported by ECB's ISO. To improve compliance, the ECB mandates consistent standards for injury and medical record keeping for the domestic game through annual Science and Medicine Audits.
A retrospective cohort analysis was used to identify players who received a helmet strike and sustained a concussion (HS-C) or received a helmet strike and did not sustain a concussion (HS-NC) during the study period. Concussions occurring without video documentation were excluded.
Performance analysis ECB Performance Analysts recorded player batting and bowling performance data for each competitive game on a purpose built central online system. Bowling performance data were analysed to separate the potential effects of changes in player behaviour following helmet strike versus changes in, for example, reaction time or motor speed.
Batting and bowling averages were obtained for 2 years prehelmet strike (from date of injury/incident), 1 and 3 months post return to play (RTP). An inclusion criterion for performance data was set with batting and bowling averages for first XI First-Class domestic County Championship cricket fixtures being included, where the player had participated in at least two innings (for batting) or overs (for bowling). First-Class County Championship performance was selected over the two other competition formats (One-Day 50 over and T20 cricket) as fixtures span the whole domestic season (from April to September inclusive), providing more opportunity to get enough performance data from fixtures that meet the inclusion criteria for the various time periods. In comparison, the other domestic competition formats (One-Day and T20) are played in intermittent 'blocks' of fixtures over a month or so. Due to the timing of incidents, complete data for each player at each time period were not always available.
Video analysis
The ECB ISO collated all available videos of helmet strikes at First-Class County teams (across County Championship, One Day 50 Over and T20 cricket) and the men's senior national side from each team's Performance
Open access
Analyst. A selection of these videos was reviewed by the ISO and the lead author to identify visible features that may be relevant to investigate further in the entire cohort of videos.
This review was made with reference to the Berlin Consensus Statement, the concussion protocols of the ICC, World Rugby, FIFA and the consensus statement on video features of concussion. This produced a list of 19 signs the player may display and three variables relating to the impact location and subsequent direction of the ball (figures 1 and 2).
The ball directions were coded as red, yellow and green corresponding to the theoretical transfer of energy in each. For example, if the ball strikes the helmet and continues behind the batsman, this is likely to represent a glancing blow (green) as compared with the ball striking the helmet and travelling directly back down the pitch (red). This list was reviewed by three experienced ECB doctors with only minor changes suggested. 4 11 13-16 The final list is included in online supplemental appendix 1.
All videos were then reviewed and coded by two researchers. Any discrepancies were discussed and if consensus could not be reached, the senior author was consulted. The mean of the two transverse plane ball angles was calculated.
Statistical analysis
Performance analysis Batting and bowling averages were converted to withinindividual z-scores and summarised with descriptive statistics before the assumption of normality was assessed with the Shapiro-Wilks test. Due to the variation in data available for each player across each time period, mean differences between the three performance time periods were assessed, with either a one-way Analysis of Variance (ANOVA) (when normality can be assumed) or Kruskal-Wallis (when normality cannot be assumed), along with suitable post hoc tests and 'Benjamini-Hochberg' adjusted p values. All estimations were made using the lme4 package with R (V.3.5.2, R Foundation for Statistical Computing, Vienna, Austria). 17 Video analysis Descriptive statistics were calculated for each feature. χ 2 test was used to compare the prevalence of each feature between the HS-C and HS-NC groups. Location of impact and ball direction after impact were assessed both alone and in combination. For these features, relative risk of subsequent concussion diagnosis was calculated. For the transverse plane ball angle, assumption of normality was assessed with Shapiro-Wilks test and then either t test or Mann-Witney U used to assess for differences between the groups. The sensitivity and specificity of video features relating to player behaviour were assessed for predicting subsequent concussion diagnosis.
Patient and public involvement
Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
RESULTS
194 male senior players, registered to compete in the ECB domestic First-Class County Championship from 2016 to 2018 inclusive, received a helmet strike, 56 HS-C and 153 HS-NC.
Performance analysis
Batting performance Adequate batting performance data were available for 24 of the 56 HS-C group and 138 of the 153 HS-NC group. Performance data were not available for each player for each time period. A summary of the available data is shown in table 1, with the individual raw batting averages for each group in online supplemental tables 1 and 2.
The mean, within-individual, batting performance z-scores for the HS-C group increased from 2-year prehelmet strike to 1-month post-RTP before decreasing between 1-month post and 3-month post-RTP ( figure 3). In the HS-NC group, mean within-individual performance decreased from 2-year pre to 1-month post incident and then decreased further from 1-month to 3-month post incident ( figure 3).
Open access
For the HS-C group, the assumption of normality was not met (W=0.88, p≤0.001), and no difference in batting performance was seen between the three time periods (H(2)=1.58, p=0.45).
For the HS-NC group, the assumption of normality was also not met (W=0.88, p=<0.001). A difference in batting performance was seen between the three time periods (H(2)=21.48, p<0.001). Post hoc tests were conducted using Wilcoxon rank sum tests and found differences in batting performance with a small effect for 2 years pre and 1 month post (p<0.05, r=0.17) and a moderate effect for 2 years pre to 3 months post (p<0.001, r=0.31). No differences were found between 1 and 3 months post-RTP (p=0.16, r=0.09).
Therefore, following a HS-NC, there was a reduction in batting average from 2 years pre incident (mean=0.24±0.75) to 1 month post (mean=−0.06±0.83) with a continued reduction in batting performance 3 months post incident (mean=−0.24±0.70).
Bowling performance
Adequate data for bowling performance were available for 12 of the HS-C group and 58 of the HS-NC group. A summary of the available data is provided in table 1, with the individual raw bowling averages for each group in online supplemental tables 3 and 4.
The mean within-individual bowling performance z-scores for the HS-C group followed a similar pattern to the batting performance, increasing from 2-year prehelmet strike to 1-month post-RTP before decreasing from 1-month to 3-month post-RTP (figure 3). For those in the HS-NC group, mean within-individual bowling performance z-scores decreased from 2 years pre to 1 month post before recovering slightly from 1 month to 3 months post but still down from their performance 2 years pre ( figure 3).
For the HS-C group, the assumption of normality was not met (W=0.80, p=<0.001), and no difference in bowling performance between the three time periods was seen (H(2)=3.00, p=0.22). Similarly, for HS-NC group, the assumption of normality was not met (W=0.88, p=<0.001), and no difference in bowling performance between the three time periods was seen (H(2)=3.04, p=0.22).
Video analysis
Video was available from 169 helmet strikes, 16 HS-C versus 153 HS-NC. Table 2 demonstrates the distribution of features regarding location of impact and ball direction. When assessing the location of the ball impact, it was difficult on the views available to distinguish between the Frontal, Peak and Peak and Grill regions and, therefore, these were combined for further analysis. The relative risk of sustaining a concussion was found to be highest in this combined region (RR 2.26) followed by the grill alone and temple.
The most common direction of ball travel poststrike was into the yellow zone. This was also the zone with the highest relative risk of sustaining a concussion (RR 1.26). No relationship was found regarding transverse plane ball angle (U=1080 p=0.53).
To investigate these features further, their combined effects were analysed. This demonstrated that the greatest relative risk is seen in strikes, which impact on Table 1 Summary of the number of players with available batting and bowling performance data points, following helmet strike for each time period between the HS-C and HS-NC groups Open access the combined frontal/peak regions and rebound into the green zone (RR 2.27) as well as those which impact on the grill region and rebound into the yellow zone (RR 2.05). Further details are provided in online supplemental appendix 2. Table 3 demonstrates the player behaviour features seen most commonly following helmet strike in the HS-C and HS-NC groups. The most sensitive features to distinguish HS-C were the player removing or checking their helmet (92.31%) and longer duration of recovery time to face the next delivery (91.67%). Meanwhile, the most specific features covered a range of behaviours relating to the responses of other participants on the field and the player's balance. During the video review, there was insufficient evidence to establish if the player retired from play following the incident or if medical staff entered the field of play uninvited, and so these were excluded.
DISCUSSION
We have attempted to describe head impact injuries in elite cricket from the perspective of both their observable characteristics and their impacts on player performance in the context of concussive and 'subconcussive' injuries.
These findings indicate a non-significant trend towards initial increased batting and bowling performance at 1-month poststrike, following by a decline in performance at 3 months poststrike in those who suffer a helmet strike with concussion. This is consistent with findings from elite baseball by Sabesan et al, 18 which showed a non-significant decline in batting and pitching performance on RTP postconcussion. However, this contrasts with Wasserman et al's 19 work in the same population. They demonstrated a significant reduction in batting performance in the initial 2 weeks post-RTP, with a nonsignificant reduction at 4-6 weeks. 18 19 Studies on playing performance in other sports postconcussion, however, provide similarly inconsistent results, even within the same sport. A possible explanation for this may be the management of RTP following concussion. In elite soccer, Ramkumar et al 7 demonstrated that players returning through more aggressive protocols suffered a decrement in performance versus those managed more conservatively.
Crucially, however, a significant reduction in batting performance was seen following a helmet strike, which did not result in concussion at both 1 and 3 months. While reductions in motor control have been described following subconcussive impacts in boxers, the effect of such impacts on 'real-world' sports performance is not well studied. 20 Possibly, this may reflect concussions, which are not detected under the current approach to diagnosis. Alternatively, the differences seen between the HS-C and HS-NC groups may be explained by the extended period of rest and graded recovery afforded by the concussion diagnosis. Whether this acts to ameliorate a lasting effect on neurocognitive/motor performance or an independent effect on player psychology and behaviour should also be considered. This is important to investigate further as it may highlight a need for further assessment of these players and a managed RTP, even in the absence of concussion.
Our findings also suggest that helmet strikes occurring to the frontal, peak and grill sections of the helmet represent the greatest relative risk in terms of subsequent diagnosis of concussion. This contrasts with accelerometry and modelling data from other helmeted and non-helmeted sports that suggest lateral impacts are more injurious. [21][22][23] In cricket, where the bowling line is permitted to contact the batsman who must react, impacts to the front of the helmet may result from a faster ball speed while a slower ball on a similar trajectory may prompt a player to turn their head in preparation for an impact. However, this relationship is clearly complex, as demonstrated by Saw et al, 24 who's work highlighted that impacts to the back of the helmet carried the highest positive predictive value of concussion.
Alternatively, the combination of helmet strike location and the subsequent direction of ball travel could be important. Counter to our intuitive colour-coding zones, impacts in the Yellow zone represented the largest concussion risk. This may be because, when the ball strikes the frontal section and rebounds laterally (Yellow zone), the rotational force on the head is greater. Rotational, rather than translational, impacts are theorised to Open access be more likely to result in concussion. This also raises a question about whether neck strengthening interventions may be beneficial for primary prevention. 24 25 Green zone impacts were also found to have higher concussion risk than expected, however, our suspicion is that this represents difficulties in judging the flight of the ball in the frontal plane. Frequently, only one view was available, usually in the frontal plane and often too narrow to view the landing of the ball after rebound. Therefore, travel into the green zone on the frontal view may in fact appear in the red zone in the sagittal view, suggesting diagnosis could be improved with access to multiple angles. This is supported by the findings of Saw et al 24 who conducted a similar exercise in Australian cricket and demonstrated that the rebound of the ball towards the source or the ball stopping dead was associated with a higher risk of concussion. Standardisation of the views and reference frame in future may assist in investigating this further.
The ECB requires all players to undergo annual baseline testing using the Standardized Concussion Assessment Tool 5 and Immediate Post-Concussion Assessment and Cognitve Test (ImPACT) (ImPACT Applications, Coralville) on a biannual basis unless helmet strikes are recorded, in which case it is annual. This data suggest that more detailed ImPACT testing should continue and that annual reviews should be considered. Jones et al 2 surveyed retired cricketers for long-term health outcomes. No cases of dementia/neurocognitive disease were recorded, but ongoing review of retired cricketers and any potential consequence of long-term helmet strikes is warranted.
Notably, observable signs of concussion in this context appear different to those from other field sports. The value of video replay in detecting concussion is clear, particularly in football codes, and the features, which suggest a transient loss of consciousness or a concussion, are well documented. However, none of those features appeared in this series and their absence may make concussion diagnosis more challenging in cricket if this is not recognised. 1 11 15 26 27 Features suggesting motor incoordination were valuable in detecting concussion in this study. However, features indicating urgency and concern from other players or officials as well as the player inspecting their helmet and pausing for >4 s before the next delivery were also important. These may be unique to cricket where rules and culture dictate the action pauses until the batsman is ready.
These findings suggest several areas for further investigation, both to understand their implications and to improve player safety. However, some important limitations should be recognised. First, our groups were weighted towards HS-NC. As a result, the concussive helmet strike element of the performance analysis was underpowered, which may explain the lack of significant findings in this group. This was unavoidable in view of the low incidence of concussive helmet strikes in Open access cricket; however, it is an important caveat. In addition, we excluded from our analysis head impacts occurring to fielding or bowling players. This was due to the low total number of these incidents, all of them resulting in concussion. Finally, we were not able to analyse differences in the clinical status of the player between those helmet strikes captured on video and those which could not be reviewed, which may introduce a potential source of bias, though we feel this is unlikely.
CONCLUSION
Helmet strikes are common in elite cricket. While the rate of concussion following helmet strike is low, these findings suggest that HS-NC may be more important than previously thought and may require management involving a supported RTP. For example, involving consideration of and interventions directed at visual motor speed and psychological readiness. The video features of helmet strikes, which may point to concussion, appear to differ in cricket when compared with other sports and this requires further investigation and dissemination to ensure that medical staff are able to recognise key features in the cricket context. | 2021-12-08T16:21:52.140Z | 2021-12-01T00:00:00.000 | {
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2684584 | pes2o/s2orc | v3-fos-license | New Synthetic Pyridine Derivate as Potential Elicitor in Production of Isoflavonoids and Flavonoids in Trifolium pratense L. Suspension Culture
The production of secondary metabolites in Trifolium pratense L. suspension culture of the family of legume plants (Fabaceae) is low, and therefore there was an attempt to increase it by elicitation. New synthetic substance, 2-(2-fluoro-6-nitrobenzylsulfanyl)pyridine-4-carbothioamide, was tested as elicitor—a substance that showed the best elicitation effect after 48-hour application of 1 μmol L−1 concentration. Maximum contents of genistin (11.60 mg g−1 DW), daidzein (8.31 mg g−1 DW), and genistein (1.50 mg g−1 DW) were recorded, and the production of these isoflavonoids thus significantly increased, when compared with the control, by 152%, 151%, and 400%. The maximum content of flavonoids (5.78 mg g−1 DW) and the increase in the production by 142%, when compared with the control, were induced by 6-hour application of 100 μmol L−1 concentration. The tested substance showed to be an effective elicitor of phenylpropane metabolism.
Introduction
Isoflavonoids and flavonoids are, due to their wide spectrum of biological effects, some of the closely studied secondary metabolites of plants [1,2]. Their major producers are plants of the legume family (Fabaceae). Trifolium pratense (red clover) contains especially the following isoflavonoids: daidzein, formononetin, biochanin A, and genistein. The contents of isoflavones were determined in the root, stem, leaf, and bloom of red clover. The highest and nearly equal concentrations were found in the root, stem, and leaf, least of all in bloom [3]. A total of 28 phenolic compounds were identified by high-performance liquid chromatography and mass spectrometry in red clover roots. The most abundant isoflavonoids in pot-grown roots were formononetin glycoside malonate (G-M) (1.51-4.26 mg g −1 DW), formononetin (2.21-3.57 mg g −1 DW), and biochanin A (1.73-2.17 mg g −1 DW), whereas field-grown roots were rich in formononetin-G-M (3.90-4.27 mg g −1 DW) and pseudobaptigenin-G-M (1.80-2.58 mg g −1 DW). Elevated ozone, cultivation regime, and growth stage affected the levels of phenolics in red clover roots, suggesting sensitivity of root phenolics to biotic and abiotic stress conditions [1].
These isoflavonoids are considered the most important phytoestrogens because of their ability to interact with estrogenic receptors and because of their structural similarity with 17 β-estradiol [4,5]. The red clover extracts are used in female menopause nonhormonal substitution therapy. They decrease the occurrence of menopausal syndromes; have positive effect in the prevention of osteoporosis; reduce the risk of the incidence of cardiovascular diseases and the breast cancer [6][7][8]. A positive impact of these isoflavonoids in the prostate gland cancer prevention was also noted [9].
In vitro methods provide opportunities for propagating and preserving endangered plant species when seed-based methods are not adequate. Tissue culture propagation methods can be used to produce such plants for reintroduction, research, education, display, and commerce. They can also be the basis for tissue banking as a way to preserve genetic 2 The Scientific World Journal diversity when seeds cannot be banked. The number of endangered species that will require in vitro methods is estimated to be at least 5,000 worldwide [10]. The plant tissue cultures are a promising source of secondary metabolites. However, their production, when compared with intact plants, is usually lower, and this is the reason why we are looking for different methods and approaches to increase the production. Many traditional strategies can be used to increase the production of secondary metabolites but elicitation is usually one of the most successful.
The principle of elicitation consists in the accumulation of the secondary substances in the plants-a process that is a part of the plant's defense reaction against the influences caused by pathogens or by the plant's environment. The factors that trigger the defensive reactions are called elicitors. Elicitation strengthens the transcription of genes, for instance, phenylpropane metabolism, thus genes that code enzymes which are necessary for synthesis of isoflavonoids and flavonoids (phenylalanine ammonia lyase, chalcone synthase, etc.) [11][12][13]. The most commonly used elicitors are, for example, fungal homogenates, heavy metals, physical factors, and phytohormones [14][15][16][17][18].
The elicitation method was also used during experiments to influence the Trifolium pratense suspension culture production. The best elicitation effects so far have been detected at mercury chloride and at jasmonic acid [18,19].
Recently, newly synthesized chemical substances have been tested as elicitors in in vitro plant cultures. Many of them have proven to possess strong elicitation effect. For instance, fluoro derivates of jasmonic acid [20] and chlorinated pyridine derivates can also be considered suitable chemical induction signals of secondary metabolism of plants [21]. In cases of the Ononis arvensis, Silybum marianum, and Genista tinctoria tissue cultures, synthetic derivates pyrazine-2-carboxamides were observed to be potential elicitors. A more suitable elicitor for the production of isoflavonoids in the callus culture of Genista tinctoria proved to be substance B (5-terc-butyl-6-chloro-N-(3-iodo-4-methylphenyl)pyrazine-2-carboxamide). The absolute maximum of genistin production was achieved by a 12-hour action of substance B in a concentration of 2.33 10 −3 mol L −1 (57 times versus control) [22]. Substituted pyrazinecarboxamides markedly influenced production of flavonolignans in Silybum marianum callus and suspension cultures. The highest increase in production of flavonoids, by 900%, was detected in the Ononis arvensis callus culture [23].
A newly synthesized benzylsulfanylpyridine derivate, 2-(2-fluoro-6-nitrobenzylsulfanyl)pyridine-4-carbothioamide), originally prepared as antimycobacterial or antifungal compound, also influences the plant metabolism-it inhibits photosynthesis in spinach chloroplasts [24]. Therefore, the aim of this study was to prove this compound as a potential elicitor of biosynthesis of isoflavonoids and flavonoids in the Trifolium pratense suspension culture. 2.2. Trifolium pretense L. Suspension Culture. A callus culture was derived from the seedling of red clover (tetraploid variety DO-8 obtained from the Breeding Station Domoradice, Czech Republic). The suspension culture was then derived from 1-year old callus culture mechanically by shaking in the Gamborg liquid nutrient medium [25] supplemented with 2 mg L −1 of 2,4-dichlorophenoxyacetic acid and 2 mg L −1 of 6-benzylaminopurine. The culture was cultivated on a roller at the temperature of 25 • C, and a 16hr light/8hr dark photoperiod. The subcultivation interval lasted 14 days. Growth and production characteristics of this culture were determined; an obvious indirect relationship between growth rate and accumulation of secondary metabolites was observed [26].
Elicitation.
For elicitation experiments, we used 6month old suspension culture and the elicitor in the concentrations of 1 μmol L −1 , 10 μmol L −1 , and 100 μmol L −1 dissolved in 96% ethanol. 1 mL of the elicitor solution was added to each flask (containing 25 mL of the suspension culture) before the end of the exponential growth phase of the culture (on the 21st day of cultivation) [26]. The durations of the elicitor applications were 6, 24, 48, and 168 hours, then the elicited cultures were gathered. 1 mL of 96% ethanol was added to the control cultures. Inspection cultures were collected after 6 and 168 hours since their production does not change notably in such short time intervals. The cells were separated from the media by vacuum filtration, rinsed with distilled water, and dried at the laboratory temperature. The nutrient medium was kept frozen to be used for subsequent analyses. All analyses were carried out in a minimum of three independent samples for each elicitation period and each concentration of elicitor.
Determining the Flavonoids and Isoflavonoids.
Spectrophotometric determination of flavonoids was carried out in the extracts of the cells and in the culture media according to the Czech Pharmacopoeia 2009 [27]. The HPLC method with fluorometric detection was used to determine isoflavonoids [28]. A 200 mg sample was extracted twice (in a water bath under reflux condenser) with 10 mL of 80% (v/v) aqueoes methanol for 20 min. The filtrated extracts were combined, and the volume was adjusted with 80% (v/v) aqueoes methanol to 25 mL. After filtration through a 0.45 μm microfilter, the extracts were injected into the HPLC system. Also samples of the culture media were filtrated through a 0.45 μm microfilter and injected into the HPLC system. The HPLC conditions were as follows: a RP-18 Lichrospher column (250 × 4 mm, particles size 5 μm) with a precolumn made of the same material; elution: linear gradient of a mobile phase A (methanol) in phase B (water containing 0.15% (v/v) of phosphoric acid) 30-80% (v/v) from 0 to 9 minutes was followed by the isocratic elution with a mixture of 80% (v/v) of phase A in phase B from 9 to 15 minutes; the flow rate was 1.1 mL min −1 ; the detection was carried out at the 260 nm wavelength. The contents of the monitored substances were quantified by using mathematical method of normalization and by comparing with the calibration curve drawn by the external standard of the same substance. The obtained results were statistically evaluated by the t-test at P = 0.05.
Results and Discussion
Successful elicitation depends on many factors that are specific for each elicitor and for each plant tissue culture. This work focused on the elicitor concentration and time duration of elicitor's effect. The results (Table 1) indicate that the elicitor used influenced favorably the production of isoflavonoids in the Trifolium pratense suspension culture. The best elicitation effect was observed after 48-hour application of 1 μmol L −1 concentration after which maximum amounts of genistin (11.60 mg g −1 DW), daidzein (8.31 mg g −1 DW), and genistein (1.50 mg g −1 DW) were determined. There was statistically significant increase in the production of the above-mentioned isoflavonoids, when compared with control, by 152%, 151%, and 400%, respectively. The positive effect of the elicitation grew with the duration time of the application (6, 24, 48 hours). On the contrary, maximum content of formononetin (7.31 mg g −1 DW) was determined after 6-hour application of 10 μmol L −1 concentration which represents a 97% increase when compared with control. The stimulation of genistin occurred even after adding the strongest, 100 μmol L −1 concentration. Only the production of biochanin A was not influenced favorably. Moreover, the longest (168-hour) application of all elicitor concentrations has decreased the production of isoflavonoids when compared with control. The production of flavonoids in the elicited Trifolium pratense suspension culture ( Table 2) was also successful. The best elicitation effect of all elicitor concentrations was recorded after 6 hours. The higher the elicitor concentration, the better the effect observed. The maximum content of flavonoids (5.78 mg g −1 DW) was induced by 6-hour application of the strongest, 100 μmol L −1 concentration of elicitor, and the increase of production was statistically significant-it increased by 142% when compared with the control. On the contrary, the positive influence of the elicitation was decreasing when the time duration of the elicitor application was prolonged. The longest, 168-hour application of all concentrations of elicitor caused (as in case of isoflavonoids) statistically significant decrease in the flavonoid content when compared with control.
The content of flavonoids and isoflavonoids was not only determined in the extracts of the cells but also in the nutrient media of Trifolium pratense suspension cultures. No detectable levels of these substances were found in any of the samples tested. There was thus no secretion of the monitored secondary metabolites into the nutrient medium after the application of this elicitor. | 2014-10-01T00:00:00.000Z | 0001-01-01T00:00:00.000 | {
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247298901 | pes2o/s2orc | v3-fos-license | FishSizer: Software solution for efficiently measuring larval fish size
Abstract Length and depth of fish larvae are part of the fundamental measurements in many marine ecology studies involving early fish life history. Until now, obtaining these measurements has required intensive manual labor and the risk of inter‐ and intra‐observer variability. We developed an open‐source software solution to semi‐automate the measurement process and thereby reduce both time consumption and technical variability. Using contrast‐based edge detection, the software segments images of a fish larva into “larva” and “background.” Length and depth are extracted from the “larva” segmentation while taking curvature of the larva into consideration. The graphical user interface optimizes workflow and ease of usage, thereby reducing time consumption for both training and analysis. The software allows for visual verification of all measurements. A comparison of measurement methods on a set of larva images showed that this software reduces measurement time by 66%–78% relative to commonly used software. Using this software instead of the commonly used manual approach has the potential to save researchers from many hours of monotonous work. No adjustment was necessary for 89% of the images regarding length (70% for depth). Hence, the only workload on most images was the visual inspection. As the visual inspection and manual dimension extraction works in the same way as currently used software, we expect no loss in accuracy.
| INTRODUC TI ON
Larval growth rate is a fundamentally important life history trait directly linked to fish population productivity and persistence. For many fish populations, fast-growing larvae have higher survival rates, largely by reducing larval stage duration in which they are exposed to higher predation risk (Houde, 2008). Also, larger body sizes are associated with increased feeding and swimming capacity of larvae, allowing them to search larger water volumes, increase encounter rates with prey, feed on larger organisms, and have better escape responses from predators (Hale, 1996;Miller et al., 1988;Munk & Kiorboe, 1985;Peck et al., 2012). Therefore, body length is an essential measurement in studies of larval fish ecology (Bils et al., 2017), development (Fuiman et al., 1998), physiology Petereit et al., 2008), evolution (Oomen & Hutchings, 2015Oomen et al., 2021), reproductive biology (Roney et al., 2018), and aquaculture (Blanco et al., 2017). Besides length, body depth is also valuable, as it can be used as a proxy for muscle development (i.e., myomere height) and potential starvation resistance Peña et al., 2021).
Body size measurements of larval fish are generally taken on individuals using a camera attached to a stereomicroscope. On the generated images, body length and depth are measured manually using a dedicated software, either open-source (e.g., ImageJ) or proprietary (e.g., Leica Application Suite). Body length is generally estimated as notochord length (tip of the snout to the end of the notochord) during the preflexion stage and as standard length (tip of the snout to the posterior extremity of the hypural plate) afterwards (Kahn et al., 2004). Body depth is generally measured as maximum depth at the head, at the anus, or at the caudal peduncle. Even with the aid of dedicated software, these manual measurements are time consuming because a typical laboratory study can generate thousands of images (e.g., 4489 by Roney et al. (2018)). This manual work also has high potential for introducing intra-and inter-observer variability, potentially leading to increased measurement errors.
Here, we introduce a novel, open-source application, FishSizer, to semi-automate measurements of larval fish length and myomere depth. FishSizer addresses the two major problems of the manual method used to estimate body size in larval fish by considerably reducing the amount of manual work and potentially decreasing technical variability. Although this application was developed specifically for use with fish larvae, it should be useful for older life stages of fish (and possibly other animals) for which satisfactory images are available.
| Overall method
All coding was done in Matlab 2020a (The MathWorks Inc.) and compiled into a single file for installation on computers without Matlab. Determination of length and depth of fish larvae from images is based on A sequence of two procedures: (1) producing a mask (a binary image the same size as the original image) segmented into "larva" and "background" and (2) determining length and depth based on this "larva" segmentation.
| Segmentation
For morphometrics assessments, pictures of anesthetized individual larvae laying on a microscope slide are taken under a stereomicroscope. During a single session, a user may typically take hundreds of pictures of individual larvae, so the zoom level is typically fixed in order to reduce the manipulation time. This procedure results in the background being mostly uniform (i.e., low in contrast) with the exception of lines arising from scratches on the glass slide and water drops. Due to this low-contrast background, segmentation is based on edge detection. Edge detection is a method of establishing regions where the contrast between neighboring pixels in an image is above a certain preset threshold. Edge detection has the advantage of being applicable across species and/or stage of the larvae being measured. More advanced methods, such as deep learning, will in most cases need retraining of the network to correctly segment species not previously encountered by the network (Kvaestad et al., 2022). Deep learning has the additional drawback of having higher hardware demands compared to this less computationally intensive approach (LeCun, 2019). In order to determine a robust edge detection threshold across a wide range of images, we determine the maximum contrast present in the image and set the threshold as a customizable fraction of this value. Many images used for larval fish length measurements contain scale bars or other high-contrast objects. To avoid basing the contrast threshold on these artifacts, this software offers the option of establishing a region of interest (ROI) within which the segmentation is contained.
Running the edge detection algorithm on an image creates a binary mask with pixel values of 1 at edges and 0 at other locations (see Figure 1 for the segmentation process illustrated). The aim is to have a complete outline of the larva and subsequentially fill in this outline. In practice, the detected outline can be incomplete, leaving small gaps and resulting in faulty segmentation. We therefore dilate . For further analysis, we extract orientation for an ellipse that best represents this region.
In order to minimize the need to adjust parameter settings, and make the software as user friendly as possible, we aimed to make the segmentation as robust as possible. We found that a Gaussian image filter with a standard deviation of four pixels followed by an image sharpening algorithm with the same standard deviation significantly improved segmentation (e.g., Figure 3). When applying a Gaussian image filter, the value of each pixel is influenced by the value of all neighboring pixels. Hence, small regions in the outline with low contrast can have increased contrast after the procedure and, therefore, a better chance of complete edge detection.
| Length and depth measurement
Length and depth estimation is done using the segmentation mask.
The first step is to rotate the mask using the orientation of the major axis of the ellipse fitted to the larva outline. This rotation is done using the imrotate.m function in MATLAB. By default, imrotate uses nearest-neighbor interpolation, setting the values of pixels in the final image that are outside the rotated image to 0. Alignment with the horizontal (X) axis allows the use of a polynomial regression for length estimation. It also allows depth estimation based solely on the Y component at a customizable location relative to the length (X axis). The next step is to establish which end is the head end by dividing the larva into two halves of equal length and determining which end contains more pixels in the segmented mask. This procedure assumes that the anterior half of the larva is larger than the posterior half.
Length estimation is based on a polynomial regression line through the larva segmentation that best describes the curvature of the larva. As larval fish are often curved, we use second-order or greater polynomial regression. Presence of a large yolk sac affects the regression, such that a larva with straight notochord and large yolk sac will yield a curved regression that does not accurately represent the larva's length. To avoid this, we base the order of the regression on the curvature of the tail alone. The greater the curvature, the higher the order of the polynomial regression. A lower-order regression on a larva predicted to be straight will result in a straighter regression and hence a more precise length estimation. Therefore, a second-order polynomial regression is computed for the tail part of the larva and, since the larva is rotated to be horizontal, both coefficients of this regression indicate the amount of curvature of the entire larva. If the absolute value of the second-degree coefficient is >0.1 or the absolute value of the first-degree coefficient is >0.5, a third-order polynomial regression is used for the entire larva. Otherwise, a second-order polynomial regression is used.
Depth is determined at a customizable location relative to the length of the larva (X axis in Figure 2, window 2). Because the segmented larvae are rotated to be horizontal, a good approximation of the depth is the difference between the maximum and minimum Y values at the user-defined X location. To make depth determination less dependent on precise point-to-point segmentation, depth is not calculated at a single point but rather as the median of all depths calculated at the user-defined location ±5% of the length of the larva.
For visual verification, length and depth estimation lines are shown in the main graphical user interface (Figure 2 window 2).
| Main GUI window description
The interface of this software is split into two main graphical user interfaces: the main GUI and the loading GUI. The main graphical user interface contains four windows and five button panels as seen in Figure
| Main GUI button description
Buttons in the GUI are grouped according to function (Figure 2). the user to manually draw lines in top left window to measure length and depth. To the right of these two buttons is a panel for setting the number of line segments needed to make the measurement. The default for measuring length is 2, as most larvae are slightly curved and hence a line consisting of two segments will give a more accurate estimate of the length than one straight line. If the number of segments is set greater than needed for a particular larva, pressing "enter" after marking fewer segments will complete the measurement. The default number of segments for manually measuring depth is 1, which usually is sufficient. To the right are buttons for cancelling the manual length or depth estimations and reverting to the automated estimations.
Below the top right window are controls for navigating the image files and exporting data to a comma-delimited file (.csv). To the left is a slider for selecting images from the loaded dataset. For optimized workflow, hotkey functions are associated with this. Pressing "a" selects the image prior to the active image, pressing "s" selects the image after the active image. To the right is the export data button. Data can be exported both with and without calibration into millimeters (mm). The "export data" button is yellow if data have not been calibrated (measurement reported in units of pixels) and green if calibration into millimeters (mm) has been applied to the dataset.
| Loading GUI description
This GUI provides the options to (1) load a test image for testing and adjusting the segmentation settings, and (2) sets the fraction of maximum contrast (see section 2.2). "Dilation" sets the number of dilations applied to each edge-detected pixel (see section 2.2)." Depth offset" determines the location of the depth measurement in percentage of total larval length from the head. If set to 50, depth is measured at the midpoint of the larva along the X axis in window 2 of the main GUI.
The ROI panel contains three buttons for: (1) setting a rectangular ROI, (2) setting a circular ROI, and (3)
| Workflow
A typical workflow with a set of images is first to confirm or change the segmentation settings on the loading GUI. It is recommended to load a test image and try settings before loading the entire data- file or by creating a new one (see manual for details). After calibration, the "Load dataset" button is green to reflect that calibration has been done. After visual validation of correct segmentation settings, images are loaded with the "Load images" button, after which a progress bar appears. When the progress bar disappears, the slider below window 2 in the main GUI, as well as hotkeys "a" and "s", can be used to navigate the dataset. If automatic estimation is satisfactory, no additional work is needed, and the user can move to the next image. If the automatic estimation is unsatisfactory, manual length and depth extraction is done in the same way as ImageJ. The "Export data" button is green if calibration has been performed and pressing the button will produce a comma-delimited file with five (without calibration) or nine (with calibration) columns containing data as described in Table 1. Time savings were calculated compared to ImageJ (Schneider et al., 2012), a commonly used software for this task. Two independent observers went through the test dataset and extracted length and depth in FishSizer, including visually verifying all measurements.
| PERFORMAN CE E VALUATION
Compared to extracting the same measurements from the same dataset in ImageJ, a time saving of 66 and 78% was observed for observer 1 (R1) and 2 (R2), respectively. Intra-observer variability is expected to be lower using FishSizer compared to ImageJ as the threshold for accepting automatic parameter extraction will remain fairly constant for each observer. For manually extracted parameters in FishSizer, variability is expected to be the same as ImageJ, as the process for manual extraction is the same in both software packages.
To examine inter-observer variability compared to ImageJ, we analyzed data from two observers to compare percentage-wise differences in length and depth measurements across 101 haphazardly chosen tuna larva images ( Figure 6). For length we found a deviation between the two observers of 2.69%±3.35% (mean±std) for FishSizer compared to 3.13% ± 4.54% for ImageJ. For depth we found a mean of 15.6% ± 14.6% for FishSizer and 19.8% ± 34.0% for ImageJ. Therefore, there were a small but not significant difference between the two software packages for both parameters.
For visual representation of the variability using a Bland-Altman graph, see Figure 6. One important aspect of variability is human error. In FishSizer, all handling of parameters and data output is automatic and linked to the image file name. Therefore, FishSizer has less potential to introduce human errors than other programs which can require manual data curation (e.g., using copy/paste), such as ImageJ.
F I G U R E 5 Length (a) and depth (b)
| CON CLUS I ON AND FUTURE DIREC TIONS
The partial automation of obtaining larval morphological measurements in this software saves a considerable amount of time compared to the manual procedure used so far. Even when all measurements obtained via our software were manually verified and corrected, we experienced a time saving of 66%-78%.
When tested across a range of species, we found the software to achieve good segmentation for a wide range of species like Atlantic cod, Atlantic bluefin tuna, Pufferfish, European plaice, and Southern flounder using a rectangular ROI, and Atlantic herring using a circular ROI. (Figure 7). As the parameter extraction relies heavily on segmentation, we expect that the accuracy for these and similar species will be the same as seen for the tuna statisti- contrast detection, we found the quality of images to be essential.
High-contrast artefacts in direct contact with the larva can hinder correct segmentation and thereby correct parameter extraction ( Figure 8).
A logical future direction for this software will be to include deep learning. This current version of FishSizer can help create large datasets of segmented larva images to be used as ground truth for training neural networks. Deep learning will not only facilitate analysis of images with more than one larva, a sought after feature for this kind of software, but it will also open the door to automatic species and/or stage identification of larval fish, which is far behind other pelagic marine organisms such as plankton (Guo et al., 2021).
ACK N OWLED G M ENTS
We appreciate the help of Fredrick Nicolai Clausen and Paul Elias Homme during the initial development of the user interface, Björn Illing for feedback that greatly improved the user experience, and Griet Nobis for early work on the topic that helped to inspire this project.
CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.
DATA AVA I L A B I L I T Y S TAT E M E N T
Compiled installable software, manual, and MATLAB files used for compiling are available at: https://doi.org/10.5281/zenodo.5833209 (https://github.com/jeppe have/FishS izer).
O RCI D
F I G U R E 8 Example of faulty segmentation of an Atlantic bluefin tuna larva. In this case, the strong contrast of the water edge to the left is connected to the contrast associated with the larva itself. As all areas directly connected to the borders of the image are ignored, a small area to the right is chosen as largest area surrounded by a complete outline | 2022-03-09T16:12:53.468Z | 2022-03-01T00:00:00.000 | {
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415673 | pes2o/s2orc | v3-fos-license | Exogenous proline stimulates type I collagen and HIF-1α expression and the process is attenuated by glutamine in human skin fibroblasts
Abundance of proline (Pro) in collagen molecule led us to investigate whether Pro supply affects collagen biosynthesis in human skin fibroblasts. Treatment of the cells with milimolar concentrations (5 and 10 mM) of Pro for 24 and 48 h contributed to increase in α1 subunit of collagen type I (COL1A1) expression in both cells and culture medium. However, the effect was more pronounced in glutamine-free medium. In such condition, Pro induced collagen expression by about twofold in the cells, while in the medium only by about 30% during 24 h incubation, compared to control. In the presence of glutamine (Gln), exogenous Pro stimulated intracellular collagen expression only by about 30% during 24 h of fibroblasts incubation, and it was not accompanied by adequate increase of collagen secretion into medium. Gln alone stimulated the processes by about 2–3 fold during the course of the experiment. Pro-dependent increase in collagen expression in Gln-free medium was accompanied by increase in prolidase activity and expression of pAkt. In both Gln-free medium and Gln-supplemented medium, Pro induced expression of p53 and HIF-1α. The data suggest that availability of Gln, as a substrate for Pro biosynthesis, determine the utilization of exogenous Pro for the collagen biosynthesis.
Introduction
Collagen is the most abundant protein in the body comprising 30% mass of all proteins. Type I collagen is the main one among about thirty discovered collagenous proteins. It contains about 20% of proline (Pro) and 4-or 3-hydroxyproline [1]. There are two sources of proline in the cells. The first one is synthesis from glutamate (Glu) catalyzed by 1-pyrroline-5-carboxylate (P5C) synthase and P5C reductase with P5C as an intermediate product. P5C is formed from both Glu and Pro, due to interconvertibility of this amino acids [2,3]. Second source of Pro constitute protein degradation products, hydrolyzed by extracellular and intracellular proteases. Among products of protein degradation are imidodipeptides containing C-terminal proline or hydroxyproline [4]. Bond between any amino acid and Pro is resistant to hydrolytic action of most proteases. Only prolidase-cytosolic peptidase is able to hydrolyze imidodipeptides and release Pro [5]. In vivo prolidase is present in many different cell types, including erythrocytes [6], contributing to maintain physiologic level of Pro in blood and extracellular matrix. However, the impact of extracellular Pro on collagen biosynthesis is not fully understood. The reason is that collagen biosynthesis assay is based on radioactive Pro incorporation into collagenous proteins and the tracer is diluted by Pro. Therefore, in this study, we employed Western blot for type I collagen, the most abundant collagen in tissues.
Pro released from imidodipeptides can be reused for protein synthesis, e.g., collagen or metabolized to P5C through proline oxidase, known also as proline dehydrogenase (POX/PRODH) [7,8]. Prolidase was found to be a rate limiting factor of collagen biosynthesis at least in certain conditions like fibrotic process [9], experimental aging of fibroblasts [10], fibroblasts chemotaxis [11], and cell surface integrin receptor ligation [12]. Interestingly, prolidase was also found as a ligand of epidermal growth factor receptor (EGFR), inducing the receptor signaling [13]. Prolidase activity is also implicated in regulation of some transcription factors. Nuclear factor-jB (NF-jB) transcriptional activity is negatively correlated with prolidase activity [14]. In opposite, hypoxia inducible factor-1 (HIF-1) transcriptional activity positively correlates with prolidase activity. The mechanism of this process is due to Pro-dependent inhibition of HIF-1a degradation [15]. It shows that Pro as a product of prolidase-catalyzed reaction may contribute to the transcriptional action of these factors. In fact, Pro is reported to increase HIF-1a expression and transcriptional activity of HIF-1 in cancer cells [15]. Both NF-jB and HIF-1a regulate collagen biosynthesis on transcriptional and posttranslational levels, respectively [14][15][16][17][18]. The specific purpose of this study was to evaluate the impact of extracellular Pro on collagen, NF-jB, HIF-1a, POX, EGFR, IGF-IR expressions as well as prolidase activity and some signaling proteins in human skin fibroblasts cultured in medium with or without Gln.
Tissue culture
All studies were performed on normal human skin fibroblasts CCD25Sk (ATCC Ò CRL1474 TM ), that were purchased from American Type Culture Collection, Manassas, VA, USA. Reference cells, endometrial adenocarcinoma cells (Ishikawa cell line), and breast adenocarcinoma cells (MCF-7) were received from Sigma Corp., USA). The cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin, 50 lg/ml streptomycin at 37°C in a 5% CO 2 incubator. Cells were counted in hemocytometer and cultured at 1 9 10 5 cells per well in 2 ml of growth medium in 6-well plates (Costar). Cells reached confluence at day 6 and in most cases such cells were used for assays. Cells were used in the 8th to 14th passages.
Cell viability assay
The assay was performed according to the method of Carmichael [19] using 3-(4,5-di-methylthiazole-2-yl)-2,5diphenyltetrazolium bromide (MTT). The cells were cultured for 24 and 48 h with various concentrations of Pro in six-well plates, washed three times with PBS, and then incubated for 4 h in 1 ml of MTT solution (0.5 mg/ml of PBS) at 37°C. The medium was removed and 1 ml of 0.1 mol/l HCl in absolute isopropanol was added to attached cells. Absorbance of converted dye in living cells was measured at a wavelength of 570 nm. Viability of Protreated cells was calculated as a percent of control cells.
DNA biosynthesis assay
To examine the effect of Pro on fibroblast proliferation, the cells were plated in 24-well tissue culture dishes at 1 9 10 5 cells/well with 1 ml of growth medium. After 48 h (1.6 ± 0.1 9 10 5 cells/well), the plates were incubated with various concentrations of Pro and 0.5 lCi of [ 3 H] thymidine for 24 h at 37°C. The radioactivity of incorporated tracer into DNA was measured in a scintillation counter [20].
Collagen production
Incorporation of radioactive precursor into proteins was measured after labeling of confluent cells (cultured in growth medium with Pro) for the last 24 h with 5[ 3 H] proline (5 lCi/ml, 28 Ci/mM) as described previously [21]. Incorporation of tracer into collagen was determined by digesting proteins with purified Clostridium histolyticum collagenase, according to the method of Peterkofsky et al. [22]. Results are shown as combined values for cell plus medium fractions.
SDS-PAGE
Slab SDS/PAGE was used, according to the method of Laemmli [23], using 10% SDS-polyacrylamide gel.
Western immunoblot analysis
After SDS-PAGE, the gels were allowed to equilibrate for 5 min in 25 mmol/l Tris, 0.2 mol/l glycine in 20% (v/v) methanol. The protein was transferred to 0.2 lm pore-sized nitrocellulose at 100 mA for 1 h by using a BIO-RAD Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell. The nitrocellulose was incubated with primary antibodies at dilution 1:1000 in 5% dried milk in TBS-T (20 mmol/l Tris-HCl buffer, pH 7.4, containing 150 mmol/l NaCl and 0.05% Tween 20) overnight at 4°C. In order secondary peroxidase conjugated antibodies were added at concentration 1:5000 in TBS-T and incubated for 30 min slowly shaking. Then nitrocellulose was washed with TBS-T (5 9 5 min). Bound antibodies were detected by enhanced chemiluminescence using Amersham ECL Western blotting detection reagents. The intensity of the bands was quantified by densitometric analysis using apparatus for gel documentation BioSpectrum Imaging System (UVP, USA) and presented in arbitral units normalized for b-actin.
Determination of prolidase activity
The activity of prolidase was determined according to the method of Myara et al. [24]. Protein concentration was measured by the method of Lowry et al. [25]. Enzyme activity was reported as nanomoles of proline released from synthetic substrate, during one minute per milligram of supernatant protein of cell homogenate.
Immunofluorescence
Fibroblasts were plated in BD Falcon 96-well black/clear bottom tissue culture plates optimized for imaging applications at 1 9 10 5 cells per well. After 24 h incubation, the cells were rinsed with PBS and fixed with a 4% formaldehyde solution at room temperature for 15 min. After fixation, the cells were washed three times with PBS and permeabilized with a 0.1% Triton X-100 solution at room temperature for 5 min. Then, the cells were washed twice with PBS, and non-specific binding was blocked (5% nonfat dry milk, 10% heat-inactivated goat serum in 0.025% Tween 20/PBS, 1 h incubation at room temperature). After that time, the cells were rinsed, incubated with anti-HIF-1a mouse monoclonal antibody (Becton, Dickinson Co., USA; 1:100) for 1 h at room temperature. Antibody was diluted in antibody dilution buffer (1% nonfat dry milk in 0.025% Tween 20/PBS). Then the cells were washed three times with PBS and incubated with a fluorescent (Alexa Fluor 594) anti-mouse secondary antibody (Invitrogen, Carlsbad, CA) for 60 min in the dark. After washing, the nuclei were stained with Hoechst 33342 (2 lg/ml) and cells were analzed using confocal microscope BD Pathway 855 using a 40 9 (0.90 NA) objective.
Statistical analysis
The results were submitted to statistical analysis using oneway ANOVA followed by Tukey test, accepting *P \ 0.05 as significant versus control.
Results
Treatment of fibroblasts with Pro at concentrations 5 or 10 mM did not influence the viability of the cells. Slight, not significant decrease in the DNA biosynthesis between treated and untreated cells was found (data not shown).
The effect of Pro on collagen biosynthesis in confluent human skin fibroblasts was determined at different time of incubation. Since glutamine (Gln) is an important substrate required for proline biosynthesis [2,26,27], we designed conditions of impaired Pro biosynthesis in fibroblasts using Gln-deprived medium. The cells were incubated with complete growth medium containing 10% serum to achieve confluency. Then the cells were pretreated with complete or Gln-deprived medium for 24 h and medium was changed with fresh, containing 5 or 10 mM Pro and 5-[ 3 H]proline (5 lCi/ml, 28 Ci/mM). Incorporation of the radioactivity into proteins susceptible to the action of purified bacterial collagenase was determined according to the method of Peterkofsky et al. [22]. However, we found that the incorporation of radioactive precursor (5[ 3 H] proline) into proteins was very low in cells growing in both media (with Gln or without Gln) in presence of Pro suggesting competition between labeled and unlabeled proline in the process of proline incorporation into collagen (data not shown). Therefore, western blot analysis of a 1 subunit of collagen type I (COL1A1) in the cell homogenates and culture media, after 24 and 48 h of fibroblasts treatment with or without Gln was performed. As can be seen in Fig. 1a, expression of COL1A1 in fibroblasts and cultured medium (Fig. 1b) is much higher in cells growing in Gln containing medium, compared to cells cultured in medium without Gln. Collagen expression in cells treated with Gln for 24 and 48 h reached 172 and 243% of control value (cells cultured without Gln), respectively. Similarly, expression of collagen in culture medium of these cells achieved 165 and 292% of control value, respectively.
Treatment of the cells with 5 or 10 mM Pro in both Glnsupplemented and Gln-deprived medium for 24 and 48 h induced expression of COL1A1 (Fig. 2a). However, removal of Gln from medium augmented Pro-dependent increase in expression of collagen type I in fibroblasts. Fibroblasts growing in medium without Gln containing 5 or 10 mM Pro produced much more (about twice) COL1A1, during the course of experiment compared to control. Increase in the expression of COL1A1 in the cells was accompanied by slight increase in secretion of collagen to the medium, but only after 24 h of incubation of the cells (Fig. 2b).
Pro-dependent stimulation of collagen production was not related to the expression of NF-jB p65 (Fig. 2c), the known inhibitor of collagen gene expression [28] in fibroblasts, growing in Gln containing medium. Moreover, in all conditions Pro was unable to decrease IjB-a expression (Fig. 2d), suggesting that canonical NF-jB activation is not induced by proline [29,30].
Since prolidase is involved in collagen biosynthesis by recycling Pro from protein degradation, the enzyme activity was evaluated. We have found that Pro increased the enzyme activity by about 15-20%, during the course of experiment and the process was more pronounced in Glndeprived medium (Fig. 3a). This increase in the enzyme activity in cells cultured in Gln-deprived medium was correlated with the enzyme expression (Fig. 3b).
Since prolidase is involved in regulation of epidermal growth factor receptor (EGFR) signaling [13], the expression of this receptor was measured by Western blot analysis. However, Pro had no significant effect on the expression of EGFR (Fig. 3c).
Proline is utilized not only in protein synthesis but also in process of Pro oxidation to P5C, catalyzed by mitochondrial proline oxidase (POX). The enzyme expression was determined in fibroblasts, MCF-7 and endometrial adenocarcinoma cells, as a reference cell lines. We found very low expression of POX in fibroblasts, using Western blot with enhanced chemiluminescence as a method of detection (Fig. 3g).
Collagen biosynthesis and prolidase activity were previously shown to be regulated due to the signal induced by activated a 2 b 1 integrin receptor [12,32] as well as IGF-IR [33]. Therefore, the expression of a 2 b 1 integrin receptor (receptor for type I collagen) and IGF-IR were measured by Western immunoblot analysis. As can be seen in Fig. 4a, b, treatment of fibroblasts with 5 or 10 mM Pro contributed to decrease in the expression of a 2 and b 1 integrin subunits, particularly in cells growing in medium with Gln. As shown on Fig. 4c, IGF-I receptor expression was increased in Pro-treated cells growing in medium with Gln, after 24 h incubation. This stimulating effect was not observed after 48 h. In fibroblasts, growing in Gln-deprived medium treatment of fibroblasts with 5 or 10 mM Pro contributed to increase in the expression of IGF-I receptor to about 109 and 121% after 24 h of incubation and 131 and 111% after 48 h of incubation, compared to the control cells (Fig. 4c).
We found that in fibroblasts growing in medium with Gln, 5 or 10 mM Pro decreased expression of phosphorylated ERK1/2 to about 93% after 24 h and to 85 and 52% after 48 h, compared to the control cells (Fig. 4d). In growth medium without Gln, 24 and 48 h treatment of fibroblasts with 5 or 10 mM Pro contributed to decrease in the expression of phosphorylated ERK1/2 to about 70% after 24 h and 87% after 48 h, compared to the control cells (Fig. 4d).
In Pro-treated cells growing in medium without Gln, an expression of pAkt was increased (Fig. 4f), suggesting that in the experimental conditions, Akt protein represent signaling molecule that respond to Pro action.
To study the specificity of proline impact on collagen biosynthesis, we used proline analogues: tetrahydro-2furoic acid (FA) and thiazolidine-4-carboxylic acid (TA). Due to structural similarity to proline, these compounds can probably substitute proline in collagen biosynthesis. It has been also documented that they impair proline oxidation by POX [34][35][36]. Based on cytotoxicity of both compounds, we selected concentration 10 mM of FA and 5 mM of TA. As shown in Fig. 5a, after 24 and 48 h incubation of fibroblasts in medium with Gln, expression of COL1A1 was decreased by analogues in similar way: to 30% by TA and to 40% by FA, compared to control. In cells growing in medium without Gln structural analogues of Pro almost completely inhibited expression of COL1A1. Thus, the inhibitory effect of Pro analogues on collagen biosynthesis is stronger in cells with impaired proline biosynthesis. Similar process was observed on expression of COL1A1 secreted to medium (Fig. 5b).
Next, we assessed whether Pro addition can affect inhibitory effect of TA and FA on collagen expression. Pro was added after 2 h pretreatment of cells with proline analogues and cultured for 24 and 48 h. We observed that in both, presence or absence of Gln in media, Pro reversed the inhibitory effect of its analogues. The effect of Pro was strongest in fibroblasts growing in medium without Gln (Fig. 5c). However, Pro-induced collagen expression in media (Fig. 5d) was less pronounced, than in the cells (Fig. 5c).
Regarding that molecules similar to Pro, like Cbz-Pro inhibit prolidase activity [14], we checked influence of FA and TA on prolidase. In cells growing in medium with Gln, both compounds did not affect prolidase activity after 48 h incubation. In cells growing in medium without Gln, after 48 h of incubation TA and FA increased the enzyme activity to 130 and 125% of control values, respectively (Fig. 5e).
Pro is reported to increase HIF-1a expression in cancer cells [15]. Heterodimeric factor HIF-1a stimulates the posttranslational hydroxylation of procollagen prolyl residues [37], necessary to obtain the correct spatial structure of collagen [1]. Recent studies indicate inhibition of transcription of type I collagen genes by HIF-1 [18]. Therefore, the expression of HIF-1a in Pro-treated cells was evaluated by bioimaging. It was found increased staining for HIF-1a in Pro-treated cells, cultured in both media for 48 h, especially with 10 mM of Pro in medium without Gln, compared to control cells (Fig. 6, panel a). An addition of TA and FA to Pro-treated cells in both medium abolishes increased staining for HIF-1a, but FA was less effective (Fig. 6, panel b, c).
Discussion
In this report, we provide evidence that extracellular Pro has significant, but relatively little impact on collagen expression in cells cultured in standard conditions in medium containing Gln. The conclusion is supported by other authors, showing that an addition of proline has failed to increase collagen biosynthesis in fibroblasts and other cells [38][39][40]. In contrast, extracellular Pro drastically increased collagen expression in the cells cultured in Glnfree medium. The mechanism of this process is due to the Pro is formed from glutamate (Glu) which is produced from Gln. The main source of Gln in the body is Glu in muscle that is converted to Gln by glutamine synthase. The enzyme is widely distributed in tissues and was reported also in fibroblasts [41].
Gln was referred as an inducer of collagen gene transcription [42]. Culture medium (DMEM) contains high concentration of Gln (4 mM) that after conversion may support also intracellular pool of Pro. Therefore, we evaluated the effect of extracellular Gln deprivation on collagen expression in human skin fibroblasts. In fact, Gln deprivation diminished collagen expression, while Pro partly counteracted this effect. The action of proline in this process cannot be linked to conversion of Pro to Gln because activity of proline oxidase (POX) in fibroblasts is very low. Therefore, we suggest that Gln deprivation impaired Pro biosynthesis contributing to relative proline deficiency that was partly normalized by Pro addition. Thus, fibroblasts need exogenous Gln, as an intermediate of proline, required for effective collagen biosynthesis.
Another source of Pro are imidodipeptides hydrolyzed by prolidase. However, the enzyme activity was only slightly but significantly increased by Pro in the cells cultured in Gln-free medium, suggesting the marginal role of prolidase for proline support in a such conditions. Therefore, we considered another function of prolidase. Previously, it has been suggested that increase in prolidase activity (that release proline from imidodipeptides) contribute to increase in NF-jB p65 expression [14]. This process is important for collagen type I biosynthesis, since transcription of genes coding type I collagen subunits is inhibited by NF-jB [16,27,28]. However, we found that extracellular Pro did not affect expression of NF-jB. Therefore, we postulate that extracellular Pro may participate in collagen biosynthesis as a substrate. The evidence that supply of Pro is essential for the biosynthesis of collagen comes from the experiment showing that proline analogues suppress collagen expression. The mechanism of proline analogues competition with Pro in collagen biosynthesis is well known phenomenon [43]. Impaired ability of fibroblasts to synthesize Pro was shown in the cells incubated in medium without Gln. An addition of exogenous Pro reversed this effect presumably as a result of competition mechanism.
Based on previous studies on exogenous glutamine-induced collagen biosynthesis [40,44] and present data showing that collagen biosynthesis is not suppressed completely in fibroblasts growing in Gln-deprived medium, we hypothesize that the effect can be associated with synthesis of glutamine/glutamate and recycling of proline by prolidase. Pro is converted in mitochondria by POX to P5C and further to glutamate, ornithine, or proline. POX is expressed ubiquitously in the body, but POX activity was found previously to be undetectable in fibroblasts [45]. In the present study we noticed very low expression of POX in fibroblasts, which suggest that the proline in fibroblasts is mainly consumed for protein biosynthesis.
Pro was referred as a HIF-1a inducing agent in colon cancer cell line RKO [15]. Pro addition prevent hydroxylation of specific proline residue in the oxygen-dependent degradation (ODD) domain of HIF-1a and prevent targeting HIF-1a for ubiquitination and proteasomal degradation via Von Hippel-Lindau (VHL) tumor suppressor protein [46]. RKO cell line expresses POX [47]; thus, it is not clear whether Pro or its metabolites affect HIF-1a expression. We presented that Pro induced HIF-1a translocation to nuclei of fibroblasts (the cells showing low expression of POX). Therefore, stimulation of HIF-1a is not an effect of metabolites of Pro. It is interesting that this process is stronger in the absence of Gln. In fibroblasts Gln is converted to Glu, which is the source of a-KG. It is known that Gln deprivation from the medium results in lowering the concentration of a-KG [48,49], whereby the cells could become more susceptible to the stabilizing effect of proline on HIF-1a. The hypothesis is outlined in Fig. 7. However, our study on the effect of proline analogues on the expression of HIF-1a provided not expected data. Pro analogues diminished HIF-1a expression in the cells and prevented its upregulation by proline addition. However, Pro-dependent stimulation of intracellular collagen expression in fibroblasts was not affected by proline analogues. Therefore, it seems that this process do not depend on HIF-1. In fact, recent studies documented inhibition of transcription of type I collagen genes by HIF-1 [18]. Nevertheless, the explanation of this effect requires further study.
Proline-dependent increase in HIF-1 expression was preceded by an increase in p53 expression. So far, stimulating effect of proline on p53 expression was not described. It is known that p53 is inducer of POX expression [31,47]; however, proline did not affect POX expression. Moreover, increased expression of p53 did not affect cell viability suggesting that apoptosis was not induced. The functional significance of proline-dependent regulation of p53 transcriptional activity requires further study.
In view of our data, the signaling pathways induced by IGF-I are also involved in stimulation of intracellular collagen expression by proline. We found that Pro-dependent increase in expression of type I collagen in fibroblasts was accompanied by activation of IGF-IR expression and Akt signaling. In fact, IGF-I is the most potent stimulator of collagen biosynthesis [33,50] and IGF-I receptor-mediated Akt-dependent signaling pathway induces collagen gene expression [51]. Collagen biosynthesis is regulated also by Fig. 7 Hypothetical mechanism for the role of exogenous proline in stimulation of type I collagen and HIF-1a expression. Glutamine contained in culture medium is converted in the cells into glutamate and further in mitochondria into a-ketoglutarate, intermediate of tricarboxylic acid cycle. Glutamate is also a source of proline required for collagen biosynthesis. This pathway of proline supply is sufficient to maintain high collagen biosynthesis rate. An addition of exogenous proline results in slight increase in collagen production. Glutamine deprivation from culture medium contributes to decrease in cellular proline content resulting in down-regulation of collagen biosynthesis. In such a condition, exogenous proline restores intracellular proline pool, providing substrate for collagen biosynthesis. The conversion of proline into glutamate is not significant due to low expression of proline oxidase (POX) in fibroblasts. This hypothesis is supported by proline-dependent regulation of HIF-1a transcriptional activity. Translocation of HIF-1a to nucleus is more pronounced in the absence of glutamine since a-ketoglutarate induces HIF-1a hydroxylation and ubiquitin dependent degradation. Therefore, in the absence of glutamine, a-ketoglutarate production is impaired, contributing to upregulation of HIF-1a transcriptional activity. In contrast, in the presence of glutamine, proline-induced HIF-1a transcriptional activity is attenuated. Pro proline; POX proline oxidase; Gln glutamine; Glu glutamate; HIF-1a hypoxia inducible factor-1a; aKG a-ketoglutarate; TCA tricarboxylic acid cycle a 2 b 1 integrin [12,32]. However, we found that stimulation of collagen expression in studied conditions is not dependent on a 2 b 1 integrin signaling.
The results of this study lead to conclusion that availability of glutamine, as a substrate for proline biosynthesis determines the utilization of exogenous proline for collagen biosynthesis. | 2017-10-08T05:33:01.144Z | 2017-05-19T00:00:00.000 | {
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225803956 | pes2o/s2orc | v3-fos-license | TRACE LEVEL DETERMINATION OF SODIUM CHLORIDE AND SODIUM SULFATE CONTENT IN SODIUM LAURETH SULFATE RAW MATERIAL USING COUNTER CATION-EXCHANGE HPLC WITH INDIRECT ULTRAVIOLET DETECTION
Objective: A novel study on a new liquid chromatographic approach has been developed and validated for simultaneous determination of trace level determination of sodium chloride and sodium sulfate measures its impurities using counter cation-exchange high-performance liquid chromatography with indirect ultraviolet (UV) detection.
Methods: Chromatographic separation is developed and validated on a Hamilton PRP-X100 column with a mobile phase contained a mixture of the para-hydroxybenzoic acid buffer with a pH of 9.0 and methanol. Chromatography is developed at a flow rate of 2.0 mL/min with an indirect UV determination at 310 nm at a sensitivity level of 0.5%. The optimized method was validated as per the ICH Q2 guidelines.
Results: The retention times of chloride and sulfate were about 2.8 and 7.6 min, respectively. The resolution between chloride and sulfate peaks is >4. Regression analysis confers a correlation coefficient for the stated compounds that are found to be >0.999.
Conclusion: A novel analytical method was validated as per the ICH method validation guidelines and found to be selective. Hence, the validated analytical method was precise, specific, and accurate, and it is more economic and simple for the determination of inorganic impurities.
INTRODUCTION
Sodium laureth sulfate (SLS/sodium dodecyl sulfate), 70% is a raw material, and the appearance of the material has the flowable paste/ powder form and transparent to yellowish color. SLS is the key material for the manufacture of liquid dishwashing and technical agents as well as a liquid light duty detergents. Due to its excellent foam characteristic and the natural thickening with salt, the product is also suited as a primary surfactant for cosmetic cleansing preparation such as shampoo, shower gel, and foam baths. During the processing of SLS, the material diluted with water to form gel structure which is typical of ether sulfates. After the addition of water, the viscosity first increases rather rapidly, and after a reduction of the active substance to a level below 30%, it decreases considerably. Liquid and stable solutions are obtained onto 28% of the active substance. At higher concentrations, the product becomes pasty. SLS has extremely low salt content and when diluted with water to the normal use concentration, which shows a very low viscosity which can be adjusted accordingly. In this way, the viscosity of dilute solutions of SLS with approximately 5-28% of washing active substances can be easily increased to the desired value. Sodium chloride is used as a thickening agent, and it is used to incorporate into the formulation in the cold state. The required amount of sodium chloride has to be diluted in the smallest quantity of water that is necessary for obtaining a solution (preparation of table salt solution). This salt solution is added to the undiluted SLS while stirring, and stirring is continued until the mixtures have become more fluid. Refer to the graphic abstract presented in GA1.
The literature describes only one method of determination using the conventional gravimetric method in United States Pharmacopeia (USP) [1] and the content of sodium chloride and sodium sulfate is determined by the potentiometric titration which involves precipitation titration leads to the endpoint when titrated with standardized silver nitrate volumetric solution. There were some instrumental methods published for chloride determinations in oil [2] and few other applications [3][4][5][6][7], namely, soil, paper devices, and food samples and similarly recorded for individual sulfate [8] determination. Simultaneous determination of chloride and sulfate is published in soil and some sodium bisulfite solution [9,10]. There is one harmonized method [11] of analysis that is published using high-performance liquid chromatography (HPLC) with charged aerosol detection technique. There was no other selective chromatographic method of analysis for simultaneous [12][13][14][15][16][17][18][19] determination of components of interest that was published in a unique HPLC method with ultraviolet detection. Hence, a method was developed and validated a high-performance liquid chromatographic technique with indirect ultraviolet detection for accurate and precise trace level determination of sodium chloride and sodium sulfate in SLS raw material using counter cation-exchange column.
Chemicals and types of equipment
The purity of sodium chloride and sodium sulfate used as an analytical grade (>95%) in chromatographic development. HPLC-grade methanol purchased from Honeywell, USA. High-purity water prepared using a Milli-Q Plus water purification system (Millipore, Milford, MA, USA). Hamilton PRP X100 (anion exchange) column obtained from Hamilton Inc. the USA. The chromatography technique developed with Waters HPLC Alliance (model 2698) equipped with ultraviolet (UV) detector.
Chromatographic conditions
The method was developed using column 250 mm, i.d-4.6 mm, 10 µm particle size with a mobile phase containing 0.005 M p-hydroxybenzoic acid buffer adjusted the pH to 9.90 (±0.05) with 15% NaOH solution
Katakam and Dongala
and methanol in the ratio of 1950:50 (v/v). The diluent used as MilliQ water. The flow rate of the mobile phase is 2.0 mL/min. The column and detector temperatures set at 40°C. The injection volume is 10 µL, and chromatographic run time is 10 min. The chromatogram shall be converted and processed from negative peak mode to positive peak mode which is called indirect UV determination.
Working standard preparation
The composite concentration of sodium chloride and sodium sulfate in the mixed standard solution was prepared with a final concentration of 0.5 mg/mL and 1.0 mg/mL, respectively, using the diluent as MilliQ water. Representative chromatograms of the blank and working standard preparation are shown in Figs. 1 and 2. The system suitability of the chromatographic analysis is presented in Table 1.
Sample preparation
Accurately weigh and transfer about 2000 mg of test substance into a 20 mL volumetric flask. To that, add 10 mL of MilliQ water and sonicate to dissolve and dilute to volume with diluent and mix well. The chromatogram of the control sample is shown in Fig. 3.
Method development
The initial method optimization is started using an anionic chromatography column which is supplied by Hamilton columns, and the columns are the chemical bonding phase of polystyrene divinyl benzene cross-linked with trimethyl ammonium which acts as a counter cation exchanger. The method development is started with weak organic salt primarily used as a parahydroxybenzoic acid at about 0.1 M and sample diluent used as water. The sample preparation is made at an absolute concentration of about 10 mg/mL. Due to the high foam characteristics of sodium lauryl sulfate, the dissolution of the sample is performed using sonication. Initial chromatographic data reveal that there are two negatively distorted peaks observed at differential retention times within the run time of 15 min as the exchange capacity in the column chemistry is dependent on the pH of the mobile phase. The selectivity of the weak buffer, from pH 3.0 to pH 11.0, was optimized to verify the resolution of peaks of interest and as well as its symmetry. As the mobile phase acidity decreases (pH increases), the peak of interest found to be less retention and vice versa. The column chemistry of the anion exchange chromatography, the stationary bed, has an ionically positive (+) charged surface, while the sample ions are of negative (−) charge. This technique is used almost exclusively with ionic or ionizable samples. The stronger the negative charge on the sample, the stronger it will be attracted to the positive charge on the stationary phase, and thus, the longer it will take to elute. From the optimized pH at 9.9, there is a good separation between the chloride and sulfate peaks and also eluted within 15 min of total retention. To further improve and sustain its peak symmetry, the addition of organic solvent (methanol) is optimized as a peak modifier, elutes the components of interest within 10 min. No other unknown peaks were observed from the subject material in the sample chromatogram.
Katakam and Dongala
From the organic phase optimization, 2.5% of the methanol found to be symmetric peaks of chloride and sulfate as determined as negative peaks with an indirect UV method. The peak integration is carried out using empower software to flip into a positive chromatographic scale.
Method precision
Method precision/repeatability was performed by analyzing six sample preparations of SLS, raw material spiked with sodium chloride, and sodium sulfate stock standard solution at a targeted specification level of 0.5% and 1.0%, respectively. A control sample preparation is analyzed to correct the % recovery in spiked samples with known amounts of sodium chloride, and sodium sulfate detected in the control sample. The results for % recovery of chloride and sulfate in spiked sample preparations are presented in Table 2. The representative chromatogram of control sample preparations from method precision study is presented in Fig. 4.
Specificity
The specificity of the method was demonstrated by injecting blank, individual injections of sodium chloride and sodium sulfate, and system suitability information presented in Table 1.
Accuracy
Accuracy was performed by preparing and analyzing three sample preparations of SLS, raw material spiked with sodium chloride stock standard, and sodium sulfate stock standard at specification levels 30% (limit of quantitation [LOQ]), 100%, and 150% to the target specification limits. A control sample preparation is analyzed to correct the % recovery in spiked samples with known amounts of sodium chloride and sodium sulfate detected. Accuracy was determined by spiking known concentrations of sodium chloride and sodium sulfate stock standards in the control sample. The obtained recoveries are presented in Table 2. Representative chromatograms for the accuracy sample preparations at LOQ and 150% level are shown in Figs. 4 and 5.
Limits of detection (LOD) and LOQ
The LOQ and LOD standards are prepared for sodium chloride and sodium sulfate as per the sample concentration. The percentage recovery and signal to the noise ratio at LOQ level for sodium chloride and sodium sulfate are found to be within the acceptance limit of 70.0-130.0% and not <10, respectively.
Linearity
The linearity of the method was demonstrated by injecting series of standard solutions of sodium chloride and sodium sulfate at five different concentration levels ranging from LOQ level (to 150% of the targeted specification limit. The correlation coefficient for sodium chloride and sodium sulfate found to be 0.99878 and 0.9994, respectively.
Stability of solutions
The stability of the solutions is determined to establish a duration, for which the components of interest are stable under specified storage conditions in the working standard and sample solution. The working standard and sample solutions were analyzed at an initial hour, 24 h, 48 h, and 72 h, which are stored at room temperature (RT) and refrigerated (RF) conditions. Based on the percentage recovery, results obtained from the aged sample solution and similarity factor results obtained from aged working standard solution concluded that the working standard and sample solution are stable for three (3) days at RT and one (1) day at RF, respectively.
CONCLUSION
A novel analytical method was validated as per the ICH method validation guidelines and found to meet the requirements. Hence, the validated analytical method was precise, specific, and accurate, and it is more economic and simple for the determination of inorganic impurities using HPLC with UV detection technique.
ACKNOWLEDGMENTS
The author expresses his gratitude to the Saptalis Pharmaceuticals LLC and Aurex Laboratories LLC, for providing laboratory facility for this research work. | 2020-08-20T10:08:54.412Z | 2020-05-29T00:00:00.000 | {
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243700538 | pes2o/s2orc | v3-fos-license | Detecting anomalous road traffic conditions using VGG19 CNN Model
Anomaly Detection on the real time road traffic has tremendous application possibilities in metropolitan road safety and traffic management. Due to the effect of numerous factors, for example: climate, viewpoints and road conditions in real-time traffic scene, Anomaly detection actually faces many difficulties. There are many reasons for vehicle accidents, for example: crashes, vehicle on flames and vehicle breakdowns, which exhibits distinctive and obscure behaviours. In this paper, we approached with a model to identify oddity in street traffic by monitoring the vehicle movement designs in two unmistakable
Introduction
An ever-increasing number of families presently have their own vehicles and going via vehicle has gotten an exceptionally normal and advantageous path in day-byday metropolitan life. The street condition consequently gets extraordinary consideration from the general population. Terrible street conditions can make monstrous misfortune to the social economy, and compromise the individual wellbeing of drivers out and about. With generally record the road situations by using traffic cameras, it is achievable. what's more, critical to build up a strategy to naturally discover the oddities on the streets utilizing PC vision procedures. A traffic checking framework furnished with these calculations will achieve numerous advantages and comforts. On one hand, when the inconsistencies occur, a programmed framework can advise the traffic police promptly, to tackle the oddities on streets at the earliest opportunity. Then again, when arranging an outing, data about street conditions can give accommodation to the two drivers and travellers. Notwithstanding, it is an exceptionally moving assignment to plan a PC vision calculation to recognize abnormalities in street traffic. One principal reason is that the development examples of vehicles on streets are generally exceptionally muddled, and distinctive irregular occasions may show complex practices. Simultaneously, the irregular occasion happens once in a while when contrasted with typical occasions. Accordingly, building up a productive and compelling clever calculation for programmed video abnormality location is a squeezing need. Numerous works of irregularity discovery in observation recordings must be applied to distinguish explicit strange occasions. For example, Mohammedi et al. build up a technique to distinguish human viciousness in recordings [4]. Additionally, the traffic finders just work in exceptionally restricted conditions [3]. With the improvement of traffic and video reconnaissance advancements, traffic the board frameworks dependent on video observation have gotten broadly utilized in rush hour gridlock the executives. In the canny preparing of traffic data, the discovery and acknowledgment of traffic inconsistencies, for example, securing, gridlock, car crashes, and unlawful driving have pulled in the consideration of numerous analysts because of its significance in rush hour gridlock the board. In any case, due to the intricacy of traffic camera feed, traffic reconnaissance video is helpless against outer factors, for example, light, climate, and checks. The constraints of existing picture preparing and investigation innovations make the traffic boundaries (flow of the traffic, density of traffic, vehicle direction, speed, and so forth) in light of traffic video extremely dubious. Looking the problem, we noticed it a bit difficult for manual surveillance. So, we came up with a proposal which is a CNN based surveillance system designed to learn for detecting the anomalies in dense traffic conditions, which handles of both the dynamic and static vehicles. Typically, the vehicles should continue to proceed onward the streets aside from certain ordinary conditions (e.g., hanging tight for traffic signals). Thusly, the static vehicles have higher likelihood for being anomalous occasions. By and large, the majority of the anomalous occasions in street deals will cause vehicle halting. For instance, vehicle slowing down, car crash or vehicle sticking. In the interim, the static vehicles can give us the exact area of the atypical occasions. To recognize the static vehicles from the moving traffic, we acquaint a static mode technique with get the running normal of the edge succession. Additionally, roused by the incredible accomplishment of deep learning in PC vision field, we send the deep learning-based strategy for the static vehicle identification. Additionally, we designed to further recognize the vehicle pictures of accidental pictures
Related Work
Vehicle monitoring, identification and tracking is the essential part in the street traffic scenario examination and assumes a significant part in many related applications, for example, driver help frameworks. Because of the extraordinary achievement of DL innovation, we have acquired a gigantic enhancement in image detection fields [2] [3] [4]. In this paper, we additionally experimenting the DL algorithms for detecting the vehicles and following. The detection of anomalies in both dynamic videos of the moving vehicles and static images of the traffic dataset have been concentrated in the previous years because of the expanding revenue in open security [3]. The customary techniques typically gain proficiency with the handcreated highlights to demonstrate the ordinary/anomalous occasion designs. As of late, deep learning innovation has been created for peculiarity detection as its accomplishment in the PC vision field [5].
In [6], the researchers present a generative adversarial network (GAN) based technique to identify the inconsistencies in pictures, utilizing just typical information to prepare the models. For observation recordings, there are a few endeavours to identify human savagery or strange occasions in group scenes. a deep peculiarity positioning system is used to foresee anomalies while testing on the recorded datasets. Since street condition assumes a significant part in our day-by-day life, recognizing peculiarities on streets has stood out from numerous researchers. For this undertaking, the main objective is to discover where and when the inconsistencies happen.
M. Schubert et al. [7] propose a mix of methods to foresee the event of street mishaps. The researchers present a visual examination structure for the investigation of ordinary social models and the detection of peculiar occasions. Be that as it may, the new works are intended to distinguish a particular strange occasion, and this present reality irregularities on streets are convoluted and assorted. Subsequently, we plan a novel double model strategy to distinguish different street traffic odd occasions in genuine scenes, which can have wide use practically speaking. Few more pulled in numerous specialists to direct top to bottom examination on traffic irregularity detection.
In, [8] the authors proposed a vehicle vision monitoring calculation dependent on PC vision, which had high tallying exactness and improved the precision of traffic surveillance. Frejlichowski et al. [9] proposed another vehicle direction design acknowledgment calculation dependent on the Cam shift calculation, which can precisely dissect and recognize the illicit leaving or unlawful turning of vehicles. Yang [11] successfully distinguish traffic occurrences of turnpike scenes by utilizing fuzzy logic (FL) by consolidating FL and improved steady examination calculations in their proposed system. The system breaks down occasions by extricating traffic flow data and vehicle speed, yet the system detection has few restrictions because of the intricacy of conditions of a traffic. In [10] the researchers utilized the GPS information of a hire taxi, which also including direction and velocity, to recognize gridlock on metropolitan streets. Albeit the precision of GPS strategy is high, also it has the limitations of significant expense, which compelled its application. In-order to identify these problems, another calculation that incorporates more traffic boundaries is proposed in this paper. The proposed calculation cannot just recognize traffic anomalies all the more precisely and carefully yet additionally be viable in various circumstances.
VGG19 Architecture
VGGNet is a CNN(Convolutional Neural Network) architecture proposed by Karen Simonyan and Andrew Zisserman from Oxford university in 2014. This paper principally centers around the impact of the CNN profundity on its precision. The contribution to VGG based convNet is a 224*224 RGB picture. Pre-processing layer takes the RGB picture with pixel esteems in the scope of 0±255 and deducts the mean picture esteems which are determined over the whole ImageNet training set. The input pictures subsequent to pre-processing are gone through these weight layers. The training pictures are gone through a stack of convolution layers. There is an aggregate of 13 convolutional layers and 3 fully connected layers in VGG16 engineering. VGG has more modest channels (3*3) with more profundity as opposed to having enormous channels. It has wound up having a similar powerful open field as though you just have one 7 x 7 convolutional layers. Another model of VGGNet has 19 weight layers comprising of 16 convolutional layers with 3 fully connected layers and a similar 5 pooling layers. In the two varieties of VGGNet, there comprises of two Fully Connected layers with 4096 channels every which are trailed by another fully connected layer with 1000 channels to foresee 1000 marks. The last fullyconnected layer utilizes a softmax layer for order purposes.
The walk through of 19 layers as follows: The initial two layers are convolutional layers with 3*3 channels, and initial two layers utilize 64 channels that outcomes in 224*224*64 volume as same convolutions are utilized. The channels are consistently 3*3 with step of 1. After this, pooling layer was utilized with max-pool of 2*2 size and step 2 which lessens tallness and width of a volume from 224*224*64 to 112*112*64. This is trailed by 2 more convolution layers with 128 channels. This outcomes in the new component of 112*112*128. Subsequent to pooling layer is utilized, volume is decreased to 56*56*128. Two more convolution layers are added with 256 channels each followed by down inspecting layer that diminishes the size to 28*28*256. Two more stack each with 3 convolution layer is isolated by a maximum pool layer.
Fig. 1. VGG19 Architecture
After the last pooling layer, 7*7*512 volume is straightened into Fully Connected (FC) layer with 4096 channels and softmax yield of 1000 classes.
ImageNet
ImageNet is a project which aims to provide a large image database for research purposes. It contains more than 14 million images which belong to more than 20,000 classes (or synsets). They also provide bounding box annotations for around 1 million images, which can be used in Object Localization tasks. It should be noted that they only provide urls of images and you need to download those images.
Traffic Net Dataset
The Traffic-Net dataset is a collection of traffic images that provide real-time monitoring, analytics, and alerts that will be used to provide a machine learning system with the data it needs to detect traffic conditions [12].
Using machine learning systems to accustom themselves to perception, understanding, and action in any environment is one of Deep Quest AI's goals [12].
Optimizer:
Adam is a deep learning algorithm that replaces stochastic gradient descent for the purpose of training models. The Adam algorithm combines the qualities of the AdaGrad and RMSProp algorithms to produce an optimization algorithm capable of handling sparse gradients on noisy problems. Adam can be configured quite simply, with default parameters that work out most of the time.
Proposed Method:
In this section, we are explaining the methodology followed to detect and classify the anomalies happened in various roads in various countries out of the varied traffic data-set. We are proposing an end-to-end deep learning architecture for traffic flow and anomalies detection and classification. We are experimenting using a VGG19 Convolutional Neural Network and ImageNet pretrained weights to discover the static vehicles on streets, as the abnormalities normally lead to vehicle halting. Typically, the majority of the inconsistencies on streets are unusual vehicle slowing down or vehicle crashes, which both make the vehicles stop in/alongside the street. In light of this perception, we acquaint a movement investigation strategy with discover the static vehicles which are met with accidents sometimes which get caught on fire on streets and further perceive the anomalous occasions dependent on that. The pipeline of peculiarity detection dependent on the static vehicle is introduced in Figure 2, 3, 4 5. The dataset which is properly bifurcated the data into two separate folders i.e., one is test dataset and another one is train data set. In each of those training and test dataset is categorised the four different scenarios labelled namely ³$FFLGHQW 6SDUVH WUDIILF ILUH DQG GHQVH WUDIILF´ ,QLWLDOO\ we took the training dataset to train the model to classify its type of class. In order to train the model, we read all the images in each category mentioned above in the training directory using the OpenCV library. After reading the whole dataset, the images are resized to same height and width in pixels. Here in this experiment, we are considering it as 300 x 300(height, width). To ease the process the whole dataset of images which has 3 channels of every image is converted into a binary scale image which means converting the image channel from RBG to binary image which is giving the range from Zero to One. In same way the test directory images also read and resized all the data into same shape and converted the images into binary images for validation purpose. After aligning the image data the pretrained VGG19 model and ImageNet weights are taken to create a model which is shown below in Fig.6.
Instead of building the whole convolutional network from scratch and initializing weights to create a model we have chosen the pretrained model which is VGG19. Any model or classifier would at the initial stage be able to detect the slant lines whatever the class we expect to classify as the first step, according to the intuition of choosing the pretrained model. The training of those in every possible opportunity to create a Neural network makes no sense. The project details that need to be trained will determine the abilities of the final layer of the network to identify classes. And also, there is one more advantage for using pretrained models which is pretrained models are always trained on big datasets or data which are not usually available to everyone. An example is ImageNet, which contains approximately 14 million images, 1.2 million of which are assigned to one of 1,000 categories. Thus, we would benefit tremendously from making use of these models´ >@ As with our previous experiment, instead of repeating the procedure for the first network and starting from scratch with random weight values, we can now use the saved weight values from the previous experiment as the initial weights for our new experiment. In this case, weights are initialized using pre-trained networks.
So, here we are taking the pretrained model and on top of it we are adding Flatten layer and dense layer. Using flatten layer function we flattened all the convoluted layers into a 1dimensional linear vector. Later drop out technique is used to dropout the nodes of the dense layer which is in our case having 1024 neurons in a dense layer out of it 30% of the neurons are dropped out at fully connected layer-1 and again out of 512 neurons of dense layer at fully connected layer 30% of them is dropped out. As the data we have chosen is having 4 different classes, so that the model has to classify the image of which class it belongs to. In order to do that the final layer has chosen a dense layer again and used SoftMax activation function which is used in the output layer of convolutional neural network for multi class classification problems. The model summary is shown in above figure-7. To compile the model, we have chosen the Adam optimization algorithm for stochastic gradient decent the AdaGrad and RMSProp algorithms to provide an optimization algorithm which adjusts the weights and bias of the network to help learning and reduces the loss. We have then selected a loss function for our optimization function, which is categorical cross entropy, which is typically used in multi-class classification problems. The purpose of this measure is to compare the probability distributions of two events. After the loss function the whole agenda is to find out the accuracy and loss mitigation of the model we have chosen. To do that we have used model.fit() function for fitting the model in which we are separating the whole training dataset into 64 batches and 10 epochs, which means the whole dataset into 64 parts and passed through the network for 10 iterations.
Results
We successfully implemented the VGG19 model to detect the anomalies of the road and achieved model accuracy of 95.62% and model loss of 13% in the eighth iteration and
Conclusion
In this paper we present a VGG19 CNN model-based traffic anomalies detection in urban traffic environments and achieved the highest accuracy of 95.6%. As this model is very feasible and accurate, when implements it in cloud environments and used for traffic surveillance purposes there will be a great chance of communicating the helpline at desperate situations and also helps to decreases the accident probability. | 2021-10-15T16:21:50.900Z | 2021-01-01T00:00:00.000 | {
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119627102 | pes2o/s2orc | v3-fos-license | Noncommutative Independence in the Infinite Braid and Symmetric Group
This is an introductory paper about our recent merge of a noncommutative de Finetti type result with representations of the infinite braid and symmetric group which allows to derive factorization properties from symmetries. We explain some of the main ideas of this approach and work out a constructive procedure to use in applications. Finally we illustrate the method by applying it to the theory of group characters.
Introduction
In our recent papers [Kös10, GK09, GK10] a systematical theory is emerging how representations of the infinite braid and symmetric group on noncommutative probability spaces give rise to noncommutative conditional independence and to factorization properties. These papers are rather long and, in parts, rather technical (and should be consulted for further background and further references), so it is timely to give a short introductory paper. This is done here. The diligent reader of the longer versions will notice that there are also some new turns and twists in this new presentation but our main objective is to give a readable guide for a main highway through the forest.
To achieve this we sacrifice generality at several points, for example we consider single operators as noncommutative random variables instead of the more general concept of algebra embeddings. Distributional symmetries such as exchangeability and spreadability are introduced in this setting and reformulated in terms of endomorphisms. We make the very important observation that instead of the representation of the infinite symmetric group S ∞ associated to exchangeability it is enough for the basic implications to consider a representation of the infinite braid group B ∞ instead. This observation enormously broadens the range of applicability when combined with further insights: a constructive procedure to produce braidable sequences, the equality of the tail algebra of the random sequence and the fixed point algebra of the representation (in the minimal situation), last but not least the noncommutative de Finetti theorem which derives conditional independence over the tail algebra from spreadability. Taken together we have a systematic way to derive structural properties, in particular factorization properties of the state, from symmetries.
To show some of these applications we concentrate on a specific and beautiful class of examples: the theory of characters of the infinite braid and symmetric group. We show how our theory can be used to study the left regular representation of B ∞ and we sketch some of the main ingredients of a new proof of Thoma's theorem on extremal characters of S ∞ . Open questions that can be studied with these methods, especially in the braid group setting, suggest themselves.
Tracial noncommutative probability spaces
Throughout this paper a (noncommutative) tracial probability space (A, tr) consists of a von Neumann algebra A acting on the separable Hilbert space H and a tracial faithful normal state tr : A → C.
An element x ∈ A is called a (noncommutative) random variable. If x 0 , x 1 , x 2 , . . . is a sequence of random variables in A, we write vN(x 0 , x 1 , x 2 , . . .) for the generated von Neumann subalgebra in A.
Example 1.1. This setting covers the classical case of bounded (complex or real valued) random variables x i on some standard probability space (Ω, Σ, P). More precisely, the x i 's are elements of L ∞ (Ω, Σ, P) and act by (left) multiplication on L 2 (Ω, Σ, P), the essentially bounded resp. squareintegrable Lebesgue-measurable functions on (Ω, Σ, P). Further the trace on L ∞ (Ω, Σ, P) is given by the expectation f → Ω f dP.
Example 1.2. An interesting class of noncommutative probability spaces arises in the following manner. Let G be a countable group. A positive definite function χ : G → C is called a character if χ is constant on conjugacy classes of G and normalized at the identity e of G. It is a folklore result (reviewed in [GK10], for example) that a character χ gives rise to a unitary representation π of G on a separable Hilbert space H and a vector ξ ∈ H such that Conversely, restricting a tracial state on a group algebra gives rise to a character of the group. It is well-known that A is a factor (in the sense of von Neumann algebras) if and only if χ is extremal. Note also that each unitary π(g) is a noncommutative random variable.
The following two choices for the group G in Example 1.2 are of particular interest in the sequel.
Example 1.3. The infinite braid group B ∞ is the inductive limit of the braid groups B n for n → ∞. It is presented by the Artin generators σ 1 , σ 2 , . . . satisfying the relations The Artin generator σ i and its inverse σ −1 i are presented as geometric braids according to Figure 1. We will later meet a special choice of the character, χ(g) = 0 for g = e. This choice yields for the q q q q q q probability space A the group von Neumann algebra L(B ∞ ), a non-hyperfinite II 1 factor.
We fix some additional notation as needed in the sequel. If B is a von Neumann subalgebra of A, then E B denotes the tr-preserving conditional expectation from A onto B. (Such a conditional expectation uniquely exists in our tracial setting. The non-tracial case would require an additional modular condition.) End(A, tr) denotes the tr-preserving endomorphisms of the von Neumann algebra A. The fixed point algebra of α ∈ End(A, tr) is denoted by A α . Similarly, Aut(A, tr) denotes the tr-preserving automorphisms of A.
Distributional symmetries
We are interested in certain distributional symmetries of infinite sequences of random variables. Given the probability space (A, tr), then two sequences (x n ) n≥0 and (y n ) n≥0 in A are said to have the same joint *-moments, in symbols: if, for every n ∈ N, for all i : {1, 2, . . . , n} → N 0 and ǫ 1 , ǫ 2 , . . . , ǫ n ∈ {1, * }. Note that this family of equations extends immediately to polynomials in the random variables such that they have the same joint *-distribution: tr P (x 1 , x * 1 , . . . , x s , x * s ) = tr P (y 1 , y * 1 , . . . , y s , y * s ) (s ∈ N, P ∈ C x 1 , x * 1 , . . . , x s , x * s ). We refer the reader to [NS06] for more detailed information on joint *-monomials and joint *-distributions.
Equivalent formulations of (i) to (iii) in Definition 2.1 are available in a minimal setting of the probability space. Here we meet again the infinite symmetric group, but now acting as automorphisms on the von Neumann algebra.
Proposition 2.3. Let (A, tr) be a tracial probability space and suppose
, also called partial shifts, such that The proof is not difficult. If required details may be found for (i) in [GK09, Theorem 1.9], for (ii) in [Kös10, Lemma 8.6] and for (iii) in [Kös10, Lemma 2.5]. Note that the partial shift α 0 is the shift α in the stationary setting.
The equivalent characterization in (i) provides us with a constructive procedure to obtain exchangeable sequences. Suppose we have a tracial probability space (A, tr) which is equipped with a representation of the infinite symmetric group, ρ : S ∞ → Aut(A, tr). Now choose an element x 0 ∈ n≥2 A ρ(σn) , hence x 0 satisfies (L). Then an exchangeable sequence (x n ) n≥0 can be constructed by using (PR).
Remark 2.4. This constructive procedure may yield an exchangeable sequence which does not generate A (this is evident from the choice x 0 = 1l). But we can always return to a minimal setting of the tracial probability space, by restricting the trace tr to vN{x 0 , x 1 , . . .}. Note that the sequence (x n ) n≥0 is also exchangeable after this restriction and thus also the representation ρ restricts to vN{x 0 , x 1 , . . .}. Thus any exchangeable sequence can be obtained from the constructive procedure.
Presently no constructive procedure is known to produce all spreadable sequences. We will see below that the constructive procedure for exchangeable sequences extends to a 'braided' setting where the role of S ∞ is taken by the infinite braid group B ∞ . In particular this provides us with an interesting class of spreadable sequences which may not be exchangeable. Next we motivate this 'braided' extension by providing an alternative proof of the simple fact that exchangeability implies spreadability, based on the equivalent characterizations in Proposition 2.3.
and (x n ) n≥0 the exchangeable sequence obtained from the constructive procedure. Then, for each N ∈ N, the map with k sufficiently large, satisfies (2.2) and extends to an endomorphism in ( A, tr), where A = vN(x 0 , x 1 , x 2 , . . .) and tr = tr | A .
A review of above proof shows that all algebraic arguments rely on the braid relations (B1), (B2), the localization (L) and the product representation (PR). We did not use the relations (S) of the infinite symmetric group. This motivates the following new symmetry for sequences in noncommutative probability spaces.
Definition 2.6. Let (A, tr) be a tracial probability space. The random variables (x n ) n≥0 ⊂ A are said to be braidable if there exists a representation ρ : B ∞ → Aut(A, tr) such that Here σ i denotes the Artin generator braiding the (i − 1)-th and i-th strand.
As in the case of exchangeability, the constructive procedure applies again to obtain braidable sequences. So given the braid group representation ρ : B ∞ → Aut(A, tr), choose an element x 0 ∈ n≥2 A ρ(σn) and use (PR) to obtain all x n 's. Most importantly, replacing S ∞ by B ∞ , the proof of Lemma 2.5 directly transfers to the 'braided' setting: It is easy to see that exchangeability implies braidability. Thus we can insert 'braidability' in the hierarchy of distributional symmetries of Lemma 2.2 between 'exchangeability' and 'spreadability'.
Remark 2.8. We should warn the reader that, in contrast to exchangeability, a braidable sequence (x n ) n≥0 may go along with a braid group representation ρ which does not restrict to vN(x 0 , x 1 , x 2 , . . .) in the non-minimal case.
As a by-product of the constructive procedure we obtain the following fixed point characterizations for braidable sequences. We remind that the tail algebra of a sequence (x n ) n≥0 ⊂ A is given by Theorem 2.9. Let (A, tr) be a tracial probability space and suppose that the braidable sequence (x n ) n≥0 generates the von Neumann algebra A. Then we have Here A T is the tail algebra of (x n ) n≥0 and A ρ(B∞) is the fixed point algebra of the representation ρ which implements braidability. Finally, A α is the fixed point algebra of the shift α satisfying α(x n ) = x n+1 for all n ∈ N 0 .
Proof. We will show that A T ⊂ A ρ(B∞) ⊂ A α ⊂ A T .
We start with the first inclusion. Let x ∈ A T be fixed. It suffices to show that ρ(σ k )(x) = x for any k ∈ N. After choosing the number k we approximate x ∈ A T by elements of s≥0 C x n , x * n , x n+1 , x * n+1 , . . . , x n+s , x * n+s for any fixed n. For k < i, from (B1) and (B2). This entails A T ⊂ A ρ (B ∞ ). The second inclusion A ρ(B∞) ⊂ A α follows from α(x n ) = ρ(σ 1 σ 2 . . . σ ℓ )(x n ) (for ℓ sufficiently large, compare Lemma 2.5) and a standard argument on the approximation of x ∈ A by elements of the *-algebra s≥0 C x 0 , x * 0 , x 1 , x * 1 , . . . , x s , x * s , which is weak*-dense in A.
The last inclusion is immediate from
where we have used again that A is generated by the x i 's.
Roughly speaking, the previous theorem allows us to upgrade joint *-distributions to operatorvalued joint *-distributions, by replacing the trace tr by the conditional expectation E T onto the tail algebra of a stationary sequence (x n ) n≥0 which generates A. To be more precise, let (A, tr) be a tracial probability space and (x n ) n≥0 , (y n ) n≥0 ⊂ A two stationary sequences which have the same tail algebra A T and each of them generates A. Then we write if, for every n ∈ N, for all i : {1, 2, . . . , n} → N 0 and ǫ 1 , ǫ 2 , . . . ǫ n ∈ {1, * }. Using Theorem 2.9 it follows from standard arguments as in [Kös10, Lemma 7.6] the following 'lifted' version of distributional symmetries.
Corollary 2.10. Let (A, tr) be a tracial probability space and suppose the stationary sequence (x n ) n≥0 generates A.
A braided noncommutative de Finetti theorem
The following general notion of conditional independence in an operator algebraic setting can actually be seen to arise from our main result in Theorem 3.2, a noncommutative version of the famous classical de Finetti theorem.
Definition 3.1. Given the probability space (A, tr) let N be a von Neumann subalgebra of A. The sequence ( for x ∈ vN{N , x i | i ∈ I} and x ∈ vN{N , x j | j ∈ J} whenever I and J are disjoint subsets of N 0 . Theorem 3.2. Let (A, tr) be a tracial probability space and suppose the sequence (x n ) n≥0 ⊂ A generates the von Neumann algebra A. Consider the following statements: is stationary and fully A T -independent; (e) (x n ) n≥0 is identically distributed and fully A T -independent.
Since we have already seen the implications exchangeable ⇒ braidable ⇒ spreadable ⇒ stationary ⇒ identically distributed (compare Lemma 2.2 and Theorem 2.7), the main difficulty is to show that spreadability implies full A T -independence. We refer to [Kös10] and give only a few hints and an example for one of the basic ideas in the proof.
We know already from Corollary 2.10 that spreadability implies spreadability with respect to the conditional expectation E T : for any increasing subsequence (n 0 , n 1 , n 2 , . . .) of (0, 1, 2, . . .). Let us study this property in an example to see how it produces factorization properties. We put E := E T for notational convenience. Notice that we have I < J (in the pointwise sense). We infer from spreadability (*) that x 8 ) for any k > 0. For the last equation we have used that spreadability implies stationarity. Now we pass to the mean ergodic average: and conclude with the von Neumann mean ergodic theorem that This example generalizes of course to any two monomials x = x ǫ1 i(1) · · · x ǫr i(r) and y = x ǫ ′ 1 j(1) · · · x ǫ ′ s j(s) as long as their index sets I = Range i and J = Range j satisfy I < J or I > J.
The general case of disjoint interlacing sets I and J is much more challenging to prove because the mean ergodic argument above fails in such a situation. A proof of this case needs an order-preserving refined version of the von Neumann mean ergodic theorem. Its formulation involves the partial shifts α N which characterize spreadability according to Proposition 2.3. See [Kös10, Section 8].
Characters
The investigation of representations of the infinite braid group B ∞ is a vast field with many deep results and many open questions. The results of the previous sections open up a new operator algebraic approach. As an illustration of our approach we concentrate in the following on the theory of characters.
We start from the setting of Example 1.2 with G = B ∞ and χ a character of B ∞ . The noncommutative probability space is (A, tr) where A = vN{π(g) | g ∈ G} is generated by the unitary representation π associated to χ. Further we consider the adjoint representation Can we find a braidable sequence here? A natural first idea is to try the constructive procedure described in Section 2 on the first Artin generator σ 1 . However we notice that, apart from very special cases, localization (L) (see Proposition 2.3) does not work because u 1 := π(σ 1 ) does not commute with u i := π(σ i ) for all i ≥ 2. But now a moment's reflection makes it clear how to overcome this difficulty: Instead of ρ we have to consider a representation obtained from it by shifting the Artin generators. We also include an inversion in its definition which is purely conventional but which helps us later to obtain sequences which are already known from other points of view. In short, we consider the representation ρ 1 determined on the Artin generators σ i , i ∈ N, by ) . In other words, for all x ∈ A we have (Note that, by definition, ρ 1 is a representation and hence, apart from special cases, it is not equal to the composition of ρ and inversion which is an anti-representation.) Now it follows from the commutation relations (B2) between the Artin generators that localization (L) works for u 1 and the representation ρ 1 , and it yields a braidable sequence v i := π(γ i ) i∈N where p p p p p p p p p The different formulas of the γ i 's follow from each other by the braid relations. It is clear that, conversely, one can solve for the Artin generators, so the sequence γ i i∈N also generates B ∞ . We refer to [GK09] for a discussion of the corresponding presentation of B ∞ and for connections with free probability arising from the fact that the squares of the γ i 's generate free groups. In view of that in [GK09] the γ i 's have been named square roots of free generators.
We have proved that the sequence v i = π(γ i ) i∈N is braidable and hence fully independent over its tail algebra. By Theorem 2.9 this tail algebra is equal to the fixed point algebra the relative commutant of the represented Artin generators excluding σ 1 . We note that A ρ1 contains the center of A. The center is trivial iff A is a factor iff the character χ is extremal.
If we can identify the tail algebra A ρ1 in a more concrete way then the independence gives us a lot of structural information about the noncommutative probability space (A, tr) and hence about the character χ. So far this program has been worked out only in some special cases. In the following we discuss these special cases, and from this discussion it should become clear to the reader what we have in mind when we talk of 'structural information'.
Recall that the group von Neumann algebra L(B ∞ ) is generated by the left-regular representation The canonical trace on the group von Neumann algebra is associated to a character χ given by χ(e) = 1, χ(τ ) = 0 for τ = e and so this fits into our scheme. We refer the reader to [GK09,Corollary 5.3] for a proof of the following result. (i) L(B ∞ ) is a non-hyperfinite II 1 -factor; (ii) L( σ 2 , σ 3 , . . . ) ⊂ L(B ∞ ) is an irreducible subfactor inclusion with infinite Jones index.
In particular we have Z(A) = A ρ = C = A ρ1 . In fact, the last equality is exactly how irreducibility for subfactors is defined. It follows that the sequence v i i∈N is fully C-independent in this case. Written out this amounts to the following factorization property of the trace. Another class of examples where a rather complete analysis has been achieved arises from the study of characters of the (infinite) symmetric group S ∞ . Recall that a presentation of S ∞ can be given by adding the relations (S) to Artin's presentation of the braid group B ∞ . In other words there exists a quotient map (surjective homomorphism) from B ∞ to S ∞ which, for all i ∈ N, maps the Artin generators σ i of B ∞ to the Coxeter generators σ i of S ∞ . (We don't expect any confusion from this double meaning because from now on we only deal with S ∞ .) If χ is a character of S ∞ then by composing it with the quotient map we obtain a character of B ∞ . Representations of S ∞ can be identified with those representations of B ∞ which factorize through the quotient map. This means that the definitions and results above are still available. But there are some special features of S ∞ which simplify our task. In fact, using our methods we can give a new fully operator algebraic proof of a famous classical result by Thoma (1964) which classifies all extremal characters of S ∞ .
Theorem 4.3 ([Tho64]
). An extremal character of the group S ∞ is of the form .
Here m k (σ) is the number of k-cycles in the permutation σ and the two sequences Our new proof is fully presented in [GK10]. In the following let us sketch the proof of one of the main features of the classification: Thoma multiplicativity. This becomes very transparent in our setting, so much so that it may be justified to think of Thoma's theorem itself as a noncommutative de Finetti type theorem.
The images of the square roots of free generators γ i (which we again call γ i ) turn out to be very special transpositions if we consider the defining action of S ∞ on {0, 1, 2, 3, ...} by permutations: Algebraists call these transpositions star generators of S ∞ : pictorially they connect the elements of N to the 'center' 0 of the 'star'. To prove that indeed we obtain this simple form under the quotient map it is enough to track the permutations induced by the braids in Figure 2 or in the corresponding algebraic definition. For later use we note the following elementary but important property of star generators.
The proof is left as an easy exercise. The importance is due to the following fact: Corollary 4.5 ( [GK10]). Disjoint cycles can be expressed by disjoint sets of star generators.
As before we need to identify the fixed point algebra A ρ1 to make our independence results more concrete. Let us denote by E 0 the conditional expectation onto A ρ1 and by E −1 the conditional expectation onto the center Z(A) = A ρ . Recall that Z(A) ⊂ A ρ1 .
Lemma 4.7 ( [GK10]). Let γ n1 γ n2 γ n3 · · · γ n k γ n1 be a k-cycle (as above). Then Note that, because the γ i 's are transpositions, the representing operators v i = π(γ i ) are idempotent unitaries. This will be used repeatedly without further comment. It follows that A 0 and the C k 's are selfadjoint contractions. We call these and similar objects limit cycles because they are weak limits (ergodic averages) of represented cycles. A systematic study of such limit cycles is undertaken in [GK10]. Here we develop just enough of this theory to be able to include a proof of the lemma.
Proof. We know from the discussion above that the sequence v i i∈N is fully A ρ1 -independent. Thus For n 1 = 0 we are done because γ 0 = e. To prepare the argument for the case n 1 = 0 choose an element x 0 ∈ A ρ1 and apply the constructive procedure with the (unshifted!) representation ρ to obtain a sequence x 0 , x 1 , x 2 , . . . Localization here amounts to the fact that x 0 commutes with u 2 , u 3 , . . ., this is true because x 0 ∈ A ρ1 . Hence the sequence x i i≥0 is exchangeable and, by the noncommutative de Finetti theorem, independent over Z(A) = A ρ . Explicitly, using again that x 0 ∈ A ρ1 , x n = ρ(σ n . . . σ 2 σ 1 )x 0 = ρ(σ n . . . σ 2 σ 1 σ 2 . . . σ n )x 0 = ρ(γ n )x 0 = v n x 0 v n .
With these results we can now evaluate E 0 (v n1 v n2 v n3 · · · v n k v n1 ) also for n 1 = 0. In fact, The first equality follows from independence over Z(A), the second equality follows from the fact that E −1 , as a tr-preserving conditional expectation onto the center Z(A), is a center-valued trace.
Based on this analysis we obtain a lot of structural information about characters of S ∞ without further work. We give a summary in the following theorem.
Theorem 4.8 ( [GK10]). The fixed point algebra A ρ1 = vN(A 0 , C k | k ∈ N) is commutative. Moreover the following are equivalent: (i) A is a factor; (ii) The C k 's are trivial; (iii) A ρ1 is generated by A 0 . Further A ρ1 = C iff the (subfactor) inclusion vN(S 2,∞ ) ⊂ vN(S ∞ ) is irreducible.
Proof. Any permutation is a product of disjoint cycles. Combining Proposition 4.6 and Lemma 4.7 we find that A ρ1 = E 0 (A) is generated by A 0 and by the C k 's. Because the C k 's are in the center it is clear that A ρ1 is commutative. The other statements are obtained by considering special cases. Thoma's theorem [Tho64] deals with extremal characters and hence with the factorial case. One of the most remarkable features of Thoma's classification is the fact that all extremal characters are multiplicative with respect to the disjoint cycle decomposition. We have reproduced this fact (Thoma multiplicativity) above. In fact, we have already achieved more. Our formula suggests that the explicit form in Thoma's theorem is now achievable with the help of a spectral analysis for the selfadjoint contraction A 0 . Noncommutative independence continues to be a guide for this part of the proof. We refer to [GK10] for the details. | 2011-02-03T23:25:31.000Z | 2011-02-03T00:00:00.000 | {
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213192129 | pes2o/s2orc | v3-fos-license | Sex-specific association of hyperuricemia with cardiometabolic abnormalities in a military cohort
Abstract Hyperuricemia has been associated with metabolic syndrome, and the association with various cardiometabolic risk factors may be affected by sex. We made a cross-sectional examination in a military cohort of 6738 men and 766 women, aged 18 to 50 years of Taiwan in 2013 to 2014. Hyperuricemia were defined as serum uric acid levels ≥7.0 mg/dL for men and ≥5.7 mg/dL for women, respectively. Multivariable logistic regression analyses were used to determine the associations between hyperuricemia and various metabolic abnormalities. In the overall population, hyperuricemia was associated with high blood pressure (odds ratio [OR]: 1.59, and 95% confidence intervals: 1.42–1.77), low high-density lipoprotein (OR: 1.75, 1.56–1.97), high triglycerides (OR: 2.14, 1.90–2.42), high low-density lipoprotein (OR: 1.71, 1.51–1.93), high fasting plasma glucose (OR: 1.29, 1.13–1.48), and central obesity (OR: 2.85, 2.55–3.18) after adjusting for age and serum creatinine concentrations. However, the associations with atherogenic lipid profiles including high triglycerides and high low-density lipoprotein were merely significant in men but not in women. In addition, there was a tendency for a sex difference in the association of hyperuricemia and raised blood pressure ≥130/85 mm Hg, which was greater in women than that in men (OR: 2.92, 1.37–6.25 and 1.54, 1.37–1.72, respectively; P for interaction = .059). Our findings suggest that the association between hyperuricemia and various cardiometabolic abnormalities in young adults may differ by sex, possibly due to a regulation of sex hormones and uneven effects of uric acid at the same levels between sexes on lipid metabolisms and arterial stiffness.
Introduction
Metabolic syndrome is characterized by presence of several cardiometabolic risk factors including central obesity, hyperglycemia, elevated blood pressure, elevated triglycerides, and decreased high density-lipoprotein cholesterol. [1,2] The prevalence of metabolic syndrome is increased largely with the obesity pandemic and affects 20% to 30% of the adult populations in most countries. [3,4] Previous studies have shown that metabolic syndrome is associated with the occurrence of type 2 diabetes, cardiovascular disease, and overall mortality. [2,5,6] Many unhealthy behaviors such as intake of cholesterol-rich foods, sedentary status, and tobacco smoking can result in these cardiometabolic abnormalities of the general populations. Rising epidemics of metabolic syndrome, particularly in young adults, is posing serious public health burden, and socioeconomic hazards. [7] Uric acid is a final enzymatic product of purine metabolism. Elevated serum uric acid (SUA) concentrations, also referred as hyperuricemia, mostly comes from high intake of purine-rich foods or is due to reduced urinary excretion. [8] Uric acid, partly produced by vascular endothelium has been considered to be an antioxidant with anti-atherosclerotic effect in plasma. [9] In contrast, higher SUA concentrations may stimulate the production of aminocarbonyl radicals which have proinflammatory effects on vascular smooth muscle cells, leading to arterial stiffness and insulin resistance. [10,11] In fact, in addition to being the primary risk factor of gout flares, [12] high SUA concentrations are associated with other metabolic disorders including metabolic syndrome, non-alcoholic fatty liver disease and type 2 diabetes. [10,11,13] Epidemiologic reports have shown that the incidence of both metabolic syndrome and hyperuricemia increase with aging. Many metabolic disorders such as obesity, dyslipidemia, and diabetes and the related comorbidities including hypertensive cardiovascular disease and chronic kidney disease are frequent among middle-toold age individuals, especially in postmenopausal women. [14] Lack of estrogen, a dominant female sex hormone, may play a key role in the development of metabolic syndrome in women. In addition, postmenopausal women on sex hormone replacement therapy had lower SUA concentrations and lower risk of gout flare than those who did not take hormone replacement therapy. [15] This provides a basis regarding sex hormone mediated pathogenesis between hyperuricemia and metabolic syndrome in middle-to-old age individuals. However, the association in young adults is unclear. Therefore, we aimed to examine the association of hyperuricemia with cardiometabolic risk factors by sex in a military cohort of primarily young men and women.
Study population
There were 9076 military participants enrolled in the cardiorespiratory fitness and hospitalization events in armed forces (CHIEF) study between January 2013 and December 2014. Participants who had missing relevant data were further excluded, leaving a sample of 7504 subjects for the present analysis. Of these, 6738 were men and 766 were women, aged between 18 and 50 years. The research design has been described in detail previously. [16][17][18][19][20][21] All participants received regular annual health examination in Hualien Armed Forces General Hospital of Taiwan. Each participant was asked to self-report a questionnaire including demographic factors, medical history, first-, second-, and third-degree relatives' family history, cigarette smoking habits, alcohol consumption status and betel nut chewing status. Body height, weight, waist circumference, and blood pressure were measured by standardized protocols. Physical examinations and face-to-face interviews were made by experienced nurses and physicians. Height was measured in meters (without shoes), and weight was measured in kilograms (with heavy clothing removed and 1 kg deducted for remaining garments). Body mass index was calculated as weight in kilograms divided by the square of height in meters. Obesity, overweight, normal-weight, and underweight were defined by a body mass index ≥30, 25-29.9, 18.5-24.9, and <18.5 kg/m 2 , respectively. Waist circumference was measured midway between the lower rib margin and iliac crest at standing position. Hemodynamic status of pulse rate and blood pressures were automatically measured once by the blood pressure monitor (Parama-Tech Co., Ltd, Fukuoka, Japan) over the right upper arm at sitting position, after taking a rest for at least 15 min. Overnight fasting blood specimens were collected to measure concentrations of fasting plasma glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and SUA. All these biochemical metabolic markers were analyzed enzymatically on an Olympus AU640 auto analyzer (Olympus, Kobe, Japan).
Definition of cardiometabolic risk factors
Metabolic syndrome was diagnosed as the existence of three or more of the following features: 1. central obesity: waist size ≥90 cm in men and ≥80 cm in women (ethnic-specifically for Han Chinese); 2. raised serum triglycerides ≥150 mg/dL or on lipid-lowering therapy; 3. low high-density lipoprotein cholesterol <40 mg/dL in men and <50 mg/dL in women; 4. elevated systolic blood pressures ≥130 mm Hg and/or diastolic blood pressure ≥85 mm Hg or on antihypertensive therapy; 5. high fasting plasma glucose ≥100 mg/dL or on antidiabetic therapy, according to an updated clinical criteria of International Diabetes Federation. [23] Other metabolic risk factors including blood pressures ≥140/ 90 mm Hg and low-density lipoprotein cholesterol ≥160 mg/dL with or without medical therapy were defined as hypertension and hyper-cholesterolemia, respectively. This study protocol was approved by the Institutional Review Broad of Mennonite Christian Hospital (No. 16-05-008) in Taiwan and written informed consent was obtained from all participants.
Data analysis
The baseline characteristics were expressed as mean ± standard deviation (SD) for continuous variables, and absolute and relative numbers for categorical variables. For continuous variables, we used the independent samples t test to compare the means between men and women. Wilcoxon signed rank test was used once the normality test, Kolmogorov-Smirnov test, was not fulfilled. In addition, the categorical variables were compared by chi square test. The Analysis of Variance (ANOVA) test for trend analysis was used to determine the characteristics of cardiometabolic risk factors according to SUA quartiles adjusted for age in both sexes. Finally, multivariable logistic regressions were used to determine the associations between hyperuricemia and various cardio-metabolic risk factors. In model 1, age was adjusted. In model 2, serum creatinine was additionally adjusted. Formal testing for interactions between men and women was performed. All data analyses were carried out with SAS statistical software (version 9.4, SAS Institute Inc, Cary, NC).
Descriptive characteristics and Sex differences
The baseline characteristics of both sexes are displayed in Table 1. Men had higher serum creatinine concentrations and higher prevalent alcohol intake and cardiometabolic abnormalities compared with women. Table 2 shows that the prevalence of hyperuricemia in men was three times higher than that in women. The mean SUA in men was also higher than that in women. In addition, the mean SUA and prevalence of hyperuricemia in both sexes were constant in every 6-year intervals from 18 to 50 years of age. In contrast, the prevalence of metabolic syndrome and related components increased with older ages in both men and women. Figure 1 demonstrates the distribution of SUA concentrations against frequency in men and women, respectively. Table 3 shows the results of linear trend analyses after controlling for age. In men, an increase in body mass index, waist circumference, blood pressure, serum creatinine, total cholesterol, triglycerides, low-density lipoprotein, and a decrease in highdensity lipoprotein cholesterol were found in parallel with an increase in SUA concentrations. In women, a similar trend was observed in body mass index, waist circumference, serum creatinine and blood pressure, but not seen in the lipid profile except high-density lipoprotein cholesterol concentrations. No correlation was found between SUA and fasting plasma glucose concentrations in both men and women. Data is presented as means ± standard deviations (SD) or percentages (%).BMI =body mass index; BP = blood pressure; FPG = fasting plasma glucose; HDL-C = high-density lipoprotein cholesterol; LDL-C = lowdensity lipoprotein cholesterol; TG = triglycerides; WC = waist circumference; Metabolic syndrome was defined as the presence of three or more of the following indexes: WC ≥90 or ≥80 cm in men and women, respectively; BP ≥130/85 mm Hg or on antihypertensive therapy; TG ≥150 mg/dL or on lipid-lowering therapy; HDL-C <40 or <50 mg/dL in men and women, respectively or on lipid-lowering therapy; and FPG ≥100 mg/dL or on antidiabetic therapy. * Independent samples t test was used to compare the means of continuous variables between men and women. Chi square test was used for categorical variables.
Hyperuricemia with various cardiometabolic risk factors and sex differences
Lin et al. Medicine (2020) 99:12 www.md-journal.com 1.77, 1.28, and 2.61, respectively). The results for men were consistent with that for the overall cohort. Notably, although the associations with fasting plasma glucose were significant in men and the overall cohort, probably due to a large sample size, the degree was very modest. However, the results for women show that hyperuricemia was not associated with high serum triglycerides, low-density lipoprotein cholesterol and fasting plasma glucose in model 1 (OR: 1.37, 1.19, and 1.70, respectively) and model 2. In addition, there tended to be a sex difference merely in the association between hyperuricemia and raised blood pressure ≥130/85 mm Hg (OR: 1.51 for men and 3.37 for women, P-for-interaction = .059) or hypertension (OR: 1.52 for men and 4.91 for women, P-for-interaction = .057). The tendency remained after additionally adjusting for serum creatinine (P-for-interaction for raised blood pressure and hypertension was .059 and .057, respectively).
Discussion
In a military cohort of young individuals, our study showed a sex difference in the relationship between SUA concentrations and various cardiometabolic risk factors. In both young military men and women, higher SUA concentrations were correlated with higher serum creatinine concentrations, greater values of body mass index or waist size, lower high-density lipoprotein concentrations, and higher blood pressure values. However, sex-stratified analysis showed that hyperuricemia was associated with higher risk of elevated fasting plasma glucose, low-density lipoprotein, and triglycerides merely in men but not in women. We further uncovered that the extent of the association between hyperuricemia and raised blood pressure significantly differed by sex, which could not be explained by age and kidney function. Obesity plays a fundamental role between SUA and other cardiometabolic risk factors in both sexes. An experimental Table 2 Sex-specific mean serum uric acid levels and prevalence of hyperuricemia, metabolic syndrome and related components stratified by age categories. Table 3 Anthropometric and biochemical characteristics according to sex-specific quartiles of serum uric acid levels. study of mouse models has uncovered that uric acid could be produced and secreted from adipocytes, which were abundant and had higher xanthine oxidoreductase (XOR) activity in obesity. [24] In another study, [25] uric acid induced monocyte chemotactic protein-1, a pro-inflammatory adipokine, production in vitro evidenced by mRNA expression within adipocytes in obesity. In addition, uric acid decreased the production of adiponectin, an anti-inflammatory agent and insulin sensitizer, for adipocytes. Using allopurinol to inhibit XOR could lower SUA concentrations in obese mice and thereby reduce macrophage infiltration in the adipose tissue and improve insulin resistance. Accordingly, obesity might be a moderator of hyperuricemia to abnormal lipid profiles, hyperglycemia, and elevated blood pressure.
Estrogens may play a critical role in modification of the relationship between hyperuricemia, dyslipidemia, and prediabetes in women. The National Health and Nutrition Examination Survey showed premenopausal women had lower serum triglycerides, non-high-density lipoproteins, and uric acid compared with men of similar ages. [26] Estrogens have uricosuric effect on kidney to excrete uric acid, [27] and regulate lipid metabolism in adipose tissues, muscle, and liver. More triglycerides-rich very low-density lipoproteins are secreted by liver to prevent hepatic fat accumulation and free fatty acids clearance rates of muscle are increased in young women, [28,29] which reduces atherogenic lipid levels and hyperuricemia mediated inflammatory process with obesity. [30] In addition, the protective effect of estrogens on hyperuricemia from prediabetes or diabetes has been reported in young women in previous studies. [31,32] Previous studies have uncovered that in young adults of the general population, the association between SUA and blood pressure was stronger in women than in men. [33] Also, the relationship was found greater in women than in men of the elderly, [34] implying that SUA might have an adverse effect on the arterial vasculatures to elevate blood pressure, which is not influenced by estrogens. [35] Uric acid inhibits nitrite oxide, and induces C-reactive protein and major reactive oxygen species productions in smooth muscle and vascular endothelial cells, resulting in cell proliferation, endothelial dysfunction, reninangiotensin-aldosterone system activation, and vascular contractility impairment. [36][37][38] Several cohort studies have shown that SUA were dose-dependently associated with maximal intima-media thickness and peripheral arterial stiffness among apparently healthy individuals and those at high vascular risk. [33,39,40] In addition, most of these studies reported that the SUA relationship with arterial stiffness or hypertension in men was null or weaker than that in women. [33,35,39,40] This sex difference could be reasoned partially by that at higher SUA levels, women may have greater xanthium oxidase activity and reactive oxygen species productions, resulting in severer renal vascular inflammation, arterial stiffness, and elevated blood pressure as compared with men. [36][37][38] There were a number of strengths in this study. First, the sample size of this study was large enough to provide sufficient power for sex-specific analyses. Second, although 1572 participants were excluded for missing data initially, the demographic characteristics were similar to the study population, reducing the selection bias (supplemental Table 1). Third, all measurements were standardly performed in one referral hospital to avoid potential bias. Several limitations should be denoted. First, our study was a cross-sectional design, which restricts the temporal relationship of SUA with cardio-metabolic risk factors. Second, although the association of SUA with triglycerides and lowdensity lipoprotein was null in women, the sex difference was not statistically significant, requiring more investigations. Third, our cohort was confined in the military of Taiwan; therefore, the results might not be proper to apply to the general public.
Conclusion
Our findings suggest that the relationship of hyperuricemia with various metabolic abnormalities in young adults may differ by sex. There tended to be a sex difference in the association between hyperuricemia and raised blood pressure, which was greater in women than in men, possibly related to a regulation of female sex hormones and uneven effects of uric acid at the same levels between sexes on lipid metabolisms and arterial stiffness. | 2020-03-21T13:06:46.855Z | 2020-03-01T00:00:00.000 | {
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256901121 | pes2o/s2orc | v3-fos-license | Metrical results on the geometry of best approximations for a linear form
Consider the integer best approximations of a linear form in $n\ge 2$ real variables. While it is well-known that any tail of this sequence always spans a lattice is sharp for any $n\ge 2$. In this paper, we determine the exact Hausdorff and packing dimension of the set where equality occurs, in terms of $n$. Moreover, independently we show that there exist real vectors whose best approximations lie in a union of two two-dimensional sublattices of $\Z^{n+1}$. Our lattices jointly span a lattice of dimension three only, thereby leading to an alternative constructive proof of Moshchevitin's result. We determine the packing dimension and up to a small error term $O(n^{-1})$ also the Hausdorff dimension of the according set. Our method combines a new construction for a linear form in two variables $n=2$ with a result by Moshchevitin to amplify them. We further employ the recent variatonal principle and some of its consequences, as well as estimates for Hausdorff and packing dimensions of Cartesian products and fibers. Our method permits much freedom for the induced classical exponents of approximation.
Best approximations in small sublattices
Let ξ = (ξ 1 , . . ., ξ n ) ∈ R n and for simplicity consider the maximum norm on R n denoted by . .For convenience we introduce the notation ξ * = (ξ, 1) ∈ R n+1 .A classical topic in Diophantine approximation is to study small absolute values of a linear form (1) q • ξ * = q 1 ξ 1 + • • • + q n ξ n + q n+1 , for non-zero integer vectors q = (q 1 , . . ., q n+1 ).Here small means compared to q , where x ∈ R n denotes the restriction of x ∈ R n+1 to its first n coordinates.We assume (1) does not vanish for any integer vector q = 0, and then call ξ totally irrational.We call a vector q ∈ Z n+1 a best approximation for ξ if where the minimum is taken over all b ∈ Z n+1 with norm of b = 0 at most q . 1 They are unique up to sign for totally irrational ξ.Considering the set of all best approximations Middle East Technical University, Northern Cyprus Campus, Kalkanli, Güzelyurt johannes@metu.edu.tr ; jschleischitz@outlook.com. 1 We follow the classical definition as for example in [14], that indeed uses the norms of the restricted integer vectors without last coordinate (constant term).When omitting the "hat", even though q ≍ ξ q , the concrete sequence may change in some cases.Our results are valid with either definition.
with norms in increasing order gives rise to the sequence q j ∈ Z n+1 , j ≥ 1, of best approximations associated to ξ with the properties Let us adapt the notation R(ξ) from [14] to denote the minimum integer R so that some tail of the best approximations (q j ) j≥j 0 lies in an R-dimensional sublattice L = L(ξ) of Z n+1 that may depend on ξ.It is well-known that for n ≥ 2 and totally irrational ξ ∈ R n , we have R(ξ) ≥ 3. See [13,Theorem 1.2] for the claim with its proof sketched in the same paper [13, § 1.3], and [14,Theorem 7] for generalizations to a system of linear forms.On the other hand, Moshchevitin [13] showed that the following sets are not empty.
Definition 1.For n ≥ 2 an integer, let Γ n be the set of all totally irrational ξ ∈ R n inducing R(ξ) = 3.
By the above observation, the set R 2 \ Γ 2 is a countable union of rational affine hyperplanes, hence we may assume n ≥ 3.More precisely, denoting by dim H and dim P the Hausdorff and packing dimension respectively, Moshchevitin's refinements [14, Theorems 12 & 13] of his own result directly imply the following fact.
Theorem 1.1 (Moshchevitin).We have dim In our first new result, relying on auxiliary results from [14] and [4,5], we determine the exact Hausdorff and packing dimension of the sets Γ n .
Theorem 1.2.The Hausdorff dimensions of Γ n are given as (2) dim and their packing dimensions as The lower bounds follow relatively easily from combining observations from [4,5,14], together with some metrical theory of Cartesian products.The upper bounds require more work, especially the three-dimensional sublattice in the definition of Γ n being arbitrary causes our proof to become technical.Define the uniform exponent of approximation with respect to a linear form as and the ordinary exponent of approximation Then by Dirichlet's Theorem for any ξ ∈ R n we have (4) ω(ξ) ≥ ω(ξ) ≥ n.
Generalizing the proof strategy of Theorem 1.2, we can obtain similar results on best approximations in sublattices of higher dimension k.It turns out that the packing dimension of the accordingly defined sets does not increase up to k = n.
The main focus of this paper is to study the following problem on best approximations for a linear form.
Problem 1.For n ≥ 2, does there exist totally irrational ξ ∈ R n so that some tail of best approximations (q j ) j≥j 0 lies in a finite union of two-dimensional sublattices of Z n+1 ?If so, determine the minimum possible number N = N(n) of sublattices.Determine/Estimate the Hausdorff and packing dimensions of these sets as functions of n.
Clearly N ≥ 2 for n ≥ 2 by the fact R(ξ) ≥ 3 for totally irrational ξ recalled above.
Remark 1.For simultaneous approximation, the answer is negative, as any hyperplane contains only finitely many best approximations as soon as ξ is totally irrational.Moreover, in either linear form or simultaneous approximation problem, clearly any onedimensional subspace contains at most one best approximation vector for given ξ, if so its (up to sign) unique integer vector with coprime coefficients.
Remark 2. As pointed out to the author in private communication by N.G.Moshchevitin, with some effort a positive answer to Problem 1 (omitting the metrical aspects) with the optimal constant N = 2 can be derived from the lemma in [10] and its proof.This lemma essentially states the following: Let G ⊆ Z n be a set of integer vectors that is not contained in a set of the form ℓ ∪ F , where ℓ is a line and F a finite set, in R n .Then there is totally irrational ξ ∈ R n such that for integer vectors within the set G * = {(g, h) : g = (g 1 , . . ., g n ) ∈ G, h ∈ Z} ⊆ Z n+1 we find very small linear forms |q • ξ * | for certain q ∈ G * (implying q ∈ G), occurring with some density that in particular admits to ask for ω(ξ) = ∞.Taking G the union of two non-collinear rational one-dimensional sublattices (lines) ℓ 1 , ℓ 2 of Z n , its embedding G * in Z n+1 lies in the union of the two two-dimensional sublattices ℓ 1 , e n+1 Z and ℓ 2 , e n+1 Z of Z n+1 .As pointed out to the author by N.G.Moshchevitin, with some cumbersome additional geometrical arguments one can guarantee that these integer points indeed form some tail of the best approximations associated to ξ.However, this is not explicitly carried out in the short note [10] (nor in later work) as it was not of relevance in that paper.In our alternative construction regarding Problem 1 below (proof of Theorem 2.1), we fix the lines ℓ i = e i Z , i = 1, 2 as the first two coordinate axes of R n and provide an explicit, rather elementary argument of this fact for certain ξ, based on Minkowski's Second Convex Body Theorem.Moreover, we address the metrical problem.
On Problem 1
2.1.Main new results.As indicated in Remark 2, we give an almost complete answer to Problem 1. Indeed, we show that for arbitrary n and the maximum norm the answer is positive, and the optimal bound N(n) = 2 can be reached for any n ≥ 2.Moreover, we may choose the two two-dimensonal sublattices so that they span a lattice of dimension only three in Z n+1 .Thereby we recover Moshchevitin's result that R(ξ) = 3 can be reached, i.e.Γ n = ∅, with a new proof, that we consider easier than the original one from [13].
To state our result in full generality, we need to introduce some notation.Let us first define the two-dimensional sublattices of Z n+1 given by i.e. the Z-span of the i-th and the (n + 1)-st canonical base vector in R n+1 .For the immediate concern of Problem 1, we will only need H 1 and H 2 .Let us define the following properties of ξ ∈ R n with induced sequence of best approximations (q j ) j≥1 : (i) ξ is totally irrational (ii) Some tail (q j ) j≥j 0 lies in the union of the two-dimensional sublattices H 1 and H 2 of Z n+1 .(iii) Some tail (q j ) j≥j 0 lies in the three-dimensional sublattice (iv) Large best approximations lie in H 1 and H 2 alternatingly, i.e. for any j ≥ j 0 (v) For j ≥ j 0 , three consecutive best approximations q j , q j+1 , q j+2 are linearly independent, thus q j , q j+1 , q j+2 R ∩ Z n+1 has full dimension three in e 1 , e 2 , e n+1 Z (vi) For w ∈ [n, ∞], we have We comment on the conditions and their mutual relations in § 2.2 below.We want to remark that we expect Theorem 2.1 below to remain true when replacing e 1 in H 1 and e 2 in H 2 by any pair of linearly independent integer vectors in the two-dimensional subspace of R n+1 defined by x 3 = • • • = x n+1 = 0.This is supported by [10], see Remark 2 above.Definition 2. Let n ≥ 2 be an integer.Define Θ n ⊆ R n as the set of ξ ∈ R n for which conditions We can now finally state the following rather satisfactory partial answer to Problem 1.
In particular Θ n = ∅ and N = 2 can be reached in Problem 1. Conversely, we have In view of (8), estimates (12) are an obvious consequence of (2) from Theorem 1.2.The bound (11) can be improved with some effort, see Remark 5 below.On the other hand, we are unable to provide a non-trivial upper bound for dim P (Θ 2 ).Comparing the bounds of Theorem 1.2 and Theorem 2.1 yields the following corollary.
The following problem remains open.
An explicit lower bound slightly exceeding n−2+1/(n 2 +1) for the Hausdorff dimension of Θ n can be readily deduced from the proof of Theorem 2.1 below, see formula (62).Inserting small n in this strengthened bound (62), the decimal expansions start with (13) dim See also Remark 8 below on bounds for Hausdorff or packing dimension when additionally restricting ω(ξ), complementing the left estimate of (9).Our method suggests the following refinements of (9) (and (62)).
See Remark 7 in § 9.4 below for more details.For small n, Conjecture 1 would yield considerable improvements of (13).While the right inequality is again strict, asymptotically our results suggest just a small improvement, with lower bound still of order n − 2 + 2/n 2 − O(n −4 ).We wonder if the special choice of H 1 , H 2 in the sets Θ n (w), Θ n are significant.This is constituted in the following more general problem.Problem 3. Do the lower bounds of Theorem 2.1 (possibly Conjecture 1) hold for the set of ξ ∈ R n with the property that some tail of best approximations (q j ) j≥j 0 lies in a union of any two fixed two-dimensional sublattices of Z n+1 ?Do the upper bounds of Theorem 2.1 (possibly Conjecture 1) hold for the set of ξ ∈ R n with some tail (q j ) j≥j 0 in a union of any two (or finite?) two-dimensional sublattices of Z n+1 , independent of ξ?
It seems the method of § 5.3 can be used to verify the second part of Problem 3 for the special case of the two sublattices jointly spanning a three-dimensional lattice in Z n+1 .This may be a necessary and sufficient criterion for the lower bounds as well.
The main substance of Theorem 2.1 are the lower bounds.In short, to prove (9), we combine a new construction for n = 2 with a result by Moshchevitin [14,Theorem 12].It follows that a "generic" vector in R n−2 , in sense of Lebesgue measure, gives rise to vectors in Θ n (w) by adding two more suitable real components.This further directly implies the lower bound n − 2 for the Hausdorff dimension of the sets Θ n (w).With some refined argument and using metrical results by Sun [16] and by Das, Fishman, Simmons, Urbański [4,5] we find the stronger lower bounds in (9).The upper bound (11) not implied by Theorem 1.2 follows independently from a classical formula by Jarník [8] and the theory of continued fractions.
By small modifications of the proof of Theorem 2.1, we can obtain best approximations ultimately lying in a union of k two-dimensional sublattices of Z n+1 , that together span a (k + 1)-dimensional space, but in no smaller number of two-dimensional subspaces.We want to explicitly state this generalization of the case k = 2 of Theorem 2.1, but avoid detailed metrical formulas for brevity.Theorem 2.2.Let n ≥ 3.For any 2 ≤ k ≤ n, there exists a set of Hausdorff dimension strictly greater than n − k, consisting of ξ ∈ R n with property (i) and (ii * ) Some tail of the best approximation sequence (q j ) j≥j 0 lies in the union . ., e k , e n+1 Z ⊆ Z n+1 .(vii) No tail (q j ) j≥j 1 lies in union of less than k two-dimensional sublattices of Z n+1 , similarly no tail is contained in a sublattice of dimension k or less.
In fact analogues of all (i)-(vi) hold for the ξ in Theorem 2.2.For fixed n, it is natural to expect that the Hausdorff dimension of the set in Theorem 2.2 increases with k.This is not reflected in the claim.For k = n, the according set has full n-dimensional Lebesgue measure (a rigorous argument for this follows from similar method in § 5.2 below).An immediate corollary of Theorem 2.2 reads as follows.
Probably Corollary 2 could be derived independently from ( 6), (7) upon determining (estimating) the involved Hausdorff dimensions.For sake of completeness, we end this section with estimating the size of the set Y n,k of ξ ∈ R n inducing infinitely many best approximations in any finite union of k-dimensional sublattices L 1 , . . ., L i(ξ) , depending on ξ, of Z n+1 .The proof is not complicated.
Theorem 2.3.Let n ≥ k ≥ 2 be integers and Y n,k be as above.Then For n = k = 2 the upper bound becomes 5/3.One may compare this with the smaller bound 7/5 from (11) for the smaller set Θ 2 ⊆ Y 2,2 dealing with a special collection of two-dimensional sublattices in which all but finitely many best approximations lie.Problem 4. Are the packing dimensions of Y n,k full?What if we instead require all large best approximations to lie in L 1 , . . ., L i(ξ) ?2.2.On the conditions (i)-(vi).Clearly (iv) ⇒ (ii) ⇒ (iii) and (iv) ⇒ (i).Moreover (iv) ⇒ (v) by the observation on one-dimensional subspaces from Remark 1.So (i), (ii), (iii) and (v) are rather stated for sake of completeness.Moreover, the theory of continued fractions and Dirichlet's Theorem (4) easily imply that conversely (ii) ⇒ (iv), see the proof in § 8.So (ii) ⇔ (iv).We want to comment on (vi) in the light of Theorem 1.1.The subset of vectors within Γ n originally constructed by Moshchevitin in [13] have the property ω(ξ) = ∞.Thus they form a set of Hausdorff dimension at most n − 2 in view of [5,Theorem 3.6], so the metrical claim in Theorem 1.1 cannot be improved in this way.However, using the refinements from [14] together with some new idea for linear forms in two variables, we will find ξ satisfying (i)-(v) and with finite uniform exponent of approximation.This enables us to surpass this treshold value n − 2 for the Hausdorff dimension even for the smaller sets Θ n ⊆ Γ n in Theorem 2.1.
A structural result on Γ n
In the proof of Theorem 1.2, we show via [14,Theorem 12] the following: For any for the smaller set Γ n with special choice of the three-dimensional lattice e 1 , e 2 , e n+1 Z ).The sets F n (ξ 1 , ξ 2 ) are hereby implicitly derived from the convergence part of the Borel-Cantelli Lemma within the proof in [14].In our last new result, we provide an explicit set for F n independent of the choice of ξ 1 , ξ 2 in terms of Diophantine properties, upon increasing the lower bound on the uniform exponent to 3n − 4.
Theorem 3.1.Let n ≥ 2. For any vector ξ = (ξ 1 , . . ., ξ n ) in the set the tail of best approximations lies in the three-dimensional sublattice L n,3 = e 1 , e 2 , e n+1 Z of Z n+1 .In particular See also Remark 10 below for refinements.From Theorem 3.1 together with metrical results from [4,5], we get another proof for the lower bound n − 1 for the packing dimension of Γ n .For its Hausdorff dimensions, using a classical result of Khintchine [9] and again [4,5], we can deduce from Theorem 3.1 the lower bound n − 2 + 2/(3n − 4) for n ≥ 3, weaker than the bound in Theorem 1.2.In fact both claims again hold for Γ n .While there are some similarities underying the fundamental ideas of Theorem 3.1 and [14, Theorem 12], the proofs differ considerably.Theorem 3.1 uses Minkowski's Second Convex Body Theorem instead of the Borel Cantelli Lemma.
The choice of the maximum norm in our new results is just for convienence, we may establish the analogous result for a large class of norms, including all p-norms ( |x i | p ) 1/p , by small modifications of the proof.In fact, at least in Theorems 1.2, 3.1, we may take any norm.
4.
1.An auxiliary result by Moshchevitin.We recall a partial result of [14,Theorem 12].In the notation of [14], its special case n = 1, m = 2 and m * equal to our present n, yields: Theorem 4.1 (Moshchevitin).Let n ≥ 3.If q j = (q j,1 , q j,2 , q j,3 ) ∈ Z 3 is the sequence of best approximations for (ξ 1 , ξ 2 ) ∈ R 2 and we have then for almost all choices of remaining entries (ξ 3 , . . ., ξ n ) ∈ R n−2 with respect to (n − 2)-dimensional Lebesgue measure, for the vector ξ = (ξ 1 , . . ., ξ n ) and some j 0 (ξ), the embedded sequence q j,1 e 1 + q j,2 e 2 + q j,3 e n+1 = (q j,1 , q j,2 , 0, . . ., 0, q j,3 is the tail of the sequence of best approximations. In fact, the logarithmic factor in ( 14) is only required when n = 3.Since the norms of best approximations grow exponentially [3], we see that the condition (14) holds as soon as for some ǫ > 0 we have It is not hard to see that this is in turn satisfied if ( 15) Note that any ξ ∈ R n arising from Theorem 4.1 is automatically totally irrational as soon as ξ 1 , ξ 2 has this property (otherwise the sequence of best approximations for ξ would terminate).
However, we will require the more general properties of Lemma 4.2 in place of (16).Conversely, the upper bounds (17) dim hold, where the non-obvious left estimates are again part of [18,Theorem 3].The full claim of [18,Theorem 3] summarizes the properties ( 16), ( 17), (18) in short as
Proof of Theorem 1.2
Let us immediately introduce a subset of Γ n of relevance below.
Definition 3. Let Γ n ⊆ R n be the set of ξ ∈ Γ n for which the three-dimensional lattice from the definition of R(ξ) can be chosen It is obvious that Γ 2 = Γ 2 which is just the set of totally irrational ξ ∈ R 2 , as well as
Proof of lower bounds.
A key observation is that the work of Das, Fishman, Simmons, Urbański [5, Here we implicitly restrict to (ξ 1 , ξ 2 ) totally irrational.They further provided a different, more complicated, explicit formula for w < 2 + √ 2 as well that we want to avoid stating.Formula (20), but not the formula for n = 3, follows alternatively from the independent paper [2] and Jarník's identity that relates one linear form with simultaneous approximation for two variables.
On the other hand, by the observations in § 4.1, via using Theorem 4.1, we may apply (I) of Lemma 4.2 with M = Γ n , and parameters n 1 = 2, t = n 2 = n − 2 and s = V(n).Note hereby that any arising ξ is indeed totally irrational by the concluding remark in § 4.1.By (19) and Lemma 4.2 we infer the lower bound √ 2 and after some simplifications of the according formula when n = 3 < 2 + √ 2, the right hand side becomes the respective values in (2).
Regarding packing dimension, it follows directly from [5, Theorem 3.10] that the packing dimension of the set involved in (20) is at least 1 for any w ≥ 2, with equality for w ≥ 3. Combined with (II) of Lemma 4.2 for the same parameters n i , t as above, indeed for n ≥ 3.For n = 2 the claim is obvious.Remark 3.An alternative proof of the bound for the packing dimension follows from (19) and the stronger claim dim P (Θ n ) ≥ n − 1 proved in § 9.5 below.
5.2.
Upper bounds: Special three-dimensional lattice.We first show the upper bounds for the smaller set Γ n where the three-dimensional lattice containing all large best approximations is just e 1 , e 2 , e n+1 Z .From the definition of Γ n and by Dirichlet's Theorem (4), we see that any Combined with (17), we get As previously noticed, by [5, Theorem 4.9] the right dimension is again V(n) as for the sets in (20) where strict inequality is imposed.This proves the reverse upper bound for the Hausdorff dimension of the sets Γ n .
Combining (21) with ( 18), we get that where the last identity is again due to [5, § 3.3].The reverse lower bound n − 1 for dim P ( Γ n ) (thus also for dim P (Γ n )) for n ≥ 2 was already shown in § 5.1, hence identity (3) is proved for the smaller sets Γ n .
5.3.Upper bounds: General case.We settle the upper bounds for the larger sets Γ n where the three-dimensional integer lattice is arbitrary.The main idea is to apply rational automorphisms of R n+1 to reduce it to the special case of § 5.2.Our proof below performing this in detail is reasonably lengthy and may not be the easiest available.
First notice that since there are only countably many three-dimensional sublattices of Z n+1 and by sigma-additivity of measures, it suffices to show that for any fixed threedimensional sublattice L of Z n+1 , the set of ξ ∈ R n inducing some tail of best approximations in L, has Hausdorff and packing dimension at most as in Theorem 1.2.Denote by Γ n (L) ⊆ Γ n ⊆ R n this set for any fixed given sublattice There is a bijective linear map f L : R n+1 → R n+1 induced by some integer matrix A L ∈ Z (n+1)×(n+1) that maps L to the particular sublattice e 1 , e 2 , e n+1 Z , as in Γ n .To see this, we extend a Z-basis of L to any vector basis of R n+1 consisting of integer vectors, then map the three Z-base vectors of L to e 1 , e 2 , e n+1 respectively, then extend it to an automorphism of R n+1 by mapping the remaining n − 2 integer base vectors to e 3 , . . ., e n , and finally multiply the arising rational matrix by the common denominator.Denote by g L : R n+1 → R n+1 the adjoint map of the inverse f −1 L of f L .Since g L is an automorphism as well, it is bi-Lipschitz and thus preserves Hausdorff and packing dimension [6].Hence if we write that just equals the ξ → ξ * map, we have We will bound the dimensions for the left hand side image sets.
Define an affine and a linear hyperplane of R n+1 , parallel to each other, by Define further a map ∆ : Note that ξ * = ι(ξ) ∈ A for any ξ ∈ R n and ∆ is just the identity on B. Obviously ∆ maps R n+1 \ B onto A. We claim that when restricting its domain to g L (A) \ B ⊇ g L (Γ L ) \ B, it is injective.Indeed, clearly g L (A) is an affine but not a linear subspace (as the image of an affine, non linear subspace under an automorphism).Thus it has only a singleton as intersection with any line through the origin, proving the claim in view of the definition of ∆.Hence, as Γ L ⊆ A and thus g L (Γ L ) ⊆ g L (A), and as ∆ is locally bi-Lipschitz on R n+1 \ B, writing g L (Γ L ) \ B as a countable union of sets with last coordinate bounded away from 0 in absolute value, by an easy sigma-additivity argument for measures, we have dim On the other hand, on B the map ∆ is just the identity.Thus together with (22) we easily conclude that Obviously the same identities hold when restricting the left hand side sets, containing only vectors with last coordinate either 0 or 1, to the first n coordinates (i.e.chopping off the last coordinate).In other words, if we let the projection that reverses ι, denoting this projected set by Therefore it suffices to bound from above the Hausdorff and packing dimensions of U L as in Theorem 1.2.
Let ξ ∈ Γ n (L) so that ξ * ∈ Γ L be arbitrary, and q ∈ L ⊆ Z n+1 .Then by definition of g L we have and by construction in particular it is an integer vector.Moreover, as bijective linear map f L is bi-Lipschitz so that (26) f L (q) ≍ q , with some absolute implied constants.Write g L (ξ * ) = (ζ 1 , . . ., ζ n+1 ) and let By (24) we have In any case, we get that where the implied constant is absolute on sets where Combining (26), ( 27) and as we may choose q best approximations for ξ and by definition of ζ, we get that ω(ζ) ≥ ω(ξ) ≥ n.
By the special form (25) of the integer vectors f L (q), it is further clear that the projection of ζ to the first two coordinates has the same property, i.e. ( For v > 1, define parametric subsets of U L given as where ζ n+1 is the last coordinate of g L (ξ * ) as above.Then ∆ is bi-Lipschitz on any X v .Thus again by the invariance of Hausdorff and packing dimension under bi-Lipschitz maps, for any v > 1, the quantities dim H (U L (v)) and dim P (U L (v)) can be estimated as in § 5.2 by precisely the same argument via (28) and ( 17), (18).Since we may write as a countable union of such sets, again by sigma-additivity of measures the same estimates hold for U L and finally in view of (23) for Γ n (L ) as well.
Remark 4. We cannot conclude that f L (q) ∈ Z n+1 are best approximations for ζ = π(∆(g L ((ξ * ))) ∈ R n .However, it suffices for the argument that they induce approximations of order at least n.
Sketch of the Proof of Theorem 1.3
By a slightly more general version of Theorem 4.1 from [14], again for any element of {(ξ 1 , . . ., ξ k−1 ) ∈ R k−1 : ω(ξ 1 , . . ., ξ k−1 ) > n}, we get some full measure set Here Γ n,k ⊆ Γ n,k is defined likewise as Γ n = Γ n,3 from § 5 with respect to the k-dimensional lattice L n,k := e 1 , . . ., e k−1 , e n+1 Z .Conversely, very similarly as in § 5.2 we get Combining these properties with (I) of Lemma 4.2 and (17) yield the claims ( 6), (7) on the Hausdorff dimension for the smaller sets Γ n,k .Very similarly as in § 5.3, via rational automorphisms that map a given k-dimensional rational lattice L ⊆ Z n+1 to L n,k , we lift the upper bound to the larger set Γ n,k .
Regarding packing dimension, we have as can be seen via [5, Theorems 3.8 & 4.9] with a short calculation.Thus, using the above observations on Γ n , we conclude with part (II) of Lemma 4.2 where n 1 = k − 1, n 2 = t = n − k + 1 (for lower bounds) and ( 18) (for upper bounds) that Finally again similarly as in § 5.3 we can lift the upper bound to the sets Γ n,k , the reverse inequality being a trivial consequence of (29), hence (5) holds.
Proof of Theorem 2.3
The lower bounds are clear by Theorem 2.1, we need to prove the upper estimates.Since there are only countably many finite subsets of sublattices of Z n+1 and by sigmaadditivity of measures, there is a subset Z n,k ⊆ Y n,k with the property that dim H (Y n,k ) = dim H (Z n,k ) and so that the finite collections of k-dimensional sublattices L 1 , . . ., L i(ξ) of Z n+1 are the same for any ξ ∈ Z n,k .So assume this set of lattices L 1 , . . ., L i is fixed.Clearly by pigeon hole principle for any ξ ∈ Z n there is some lattice L j , j = j(ξ) ∈ {1, 2, . . ., i} containing infinitely many best approximations.Again by additivity of measures, it suffices to treat the case where this lattice is the same for any ξ ∈ Z n,k .Without loss of generality we can assume it is L := L 1 .However, then within L we find infinitely many vectors inducing approximations of order > ω(ξ) − ε.If L = H 1 = e 1 , . . ., e k−1 , e n+1 Z , then it follows from Dirichlet's Theorem (4) that ω(ξ 1 , . . ., ξ k−1 ) ≥ ω(ξ) ≥ n for any ξ = (ξ 1 , . . ., ξ n ) ∈ Z n,k .Otherwise, we extend any rational linear map sending any base of L bijectively to e 1 , . . ., e k−1 , e n+1 to a rational automorphism of R n+1 and argue very similarly as in § 5.3 to get a set of the same Hausdorff dimension dim H (Z n,k ) where this is the case.Hence (17) and a formula generalising a classical result of Jarník [8] to higher dimension, see for example [1], imply
Proof of Theorem 2.1: Upper bounds
The upper bounds in ( 12) and ( 10) follow immediately from Theorem 1.2 and (8).We are left with the proof of (11).For this we use a different strategy.We show that for any If this is true then a classical metrical formula by Jarník [8] and (17) indeed imply Let q = (q 1 , q 2 , q 3 ) ∈ Z 3 be a best approximation of large norm for ξ.Without loss of generality we can assume q ∈ H 2 , thus q 1 = 0. Let µ be implictly defined by Then by Dirichlet's Theorem (4) and since q is a best approximation, we have µ ≥ 2. Then −q 3 /q 2 is a convergent to ξ 2 .Note further that by (32) and the theory of continued fractions, the next convergent has denominator at least q µ /2 (see [15,Proposition 5.2]).Hence, any integer linear form aξ 2 + b with max{|a|, |b|} < q µ /2 satisfies |aξ 2 + b| ≥ q −µ .In other words, there is no better approximation for ξ within H 2 up to norm q µ /2.On the other hand, by Dirichlet's Theorem there is some p = (p 1 , p 2 , p 3 By (32) clearly p = q.We may assume p is a best approximation for ξ, so since (ξ 1 , ξ 2 ) ∈ Θ 2 and the above argument excludes p ∈ H 2 , we must have p ∈ H 1 .Clearly p > q .Let r ∈ Z 3 be the best approximation for ξ following p.By a very similar argument as above based on Dirichlet's Theorem for p in place of q, we can now exclude r ∈ H 1 , so we must have r ∈ H 2 .But then r ≥ q µ /2 by the above observation.Hence there is no other best approximation between p and q µ /2, so p minimizes |u • ξ * | among all integer vectors u ∈ Z 3 with û < q µ /2.On the other hand, again by Dirichlet's Theorem has a solution v ∈ Z 3 .Again we can assume v is a best approximation, hence the above observation that there is no best approximation with norm in ( p , q µ /2) implies v = p.
Combining the right estimate from (34) with the left bound from (33) and this happens for infinitely many p ∈ H 1 as above, we see that ω(ξ 1 ) ≥ 4.An analogous argument yields ω(ξ 2 ) ≥ 4 as well, hence (30) is proved.
Remark 5.The argument in fact shows that the best approximations in H 1 and H 2 must occur at some high rate when (30) is close to optimal.Using the variational principle [4,5], with some effort some stronger bound in the interval (1, 7/5) can be obtained, however we omit its slightly techincal explicit calculation.
Remark 6. An analogous argument shows in general that
We can conclude and by ( 17) go on to estimate However, by [15, Theorem 3.3], for n ≥ 3 the right hand side in (35) is at least n − 1, in particular the right expression exceeds twice the single dimension of its factors.Hence it seems the bound in ( 12) cannot be reached with this method.This argument is most likely true for n = 2 as well (see [15,Conjecture 3]), so just (30) may be insufficient to improve on (31) either and the bound 1 seems to be the optimal outcome of the method.9. Proof of Theorem 2.1: Lower bounds 9.1.Outline.We first show in § 9.2 that for n = 2, there exist vectors (ξ 1 , ξ 2 ) ∈ R 2 with properties (i)-(v), and with a slight twist of (vi).The transition to general n as well as the weaker lower bound n − 2 for the Hausdorff dimension in § 9.3 will then be an easy consequence of Theorem 4.1 above obtained in [14].By modifications of the method, the stronger metrical claims will be proved in § 9.4, 9.5.
Existence claim:
Case n = 2.In this section, we prove.
Theorem 9.1.There exist uncountably many totally irrational ξ ∈ R 2 for which the tail of the best approximation sequence with respect to the maximum norm lies in the union of the two 2-dimensional sublattices of Z 3 given by Moreover, large best approximations alternately lie in H 1 and H 2 .Furthermore, for any given w ∈ [2, ∞] we can choose ξ so that additionally ω(ξ) = w.
Note that the condition ω(ξ) = w 2 in (vi) is missing for a full analogue of Theorem 2.1.Indeed, the vectors ξ constructed in this section satisfy ω(ξ) = w 2 − 1 instead.We construct our real vector.Let τ > 1 + √ 2 be a parameter.Let α j , β j be increasing positive integer sequences and derive the integers Clearly such choices are possible.Then in particular (37) Then, upon changing initial terms if necessary, we can assume We claim that it satisfies the assertions of the theorem. Put Then F j ≡ 1 mod 2 and G j ≡ 1 mod 3 imply the coprimality assertions Then obviously Thus by ( 38) clearly Then by (37) moreover , are small linear form for j ≥ 1, when τ is large.We show that v j and w j precisely comprise all large best approximations.This obviously finishes the proof.
Let b be any best approximation.Then by (41) there is an index j such that either vj ≤ b < ŵj or ŵj ≤ b < vj+1 .We show that in the first case b = ±v j , and in the latter case b = ±w j .Assume the first case, so the latter works very similarly by symmetry.First observe that since b is a best approximation of norm at least vj , we know that We distinguish two cases.
Case 1: b lies in the two-dimensonal subspace of R 3 spanned by v j , w j , i.e. b ∈ v j , w j R ∩ Z 3 .The special form of A j , B j and (39) imply the following crucial result on integer vectors in the two-dimensional lattices v j , w j R ∩ Z 3 .Proposition 9.2.For v j , w j as above, if a linear combination gv j + hw j is an integer vector, then in fact g ∈ Z and h ∈ Z.In other words, v j , w j R ∩ Z 3 = v j , w j Z .
Proof.Clearly we must have g, h ∈ Q.If we write (p 1 /q 1 )v j + (p 2 /q 2 )w j with p i /q i in lowest terms, then it is clear that q 1 must be a non-negative integer power of 2 and q 2 a non-negative integer power of 3 to make the first two coordinates (p 1 /q 1 )A j = (p 1 /q 1 )2 α j resp.(p 2 /q 2 )B j = (p 2 /q 2 )3 β j of gv j + hw j integers.But then by (39) clearly the third coordinate (p 1 /q 1 )F j + (p 2 /q 2 )G j is not an integer unless q 1 = q 2 = 1.
By the proposition applied to b and since b < ŵj obviously we must have h = 0. Hence b = gv j is an integer multiple of v j , but since b is a best approximation and thus primitive this integer must be g = ±1.Hence indeed b = ±v j .The case ŵj ≤ b < vj+1 works very similarly by symmetry and yields for the best approximation the only candidates ±w j .
Case 2: b does not lie in the space spanned by v j , w j .For this case we use an easy consequence of Minkowski's Second Convex Body Theorem.Lemma 9.3.There exists a constant c > 0 such that for any ξ ∈ R 2 and any parameter Q ≥ 1, the system Proof.Consider the integer lattice Z 3 and the box of (x 1 , x 2 , x 3 ) ∈ R 3 with coordinates It has volume 8c, independent of Q and ξ 1 , ξ 2 .Hence, by Minkowski's Second Convex Body Theorem, the product of the induced successive minima is ≪ c, hence choosing c small enough the third successive minimum is smaller than 1.This means there cannot be three linearly independent integer points within the box, which in turn is equivalent to the claim.
We first notice that both v j and w j induce approximations of order greater than two.By (40), (42) and as our choice of τ > 1 + √ 2 that implies (τ 2 − 1)/τ > 2, for some ǫ = ǫ(τ ) > 0 we get (46) and for w j by (43) we have a stronger estimate that also yields (47) Combined with (41), ( 44), (45), we have By the assumptions of Case 2, the three vectors v j , w j , b are linearly independent.So we get a contradiction to Lemma 9.3 for Q = ŵj , as soon as ŵj is sufficiently large.Hence in total Case 2 provides only finitely many best approximations, of small norm.
Combining our observations from Case 1 and Case 2, we see that v j and w j comprise all best approximations of large enough norm, as desired.Moreover it is clear that any ξ as above is totally irrational and by the freedom in the choice of A i , B i the set of induced ξ is uncountable.Finally it is easy to check from (46), (47), the fact that v j , w j comprise all the best approximations and (40) that ( 48) So choosing τ > 1 + √ 2 appropriately, we can realize any uniform exponent in (2, ∞).Finally, small modifications of the construction allow for obtaining the endpoints 2 and ∞ as well.9.3.General case and lower bound dim H (Θ n ) ≥ n − 2. As indicated before, the extension to the general case works with Theorem 4.1.In view of the sufficient condition ( 15) and (48), the lower bound dim H (Θ n ) ≥ n − 2 follows by taking any ξ 1 , ξ 2 constructed in § 9.2 upon increasing τ if necessary, and extending it to n-dimensional real vectors via Theorem 4.1 (we do not need Lemma 4.2 here).As noticed in § 4.1 any arising ξ ∈ R n is automatically totally irrational since (ξ 1 , ξ 2 ) has this property.In fact the same bound holds when restricting to vectors with arbitrary uniform exponent ω(ξ) = w ∈ [n, ∞], similar as in Θ n (w) but with some altered value for the ordinary exponent ω(ξ) in terms of w.
To improve the bound n − 2, in the next section we generalize the construction of § 9.2 to obtain some Cantor type set with the properties of Theorem 2.1, and determine a stronger lower bound for its Hausdorff dimension using results from [16,4,5].9.4.Proof of (9), up to strictness.In this section, we show the improved lower bound and thus as w can be arbitrarily close to n also dim H (Θ n ) ≥ n − 2 + 1/(n 2 + 1).Up to the latter inequality not being strict yet, this agrees with claim (9) in Theorem 2.1.
For τ > n, we now consider modified ξ 1 , ξ 2 , with sequences of continued fraction convergents (r i,j /s i,j ) j≥1 , i = 1, 2, with the following denominator growth properties: We then follow the proof above with A i resp.B i replaced by s 1,j resp.s 2,j , and F i resp.G i replaced by r 1,j resp.r 2,j , and we replace v j , w j by (52) In order to establish an analogue of Proposition 9.2, we need coprimality conditions for the denominators, concretely it suffices to guarantee Write K(τ ) for the set of (ξ 1 , ξ 2 ) ∈ R 2 satisfying (50), ( 51), (53).
Note that now by the theory of continued fractions 2,j , slightly stronger than in § 9.2 where the approximations were of order τ 2 − 1. Together with (50) it follows easily that ω(ξ 1 , ξ 2 ) ≥ τ 2 /τ = τ .Thus if τ > n, we can apply Theorem 4.1 again to see that for any (ξ 1 , ξ 2 ) ∈ K(τ ) there is a full measure set F n (ξ 1 , ξ 2 ) ⊆ R n−2 , so for any (ξ 3 , . . ., ξ n ) ∈ F n (ξ 1 , ξ 2 ) the vector (ξ 1 , . . ., ξ n ) has essentially the same best approximations as (ξ 1 , ξ 2 ) (with zeros added for entries at positions 3, 4, . . ., n).As in Case 1 in § 9.2, via an analogous claim to Proposition 9.2 for g j , h j , we can conclude that any best approximation within the space spanned by a pair g j , h j or a pair h j , g j+1 is actually equal to g j or h j .Moreover, similar to Case 2 in § 9.2 by ω(ξ 1 , ξ 2 ) ≥ τ > n ≥ 2 via Lemma 9.3 we see that large best approximations must lie in such spaces, hence the large best approxmations are precisely the g j , h j .
Thus we have that the g j and h j again comprise all best approximations for any ξ as above, i.e. (ξ 1 , ξ 2 ) ∈ K(τ ) and (ξ 3 , . . ., ξ n ) ∈ F n (ξ 1 , ξ 2 ).Moreover it is clear that hence ω(ξ) = τ and ω(ξ) = τ 2 , so (vi) holds as well.Thus any vector of the form To finish the proof of (49), we identify w with τ and show As τ can be taken arbitrarily close to n, the claimed lower bound for Θ n follows as well.
Then, since the projection of a set has at most the Hausdorff dimension of the original set (by Lipschitz property of projections [6]), the claim (54) follows via We verify (55) to finish the proof.We need to show that for any ξ 1 as above (i.e. with condition (51) for i = 1), there exists ξ 2 ∈ R as above, that may depend on ξ 1 , i.e. so that conditions (50) and (53) hold as well (then (51) holds for i = 2 as well; in fact there is set identity in (55)).So let arbitrary ξ 1 with property (51) for i = 1 with convergent sequence (r 1,j /s 1,j ) j≥1 be given.Assume we have constructed the partial quotients of ξ 2 up to convergent r 2,j−1 /s 2,j−1 = [a 2,0 ; a 2,1 , . . ., a 2,j−1 ] for given j, and let a 2,j be the next partial quotient for ξ 2 to be fixed.We may assume s 2,j−1 < s 1,j in view of (50).From the recursion (57) s 2,j−2 + a 2,j s 2,j−1 = s 2,j for convergent denominators, there are many consecutive partial quotients a 2,j that induce s 2,j−2 + a 2,j s 2,j−1 = s 2,j ≍ s τ 1,j , as we need for (50) in the current step.We must show some of them induce (53) as well.
On the other hand, by (51) for i = 1, there are ≪ log(s 1,j s 1,j+1 ) ≪ log s 1,j many primes dividing either s 1,j or s 1,j+1 .Denote S j = {p ∈ P : p|(s 1,j s 1,j+1 )} the set of such primes.For condition (53) to hold in the current step, it suffices that any p ∈ S j does not divide s 2,j .Now, for any such prime p ∈ S j , again by the recursion (57) and (s 2,j−2 , s 2,j−1 ) = 1 we see that at most one congruence class modulo p for a 2,j will induce p|s 2,j .Hence by Chinese Remainder Theorem and estimate (58), there exist many partial quotients a 2,j for which s 2,j is not divisible by any such prime.If this is at least 1 for large j, we are done.However, again by (51) and a standard estimate for the number of prime divisors of an integer, the cardinality of S j can be bounded Hence we may estimate the latter factor of (59) which gives p∈S j (1 − p −1 ) ≪ exp(− log log s 1,j ) = (log s 1,j ) −1 .
Hence as δ > 0 indeed there remain many suitable a 2,j ∈ S j in each step so that condition (53) holds as well, and thus (55) is true.We have proved the claims of the section.
Remark 7. The right equality of (56) and Lemma 4.2, and as it is reasonable to expect that condition (53) is metrically negligible, suggest the bound n − 2 + 2/(n 2 + 1) stated in Conjecture 1.A slightly stronger conjectural bound, still of order n − 2 + 2n −2 − O(n −4 ) for large n, is motivated in § 9.5 below.9.5.Proof of strict inequality in (9) and lower bounds in (10).A small improvement of the bound compared to § 9.4 (and likewise presumably for Conjecture 1) can be made when extending the sets K(τ ) ⊆ R 2 to larger sets.We then instead of the formula from [16] apply the variational principle from [4,5] for m = n = 1 (approximation to a single real number) to estimate their Hausdorff and packing dimension, and finally conclude with Theorem 4.1 and Lemma 4.2 again.We use the template formalism from [4,5].The general definition of templates can be found in [5,Defintion 4.1], however in our easiest setting, a template f = (f 1 , f 2 ) consists just two piecewise linear, continuous functions f i (t) : [0, ∞) → R with the following properties: for all t ≥ 0, slopes among {−1, 0, 1}, where 0 is only possible on intervals where f 1 (t) = f 2 (t) = 0.Moreover, any local maximum of f 1 is a local minimum of f 2 , hence f 1 (t) = f 2 (t) = 0 at such points.
We evaluate the lower limit δ(f τ,ǫ ) of the average local contraction rate in t ∈ [0, T ] as T → ∞ according to the variational principle [5,Theorem 4.7].As ǫ → 0, a short calculation and (61) lead for any τ > n to the bound As τ > n can be arbitrary, we get Note that inserting τ = n, we obtain the left bound n − 2 + 1/(n 2 + 1) of ( 9).The maximum over τ is taken at some slightly larger value, thereby confirming the strict inequality in (9).
For the packing dimension, as ǫ → 0 we evaluate the upper limit δ(f τ,ǫ ) of the average contraction rates for the template in Figure 1 as The quantity tends to 1 as τ → ∞.Thus, for any ε > 0 (note ε = ǫ), choosing τ large enough and ǫ > 0 small enough, by means of (II) from Lemma 4.2 and as coordinate projections as Lipschitz maps again do not increase the packing dimension of a set [6], the variational principle [5,Theorem 4.7] gives the lower bound As ε > 0 can be arbitrarily small, we obtain the desired bound n − 1.
Remark 8.As remarked in the proof, for ξ ∈ R n obtained via ξ 1 , ξ 2 as constructed above, we have Thus, it is possible to find lower bounds for the Hausdorff and packing dimension of sets with a slightly altered definition compared to Θ n (w), for example in place of (vi) just restricting to ω(ξ) = w and omitting the claim on the ordinary exponent.
similar argument a lattice of dimension k is insufficient as well: As we find infinitely many best approximations in each H i , thus any lattice containing a tail of best approximations must have 2-dimensional intersection with each of them, by Remark 1 again.But this is only possible if its dimension exceeds k.Moreover, the analogue of (48) that reads holds.Since k = n, we have found a suitable vector (ξ 1 , . . ., ξ n ) = (ξ 1 , . . ., ξ k ).Now assume n > k.Increase τ if necessary, so that in addition to (64), we have (τ k − 1)/τ > n as well.Take any (ξ 1 , . . ., ξ k ) as in the case n = k above.We see that Hence we may apply a more general version of Theorem 4.1 from [14], stating that for almost all vectors (ξ k+1 , ξ k+2 , . . ., ξ n ) ∈ R n−k with respect to Lebesgue measure, the magnified vector ξ = (ξ 1 , . . ., ξ n ) has the same tail of best approximations up to lifting, i.e. again lie in the according lattices H i = H i (n).The claims (ii * ), (vii) follow analogously to the case n = k.The lower bound n − k for the Hausdorff dimension is clear, finally the inequality being strict can be shown by the method from § 9.4, we omit details.
11. Proof of Theorem 3.1 We first point out that in § 9.2 above, essentially we only require the special form of ξ 1 , ξ 2 to exclude in Case 1 other best approximations within the lattices v j , w j Z and w j , v j+1 Z , sublattices of e 1 , e 2 , e n+1 Z .In context of Theorem 3.1, this will not be an issue when we simply lift the sequence of best approximations of some (ξ 1 , ξ 2 ) to Z n+1 .However, it turns out that our method below requires the property ω(ξ 1 , ξ 2 ) > 3n − 4 to eliminate the dependence of the derived set F n from ξ 1 , ξ 2 .So let ξ 1 , ξ 2 be any such numbers, totally irrational.To simplify the argument below, we fix any constant (65) σ ∈ (3n − 4, ω(ξ 1 , ξ 2 )).
Note that any v j lies in the fixed three-dimensional sublattice e 1 , e 2 , e n+1 Z of Z n+1 as in the definition of Γ n .
Here we cannot use Theorem 4.1, so we follow another strategy.Again assume b is any best approximation of large norm.There is a unique j such that (67) vj ≤ b < vj+1 .
We will show that b = v j to finish the proof.First observe that since b is a best approximation of norm at least vj , we know that Besides, a simple application of Minkowski's Second Convex Body Theeorem implies Lemma 11.1.Let ξ 3 , . . ., ξ n satisfy (66) and ε > 0. For any Q ≥ Q 0 (ε), there exist n − 1 linearly independent integer vector solutions u 1 , . . ., u n−1 in Z n−1 to where again ûi omits the last coordinate of u i .
Remark 9.If we would restrict to c-badly approximable vectors in R n−2 whose Hausdorff dimension tends to n − 2 as c → 0 + , then we could sharpen the right hand side estimate to ≪ c,n Q −(n−2) , which would simplify the proof below a little.
Proof.Consider the integer lattice in Z n−1 and for Q ≥ 1 the convex body consisting of (x 1 , . . ., x n−1 ) ∈ R n−1 with max 1≤i≤n−2 The volume is 2 n−1 , so by Minkowski's Second convex body theorem the product of the successive minima are of order ≍ n 1 as well.The condition (66) tells us that the first successive minimum is ≫ Q −ǫ for any ǫ > 0 and all Q ≥ Q 0 (ǫ).Hence all successive minima are ≪ n Q ǫ/(n−2) .This easily yields the claim by slightly modifying ǫ to some ε if necessary.
does not have three linearly independent solutions in integer vectors b = (b 1 , b 2 , b 3 ).
4.2.Metric results for Cartesian products and fibers.Part (I) of the following is partial claim of [12, Proposition 2.3], originally due to Marstrand [11] when n 1 = n 2 = 1, see Federer [7, § 2.10.25] for arbitrary dimension.Part (II) can be obtained by slightly generalizing the proof of part (c) in the proof of Tricot's [18, Theorem 3].
Lemma 4.2 (Marstrand; Tricot).Let n 1 , n 2 be positive integers and M ⊆ R n 1 +n 2 be measurable.Denote fibers by M | 2023-02-17T06:42:24.110Z | 2023-02-16T00:00:00.000 | {
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149446626 | pes2o/s2orc | v3-fos-license | Effect of lipid metabolism disorder on liver function in patients with malignant tumors after chemotherapy: a case-control study
Background This study aims to investigate the effect of lipid metabolism disorder on liver function in patients with malignant tumors after chemotherapy. Method A total of 428 patients with malignant tumors with normal liver function in our hospital between May 2013 to June 2018 were divided into an observation group (lipid metabolism disorder, n = 265) and control group (normal lipid metabolism, n = 163). The lipid metabolism levels and liver damage of the two groups were compared before and after chemotherapy. Results No significant differences in age, gender, body mass index, tumor types, history of surgery, levels of alanine aminotransferase (ALT; an indicator of liver function), and chemotherapy regimen were observed between the two groups. However, the observation group showed increased levels of total cholesterol (P = 0.000), triglycerides (P = 0.000), and low-density lipoprotein (P = 0.01), as well as decreased levels of high-density lipoprotein (P = 0.000) before chemotherapy compared with the control group. Furthermore, patients with lipid metabolism disorders were more likely to develop abnormal liver function after chemotherapy. Moreover, mixed lipid metabolism disorder was more likely to cause severe liver damage after chemotherapy. Additionally, the number of patients with lipid metabolism disorders after chemotherapy (n = 367) was significantly increased compared with before chemotherapy (n = 265) (P < 0.01), indicating that chemotherapy might induce or aggravate an abnormal lipid metabolism. Conclusions After receiving chemotherapy, patients with malignant tumors presenting lipid metabolism disorders are more prone to liver damage and lipid metabolism disorders than patients with a normal lipid metabolism.
Background
Malignant tumors are a serious threat to human health, and chemotherapy remains one of the main treatments. However, chemotherapy has many toxic side effects [1]. For instance, as most chemotherapeutic drugs are metabolized by the liver, liver toxicity is one of the most common side effects [2]. Clinical manifestations of chemotherapy-induced liver damage are diverse, ranging from slight liver function abnormalities to severe cases of toxic hepatitis or fulminant hepatic failure. Hepatic toxicity is generally reversible [3], but may still cause fibrosis or cirrhosis if discontinued. In addition, liver damage caused by chemotherapeutic drugs may lead to lipid metabolism disorders and even hyperlipidemia [4,5].
High-fat, high-energy diets have resulted in an increase in the incidence of lipid metabolism disorders [6]. Under this condition, reduced transport of fat causes lipids to accumulate in liver cells, resulting in a fatty liver [7]. Fat accumulation in the liver can affect the function of or destroy liver cells and cause connective tissue hyperplasia and cirrhosis, which affects drug metabolism [8]. Because chemotherapeutic drugs can cause liver damage and liver damage is associated with lipid metabolism disorders, we suspect that lipid metabolism disorders may be related to liver damage caused by chemotherapeutic drugs, but no research on this topic has been reported.
In the present study, we examined lipid metabolism in patients with malignant tumors who were treated at our hospital in recent years and assessed impairments in liver function after chemotherapy to investigate the effect of lipid metabolism disorders on liver function in patients undergoing chemotherapy.
General information
This study included patients who were undergoing chemotherapy at our hospital from May 2013 to June 2018. Exclusion criteria were patients with diseases associated with liver damage, such as liver metastasis, viral hepatitis, drug-induced liver disease, total parenteral nutrition, and hepatolenticular degeneration. Inclusion criteria were patients diagnosed with malignant tumors via a pathological or cytological examination who required chemotherapy, had no chemotherapy contraindications, were aged≥18 years, had normal liver function (alanine aminotransferase (ALT) level ≤ 50 IU/L), and had a Kamofsky performance status ≥70 points. Before chemotherapy, tests of blood lipids and liver function were performed on all patients.
Procedure
The blood lipid levels and liver function of each patient were assessed before chemotherapy. All indicators were in the normal range before chemotherapy. All patients underwent chemotherapy with a combination regimen according to the pathological type of malignancy. Common chemotherapy drugs included oxaliplatin, 5-fluorouracil, paclitaxel, cyclophosphamide, epirubicin, cisplatin, platinum, vinorelbine, gemcitabine, capecitabine, vincristine, and irinotecan, among others. Blood lipid levels and liver function were also measured approximately 1 week after chemotherapy was initiated. If abnormal liver function was observed during chemotherapy, chemotherapy was suspended and relevant drugs were administered as a protective treatment. Liver function was reassessed every 5-7 days until it returned to normal levels, after which chemotherapy was continued. After chemotherapy, liver function and blood lipid levels were analyzed again. In this study, liver function and blood lipid levels were all measured approximately 1 week after chemotherapy was initiated. Venous blood was collected in the morning from all patients who had fasted for more than 8 h, and total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were determined. Lipid indicators, liver function and ALT levels were assessed using enzymatic methods.
Types of lipid metabolism disorders
Based on the recommendations for the prevention and treatment of dyslipidemia, lipid metabolism disorders are classified as follows [9]: 1. hypercholesterolemia (fasting serum TC level exceeding 5.72 mmol/L); 2. hypertriglyceridemia (fasting serum TG level exceeding 1.70 mmol/L); 3. mixed hyperlipidemia (increased fasting serum TC and TG levels); and 4. low high-density lipoproteinemia (fasting serum HDL level < 0.9 mmol/L).
Criteria for evaluating liver function
According to the World Health Organization standards for the toxicity and side effects of anticancer drugs, the evaluation of liver function was defined using the following criteria: 1. normal liver function, serum ALT level ≤ 1.25 N (N = 40 IU/L); 2. liver function damage I level, 1.25 N < serum ALT level < 2.5 N; 3. liver function damage II level, 2.5 N < serum ALT level < 5 N; 4. liver function damage III level, 5 N < serum ALT level < 10 N; and 5. liver function damage IV level, serum ALT level > 10 N.
Non-alcoholic fatty liver disease (NAFLD)
According to the guidelines for the diagnosis and treatment of NAFLD [10], the disease is divided into non-alcoholic simple fatty liver disease, non-alcoholic fatty liver hepatitis and non-alcoholic fatty liver cirrhosis.
Statistical analysis
Statistical analyses were performed using SPSS 20.0 statistical analysis software. The count data are presented as means±standard deviations. T-tests were used for comparisons between groups. Dichotomous variables were analyzed using the χ 2 test. If the number of observations was less than 5, Fisher's exact probability test was used to calculate the P value. P < 0.05 was considered statistically significant.
Results
The observation group showed increased levels of TC, TG, and LDL and decreased levels of HDL before chemotherapy A total of 428 patients with malignant tumors with normal liver function (199 males and 229 females, with an average age of 62.4 ± 9.2 years) were enrolled in our hospital from May 2013 to June 2018. These patients were divided into the lipid metabolism disorder group (observation group, n = 265) and normal lipid metabolism group (control group, n = 163) according to the lipid metabolism status before chemotherapy. In the observation group, 69 patients (26.0%) had high cholesterol levels, 98 patients (37.0%) had high TG levels, 61 patients (23.0%) had a mixed type, and 37 patients (14.0%) had a low HDL-C type. General characteristics of the two groups are shown in Table 1. No significant differences in age, gender, body mass index (BMI), tumor types, and history of surgery between the two groups.
Furthermore, significant differences in ALT levels (a sensitive indicator of liver function) before chemotherapy were not observed between the observation (26.2 ± 7.8 IU/L) and control groups (25.4 ± 9.4 IU/L). However, the observation group showed increased levels of TC (P = 0.000), TG (P = 0.000), and LDL (P = 0.01) and decreased levels of HDL (P = 0.000) before chemotherapy compared with the control group. Moreover, significant differences in the chemotherapy regimen were not observed between the two groups ( Table 2).
The observation group showed a higher incidence of liver damage after chemotherapy The levels of liver damage after chemotherapy in the two groups are shown in Table 3. The incidence of liver damage after chemotherapy was 47.2% in the observation group (125/ 265), with 111 patients presenting level I damage (41.9%), 8 patients presenting level II damage (3.0%), and 6 patients presenting level III damage (2.3%), whereas the incidence in the control group was 20.9% (34/163), with 29 presenting level I damage (17.8%), 3 patients presenting level II damage (1.8%), and 2 patients presenting level III damage (1.2%). The incidence of liver damage after chemotherapy in the observation group was significantly greater than in the control group (P < 0.01). Based on these results, patients with lipid metabolism disorder are more prone to liver damage after chemotherapy than patients with normal lipid metabolism. Types of lipid metabolism disorders before chemotherapy correlated with liver function after chemotherapy The correlations between the types of lipid metabolism disorders before chemotherapy and liver function after chemotherapy are presented in Table 4. No significant differences in the percentages of patients with normal liver function and liver damage at levels I and level II were observed among the hypercholesterolemia group, hypertriglyceridemia group, the mixed hyperlipidemia group, and low-HDL group. However, liver function at damage level III after chemotherapy was observed in 6 patients in the mixed hyperlipidemia group, but was not observed in the other groups. Moreover, a significant difference in the percentage of patients presenting function damage level III after chemotherapy was observed among the four groups (P = 0.000). In addition, a statistically significant difference in the degree of liver damage after chemotherapy was observed among the four groups (P = 0.002). Thus, a significant correlation exists between the type of lipid metabolism disorder before chemotherapy and liver function after chemotherapy.
Changes in lipid metabolism before and after chemotherapy
In addition to investigating the effect of lipid metabolism disorders on damage to liver function in patients with cancer after chemotherapy, we also evaluated the effect of chemotherapy on lipid metabolism in patients. Two hundred sixty-five patients presented with dyslipidemia and 163 patients presented with normal lipid metabolism before chemotherapy. Interestingly, the number of patients with dyslipidemia (n = 367) after chemotherapy was significantly increased compared with before chemotherapy (P < 0.01) ( Table 5). Based on these data, chemotherapy might induce or aggravate lipid metabolism disorders.
Discussion
Clinically, many patients receiving chemotherapy develop lipid metabolism disorders. In addition, the administration of chemotherapy to patients with lipid metabolism disorders may increase the risk of liver damage. However, currently, evidence for the relationship between lipid metabolism disorders and liver damage after chemotherapy is limited. The results from the present study showed increased levels of TC, TG, and LDL and decreased levels of HDL in the patients with lipid metabolism disorder before chemotherapy compared with the patients with normal lipid metabolism (Table 1). Furthermore, patients with lipid metabolism disorders are more likely to develop abnormal liver function after chemotherapy (Table 3). Moreover, mixed lipid metabolism disorder is more likely to cause severe liver damage after chemotherapy (Table 4). In addition, the number of patients with lipid metabolism disorders after chemotherapy (n = 367) was significantly increased compared with before chemotherapy (n = 265) ( Table 5), indicating that chemotherapy potentially induces or aggravates abnormal lipid metabolism. According to the evidence, chemotherapy drugs can cause lipid metabolism disorders. Paclitaxel and platinum-based chemotherapy caused transient dyslipidemia in three patients with cancer [11]. The chemotherapy drug 5-fluorouracil induces steatosis by generating reactive oxygen species [12]. Lipid metabolism disorders may be induced by chemotherapy due to drug toxicity [13]. Consistent with this finding, chemotherapy induced or aggravated abnormal lipid metabolism in the present study, as indicated by the increased number of patients with lipid metabolism disorders after chemotherapy. Compared with the control group, * P < 0.05, ** P < 0.01 Chemotherapeutic drugs have been shown to induce liver damage [12,14,15]. Almost all chemotherapeutic drugs are metabolized by the liver, the metabolic center of the body, and thus easily lead to drug-induced liver damage. Chemotherapeutic drugs mainly cause liver damage mainly through the following three pathways: (1) drugs and their metabolites directly damage liver cells; (2) chemotherapy may aggravate liver damage in patients with pre-existing liver diseases; and (3) pre-existing liver diseases might reduce the activity of drug-metabolizing enzymes and prolong the duration of drug action, thereby increasing the toxicity of chemotherapeutic drugs.
As shown in recent studies, lipid metabolism is closely related to the occurrence and development of tumors. Patients with tumors tend to have lipid metabolism disorders. In the present study, compared with patients with normal lipid metabolism, patients with malignant tumors presenting dyslipidemia showed an increased incidence of hepatic impairment after chemotherapy. Thus, hepatotoxicity should be more carefully assessed when chemotherapeutic drugs are administered to patients with lipid metabolism disorders.
The mechanisms by which lipid metabolism disorders affect liver function after chemotherapy are not yet completely understood, but may be related to the factors described below. (1) Excess lipids directly exert adverse effects on liver function. When a lipid metabolism disorder occurs, the liver is unable to transport fat in a timely manner, thus causing fat to accumulate in liver cells and resulting in fatty liver. Fat accumulation in the liver affects liver function and destroys liver cells, subsequently reducing the liver compensatory capacity and causing connective tissue hyperplasia, cirrhosis, and liver damage [16]. (2) Pre-existing dyslipidemia indirectly contributes to liver damage after chemotherapy. Most chemotherapeutic drugs are cytotoxic and immunosuppressive agents, causing a certain degree of damage to the liver themselves [17]. Chronic liver damage is present in patients with lipid metabolism disorders, resulting in a decreased drug metabolism capacity and aggravation of liver damage. (3) Pro-inflammatory cytokines exacerbate liver damage. For example, tumor cell necrosis after chemotherapy causes the release of tumor necrosis factor, one of the main factors involved in liver cell damage and even hepatic necrosis [18]. (4) The oxidative stress response also contributes to liver damage. Excess intake of free fatty acids causes oxidative overload in the mitochondria of hepatocytes, producing more reactive oxygen species (ROS). The expression of uncoupling protein 2 is compensatorily increased to reduce ROS production, and a large amount of adenosine triphosphate is consumed, leading to the apoptosis or even necrosis of hepatocytes [19]. Hepatocyte necrosis produces a large amount of inflammatory factors, further increasing the production of free radicals.
Chemotherapy-induced liver damage in patients with cancer may occur during chemotherapy or after the total course. Although liver function impairment may be reversed in some patients after treatment, abnormal liver function may lead to an interruption of anti-tumor therapy and jeopardize the prognosis of patients [20]. The clinical consequences of liver damage after chemotherapy in patients with cancer vary. Mild cases may only produce an asymptomatic increase in ALT levels, spontaneous reversal may occur in some patients, and severe cases may involve jaundice, ascites, coagulation abnormalities, encephalopathy and other signs of liver failure, with a high mortality rate if not treated effectively [21]. The symptoms experienced by these patients are mild, with most having no symptoms or signs, and abnormalities are only observed when liver function is measured. For patients with symptoms and signs, except for jaundice, the main symptoms are fatigue, anorexia, bloating, abdominal pain, and fever. The clinical manifestations of patients are similar to the original disease; they are not specific and are difficult to identify. Thus, the clinical manifestations of liver damage after chemotherapy are subtle in some patients and may not be detected in a timely manner, with a risk of worsening adverse reactions during continued treatment. Therefore, transaminase levels should be regularly monitored in patients with cancer who are undergoing chemotherapy. Once abnormal liver function is detected, a hepatoprotective enzyme treatment should be administered in a timely manner to prevent the further aggravation of the disease and ensure the successful completion of chemotherapy. Lipid metabolism disorders are major risk factors for cardiovascular and cerebrovascular diseases and exert important effects on the metastasis and prognosis of some tumors, particularly breast and gastrointestinal tumors. Therefore, blood lipid levels must be controlled in patients with tumors. Patients with cancer should pay special attention to their eating habits and avoid high-fat diets to prevent abnormal lipid metabolism. Routine screening of blood lipid and transaminase levels before chemotherapy is recommended for patients with cancer, as are preventive measures to regulate lipid metabolism and protect liver function to reduce the incidence of hepatic damage, liver failure and tumor-related mortality. Improvements in the quality of life of patients with cancer will have a positive impact on the prognosis.
Conclusions
In conclusion, patients with malignant tumors presenting lipid metabolism disorders are more prone to liver damage after chemotherapy than patients with a normal lipid metabolism. Furthermore, mixed lipid metabolism disorders are more likely to cause severe liver damage. | 2019-05-11T13:46:54.225Z | 2019-05-10T00:00:00.000 | {
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12387318 | pes2o/s2orc | v3-fos-license | doi:10.5455/vetworld.2013.39-41 Mortality pattern in non human primates in Assam, India 1
Aim: The study was conducted to know the mortality pattern in non human primates in Assam. Materials and Methods: A total of 27 deaths were recorded in six different species of non human primates of Assam State Zoo and Department of Forest and Environment, Government of Assam during the period from August, 2009 to December, 2009. The cause of death was determined on the basis of gross and histopathological examinations conducted at Department of Pathology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, Assam. Results: The causes of death attributed to specific diseases in non human primates were tuberculosis (22.22%), pneumonia (18.57%), enteritis (11.11%), encephalitis (11.11%), nephritis (11.11%), septicaemia (03.7%), malignant neoplasm (03.7%), zygomycotic gastritis (03.7%), traumatic injury (03.7%), poisoning (03.7%), stress (03.7%) and senility (03.7%). Conclusion: The study viewed that it is important to know the causes of death of non human primates for preservation and conservation of these endangered wild species.
Introduction
with high mortality in some primate colonies [3].
For conservation of these wild species, many of Northeast India comprising the states of Arunachal which are globally endangered, developing long-term Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland, strategy is of utmost importance.Although, a majority and Tripura is the richest in terms of primate diversity of the population is protected in the various parks and with 9 confirmed species records and 3 other species sanctuaries, the coverage is still inadequate.To reduce whose sightings need confirmation [1].The pattern of the mortality of wild animals, the study of their distribution of different species and the role of rivers, pathological condition is necessary to take preventive both large (Brahmaputra and Dibang) and small measures and control programmes.Keeping the above (Sankosh, Manas and Barak) as zoogeographic barriers facts in view, the present study recorded the causes of in dispersal is intriguing.Except for the golden langur death of non human primates in Assam.(Trachypithecus geei) and phayre's langur (Trachypithecus
Materials and Methods
phayrei), all other species have a large extent of occurrence in the region.The rhesus macaque (Macaca The study recorded mortality of non human mulatta), capped langur (Trachypithecus pileatus) and primates of Assam State Zoo and Department of Forest the Assamese macaque (Macaca assamensis) are the and Environment, Government of Assam during the most abundant species in this region [1].
period from December, 2007 to November, 2009.A Mortality patterns in non human primates are total of 27 deaths of six different species of non human influenced both by diet and degree of environmental primates were recorded.Data pertaining to history, age, seasonality [2].Emerging and reemerging infectious sex, species, date and cause of death were examined.diseases remain a major threat to these animal colonies.
The cause of death was ascertained on the basis of gross Due to the close genetic relationship between non human and histopathological examinations conducted at primates and humans, disease causing organisms are Department of Pathology, College of Veterinary easily exchanged between them.Often these animals Science, Assam Agricultural University, Khanapara, carry and transmit diseases without any visible signs.
Guwahati, Assam.They are more likely to contract hepatitis A, measles or
Results and Discussion
poliomyelitis from humans or as part of laboratory experiment to transmit these diseases to humans.
In the present study majority (22.22%) of animals Diarrhoea and respiratory diseases were major causes died due to tuberculosis (Table-1).Tuberculosis was a of morbidity in non human primate and were associated common as well as a major killer disease in non human primates [4].Rajknowar et al. [5] made a detail study and capture injury was recorded.Chemical immobilizations on tuberculosis of non human primates of Assam State for restraint might have been another factor in decreasing Zoo and recorded tuberculin positive in captive and the number of deaths due to capture injury.In accordance free living animals to be 25% and 15% respectively.
of the present findings, Pathak [14] also reported The presence of tuberculosis infection in zoo animals is organochlorine poisoning in free living golden langurs.not only a potential danger to the workers and One slow loris died due to stress associated with veterinarians working there, but also to the general capture myopathy.Stress related diseases were an public who visit the zoo.
important cause of death in many animals, including The study recorded 11.11% pneumonic death.
humans.During capture of wild animals, physical, Pneumonia always plays a significant role in the environmental and psychological factors effect upon mortality of captive animals as these animals always the animal simultaneously and the effects were remain under stress.Enteritic disease recorded in the accumulative.Death due to senility was recorded in a present study was 11.11%.Enteric diseases, specifically rhesus macaque in the study.Senility cause of deaths of diarrhea, are frequently associated with morbidity and animals was also recorded by former researchers in mortality in nonhuman primates in captivity.Enterodifferent zoos.pathogenic E.coli may be a significant pathogen for Conclusion nonhuman primates to cause enteritis.In addition to the impact these bacteria may have on the health of Non human primates can carry a variety of zoonotic diseases.Therefore proper care should be taken by colonies of animals held in captivity, there is also the anyone handling these animals to prevent potential potential risk of transmission to humans, which exposure to zoonotic pathogen.To facilitate the detection characterizes the zoonotic potential of these infections.and control of potential pathogens, all facilities that In concurrence of the present exploration, encephalitis house non human primates should implement was recorded earlier in barbary macaque (Macaca comprehensive microbial quality programs.This will sylvanus) [6] and in chimpanzee (Pan troglodytes) [7].
help to reduce the morbidity and mortality of these wild In the present investigation, 3 animals (11.33%) died animals.due to nephritis.Similar reports of nephritis in pigtailed macaque (Macaca nemestrina) [8] and in woolly Author's contribution monkey (Lagothrix lagotricha) [9] had been published.
BGN done this study under the guidance of AC.This Death due to septicaemia with isolation of E. coli study is the part of MVSc thesis of BGN and AC was had been encountered in non human primate in the the major advisor.Both author read and approved the present study.Zygomycotic gastritis recorded in a free final manuscript.living golden langur was in conformity with the | 2014-10-01T00:00:00.000Z | 2013-01-01T00:00:00.000 | {
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10594752 | pes2o/s2orc | v3-fos-license | Structural Variation of Type XII Collagen at Its Carboxyl-terminal NC1 Domain Generated by Tissue-specific Alternative Splicing*
This paper reports the identification of two structural variations in the NC1 domain of rat and mouse type XII collagen. The long NC1 domain encoding 74 amino acids showed homology to chicken type XII and XIV collagens. The short NC1 domain was composed of 19 amino acids. Through genomic DNA analyses, two alternative exons were identified, each of which contained the variable NC1 sequence. With the amino-terminal NC3 splicing alternatives, we propose here a new descriptive nomenclature: types XIIA-1 and XIIB-1 which include a long NC1 sequence encoded by exon 1 (from the 3′-end), and types XIIA-2 and XIIB-2 which include a short NC1 sequence encoded by exon 2. Types XIIA-1 and XIIB-1, the predominant transcripts in 15-day old mouse embryos, showed decreased expression in 17-day old embryos when type XIIB-2 expression was sustained at constant levels. In adult mice, type XIIB-1 associates with ligament and tendon, whereas type XIIB-2 is expressed in various other tissues. The long NC1 domain contains an extended acidic region (pI = 3.4) followed by a terminal basic region (pI = 13.8). Because the short NC1 domain lacks these features, structural variations in the type XII collagen NC1 domain suggests different functional roles in a tissue-specific fashion.
The primary structure of chicken and mouse type XII collagen has been determined from overlapping cDNAs and suggests that type XII collagen may interact in several ways with other matrix molecules (1)(2)(3). For example, the amino-terminal region of type XII collagen forms three "finger-like" projections, the NC3 domains (see Fig. 1) (4) which contain multiple repeats of fibronectin type III-like subunits, the domain A of von Willebrand factor, a region homologous to the NC4 domain of type IX collagen, as well as RGD sequences. Because of its adhesive structure, it has been proposed that this region of type XII collagen may play a role in the interactions between extracellular matrix molecules and cells, thus contributing to the phys-ical state of the extracellular matrix (5).
The carboxyl-terminal region of type XII collagen contains triple-helical domains (COL1 and COL2) interrupted and flanked by non-triple-helical domains (NC1 and NC2) (Fig. 1). This structural feature is characteristic of a class of collagens, the Fibril Associated Collagen with Interrupted Triple helices or FACIT 1 (4,6). Fibril association through the carboxyl-terminal region has been postulated for members of the FACIT class of molecules. The details of such association with cartilage type II collagen fibrils have been characterized for type IX collagen (7)(8)(9)(10). In type XII collagen, the "fibril association region" makes up less than 10% of the molecule and is thought to be integral to molecular interactions with type I collagen fibrils. However, although there is indirect evidence available to support such an interaction (5,(11)(12)(13), the details of type XII collagen "fibril association " have not yet been fully elucidated.
One obstacle is the considerable difference in the NC1 domain structure of chicken and mouse type XII collagens. Chicken type XII collagen contains a long NC1 domain composed of 76 amino acids (1, 2), whereas mouse type XII collagen has only a 22-amino acid NC1 domain (3). The purpose of this study was to determine the structure of the carboxyl region of mammalian type XII collagen. In this report, we present evidence for two structural variations in the NC1 domain of type XII collagen that are generated by alternative splicing in a tissue-specific fashion.
EXPERIMENTAL PROCEDURES
cDNA Cloning and Sequencing-To obtain cDNAs encoding the COL2, NC2, COL1, and NC1 domains of rat and mouse type XII collagen, the 3Ј-rapid amplification of cDNA ends was adapted (14) using poly(A)ϩ RNA isolated from adult rat and mouse maxillary dentoalveolar tissues or total RNA from neonatal mouse skin. Site-directed coding and nested primers were designed based on the previously reported sequence of rat (15) and mouse cDNAs (3). Primers 5Ј-GGT CCT CCA GGC CCT CAG GG-3Ј and 5Ј-(CAU) 4 CCA GGA GAA CCG GGT CGC CA-3Ј were used to clone rat type XII collagen cDNAs. Primers 5Ј-TGG CAG TGC TGG AGC CAG AGG AG-3Ј, 5Ј-(CAU) 4 TAT TGT GAT TCA TCC-3Ј, and 5Ј-(CUA) 4 TGA AAA ATG CCT CTT TTG-3Ј were used to clone mouse type XII collagen cDNAs. PCR products were analyzed on Southern blots and cloned into the pAMP 1 vector (Cloneamp system, Life Technologies, Inc.) or the T vector (pT7 Blue vector, Novagen). The DNA sequences were determined by the chain-termination method (16).
Exon-Intron Structure Analysis and Sequencing of Genomic DNA Clones-Tail tissue from rats and mice were used to isolate genomic DNA samples. Two separate 3Ј-PCR primers (5Ј-CAA ACT GTA AGC AGC ACT-3Ј and 5Ј-TGA AAA ATG CCT CTT TTG-3Ј) were designed to * This study was supported in part by National Institutes of Health Grants DE07010 (to A. M. K.), DE00351 (to N. Y. K.), AR36820 (to B. R. O.), and ITI Foundation (to I. N., and N. Y. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM encode the reverse and complementary sequences of each of the different 3Ј-untranslated regions of type XII collagen cDNAs. Common 5Јprimers were designed from the coding sequence of the COL1 and NC1 domains: 5Ј-TAT TGT GAT TCA TCC-3Ј and 5Ј-ATT CAT CCC AGT GTG CCA GCA T-3Ј. Either a conventional PCR or long range PCR protocol (TaqPlus system, Stratagene) was used to amplify the ␣1(XII) collagen gene fragments, which were then confirmed by Southern blots. The initial experiments suggested that the exon involved in the short NC1 variant was located on the 5Ј-side of the exon involved in the long NC1 variant. To confirm this exon orientation, a separate PCR exper- iment was performed using a coding primer within the "short NC1" exon, 5Ј-GGT TCC GGC TAA CAA ACT-3Ј, and the anti-coding primer in the "long NC1" exon, 5Ј-GCA GCG ACT CTA TGT TCA TAG TCT TGC AAA-3Ј. These PCR products were cloned and sequenced to identify the exon-intron junctions. RNA Transfer Blot Analysis-A multiple tissue RNA transfer blot with poly(A)ϩ RNA from whole mouse 7-, 11-, 15-, and 17-day old embryos was purchased from CLONTECH. Hybridization probes were generated from the subcloned fragments of exon 1 and exon 2, separately encoding "long NC1" and "short NC1" specific sequences. In some experiments, an antisense-rich linear amplification PCR was used to prepare the hybridization probes. As a positive control, cDNA of a 370-bp HindIII fragment (MRK) encoding the mouse type XII collagen NC3 domain was used (3). A mouse -actin probe was used as a housekeeping gene control.
Ribonuclease Protection Assay-Mouse clones SB#2 and SB#3, containing a 153-bp insert of the long NC-1 variant and a 102-bp insert of the short NC-1 variant, respectively, were used to synthesize riboprobes labeled with [␣ 32 P]UTP (MAXIscript, Ambion). The MRK clone containing a 370-bp insert of mouse type XII collagen NC3 domain sequence and mouse -actin were used as a positive control and as a housekeeping gene control. The ribonuclease protection experiment was performed with 20 g of total RNA from each of the following mouse tissues: sternal cartilage, dento-alveolar tissue, eye, skin, and tail; the protected bands were analyzed using a computerized program (Kodak Digital Science 1D).
The isolated mouse type XII collagen cDNA, ER#K (Gen-Bank TM U57095), exhibited a sequence highly homologous to the rat AK#G clone with a short NC1 domain (19 aa; 100% homology) and a short 3Ј-untranslated region (63 bp long) ( Table I and Fig. 2). Four of four mouse clones, which were amplified from the neonatal mouse skin total RNA sample, contained identical sequences, with the exception that the poly(A) tail varied from A 19 to A 60 . The separately isolated mouse cDNA from the dento-alveolar tissue total RNA sample, SB#1, contained a long NC1 sequence (74 aa long) that was (Table I and Fig. 2).
The data, based on these cDNAs, suggested the presence of alternative exons in the 3Ј-end of the ␣1(XII) gene. Rat and mouse genomic DNAs were examined by PCR analyses; two alternative exons were identified (Fig. 3). Exon 1 (from the 3Ј-end) encoded the variable sequence of the long NC1, whereas exon 2 (from the 3Ј-end) encoded the variable sequence of the short NC1. Both exons 1 and 2 contained separate translational termination codons (TAA and TGA, respectively) and separate 3Ј-untranslated regions. At the terminal end of exon 2, we detected the poly(A) signal consensus sequences-AATACT in the rat gene and AATACA in the mouse gene (Fig. 3). An exon-intron junction was detected at the beginning of each of the variable sequences. The split codon exon junction structure generates G AG (Glu) at the 17th amino acid residue of the long NC1 domain and G GT (Gly) at the 17th amino acid residue of the short NC1 domain (Figs. 2 and 3). Thus, an alternative splicing mechanism is strongly suggested to generate the long and short NC1 domains of type XII collagen. Exon 3 (from the 3Ј-end) encoded the common NC1 sequence and part of the COL1 domain. The intron sizes between exons 1 and 2 and between exons 2 and 3 were estimated at 1.4 and 1.1 kilobase pairs, respectively. DNA sequence analyses revealed that the previously reported mouse NC1 domain sequence (3) matched the intron sequence immediately to the 3Ј-side of exon 3 (Fig. 3).
Type XII collagen transcripts also contain separate alternative splicing variants, at the 5Ј-region of the ␣1(XII) gene, that generate the long and short NC3 domains, or type XIIA and XIIB, respectively. Poly(A)ϩ RNA from mouse embryos at days 15 and 17 showed the presence of both type XIIA (approximately 10 kb) and XIIB (approximately 7-8 kb) collagen mRNAs using MRK (mouse type XII collagen NC3 domain) as a probe. When a fragment of mouse genomic DNA containing the exon 1 sequence (long NC1) was used as a probe, both the 10-and 7-8-kb forms of type XII collagen mRNA were positively recognized. The expression pattern of long NC1 type XII collagen was similar to the pattern of the mRNA recognized by the MRK probe, which peaked in 15-day old mouse embryos and decreased in 17-day old embryos (Fig. 4). The exon 2 probe (short NC1) recognized primarily the 7-8-kb form of type XIIB collagen mRNA but only weakly hybridized with a 10-kb type XIIA collagen mRNA. The expression of short NC1 type XIIB collagen mRNA remained constant during the late stages (15-and 17-day old) of mouse embryo development (Fig. 4).
The relative mRNA levels of type XII collagen varied among the different tissue specimens (cartilage, dento-alveolar tissue, eye, skin, and tail). The ribonuclease protection assays revealed an exceedingly high level of expression of type XII collagen with the long NC1 domain in dento-alveolar and tail tissue specimens compared with other mouse tissues and revealed moderate expression in cartilage (Fig. 5). Expression of type XII collagen with the short NC1 domain in the mouse was noticeably present in all tissues tested (Fig. 5). DISCUSSION The newly discovered sequence variations in the NC1 domain of rat and mouse type XII collagen indicated the following characteristic substructures. At the amino-terminal end of the NC1 domain immediately adjacent to the COL1 domain, there is a "common region" that contains 16 amino acid residues, which are 100% conserved in rat and mouse NC1 variants (Fig. 6). The short NC1 domain found in the rat AK#G and mouse ER#K clones extends three additional amino acid residues from the common region prior to the termination codon. The short NC1 domain sequence, with a total of 19 amino acid residues, shows great similarity to the previously reported 22-amino acid mouse NC1 domain sequence deduced from cDNA mc102T (3) (Table I). However, based on the differences between the amino acid and nucleotide sequences at the carboxyl end of the coding region and the 3Ј-untranslated regions, the newly discovered short NC1 sequence should be considered distinct. These short NC1 sequences contain two conserved locations of cysteinyl residues, one at the end of the COL1 domain and the other at the fifth position in the NC1 domain. This structure is homologous to the corresponding domain of type IX collagen chains, which were recently shown to possess all the necessary infor-mation for chain selection and trimer assembly in vitro (17).
The long NC1 sequence of rat and mouse type XII collagen exhibited structural homology with chicken type XII collagen. Following the common region, a stretch of amino acids containing aspartic acid and glutamic acid residues forms the "acidic extension region" (Fig. 6), with a calculated pI of 3.4. The acidic extension region is further followed by a short polypeptide composed of nearly 40% basic amino acid residues, the "basic terminal region," with a calculated pI of 13.8. These characteristic sub-structures of the long NC1 domain of type XII collagen seem to be shared with the corresponding domain of type XIV collagen. Type XII and XIV collagens belong to the FACIT family and show significant structural homology. However, histolocalization of these collagens appears to be somewhat different (18 -21). In vitro experiments indicate that type XIV collagen appears to interact with a dermatan sulfate glycosaminoglycan (GAG) side chain of decorin (22). Because the type XIV collagen NC1 domain contains a typical sulfated GAG binding sequence, it has been postulated that type XIV collagen interacts with type I collagen fibril through decorin. In contrast, the molecular interaction between type XII collagen, type I collagen, and proteoglycans is not yet fully understood (13). The newly discovered NC1 domain isoforms may provide a long awaited clue toward elucidating the molecular interaction of type XII collagen.
Our rat and mouse ␣1(XII) collagen genomic DNA sequence data further defined two alternative exons 1 and 2 that encode the long and short NC1 domain isoforms, respectively (Fig. 3). The previously reported NC1 domain sequence in mice (3) did not match the NC1 domain sequences found in this study, rather it matched the sequence of the intron immediately following exon 3. FIG. 4. A, poly(A)ϩ RNAs from 7-, 11-, 15-, and 17-day old mouse embryos were analyzed by RNA transfer blots. The mouse ␣1(XII) collagen probe, MRK, primarily hybridized two species: 10 kb (type XIIA collagen) and 8 -9 kb (type XIIB collagen) (Common Probe). Two separate genomic DNA fragments each containing either the exon 1 sequence or the exon 2 sequence hybridized to type XIIA and type XIIB collagen mRNAs, respectively. B, diagrammatic sketches depicting the alternative type XII collagens and proposed nomenclature: type XIIA-1 contains long NC3 domain and long NC1 domain; type XIIA-2 contains long NC3 domain and short NC1 domain; type XIIB-1 contains short NC3 domain and long NC1 domain; and type XIIB-2 contains short NC3 domain and short NC1 domain.
RNA transfer blot analyses indicated that the poly(A)ϩ RNA sample isolated from 15-and 17-day old mouse embryos contained both type XIIA (10 kb) and type XIIB (7-8 kb) mRNAs that are due to alternative splicing in the NC3 domain ( Fig. 1) (2,23,24). It was noted that the exon 1 and exon 2 type XII collagen genomic DNA probes hybridized to the types XIIA and XIIB mRNAs to different degrees (Fig. 4). In light of the NC3 splicing alternatives (type XIIA and XIIB), we propose here a new descriptive nomenclature: 1) type XIIA-1, contains long NC3 domain and long NC1 domain; 2) type XIIA-2, contains long NC3 domain and short NC1 domain; 3) type XIIB-1, contains short NC3 domain and long NC1 domain; and 4) type XIIB-2, contains short NC3 domain and short NC1 domain.
Types XIIA-1 and XIIB-1 include a long NC1 variable sequence encoded by exon 1 (from the 3Ј-end), and types XIIA-2 and XIIB-2 include a short NC1 variable sequence encoded by exon 2. These 5Ј-end (NC3) and 3Ј-end (NC1) alternative splicing mechanisms seem to be coordinately regulated during ␣1(XII) gene transcription. Type XIIA collagen (long NC3) is expressed primarily in embryonic tissues, whereas type XII collagen in adult tissues is predominantly the type XIIB species with the short NC3 domain. The ribonuclease protection assay examining adult mouse tissues further reveals that the type XII collagen long NC1 domain probe is protected in dentoalveolar tissue and tail tissue, which contain periodontal ligament and tail tendon, respectively. Because chicken type XII collagen derived from tendon is homologous to type XIIB-1, we speculate that the long NC1 domain may play a tissue-specific role essential to ligament/tendon extracellular matrix organization.
In contrast, type XIIB-2 collagen with the short NC1 domain appears to be more widely expressed (Fig. 5). Sugrue et al. (25) reported that when monoclonal antibody 75d7 (generated against a synthetic peptide encoding a portion of the long NC1 domain of chicken type XII collagen) was used, the bone tissue including periosteum lacked any immunostaining. However, when another monospecific antibody (generated against a fusion peptide containing a nonvariant-region of the NC3 domain of mouse type XII collagen) was used, the entire periosteum of the long bone and membranous bones was stained (26). The implication of these previously published conflicting results is that newly defined type XIIB-2, which lacks the 75d7 epitope sequence in the long NC1 domain, may be predominantly present in periosteum. It is interesting to note that in nonligament/ tendon tissues, type XII collagen localizes at the tissue interface zones such as the perichondreum of cartilage (18,19,25), the periosteum of bone (20,26), the epimysium, perimysium, and endomysium of skeletal muscle (20), and Descemet's membrane of cornea (26,27). The short NC1 domain of type XII collagen may play a functional role at these tissue interface zones. The deduced sequence of the short NC1 domain contains a potential recipient site for O-serine-linked oligosaccharides at the carboxyl end. It is unknown, however, if this site is posttranslationally modified.
These results seem to suggest that the selection of the 3Ј-end alternative splicing is dependent on the tissue type and func-FIG. 5. The ribonuclease protection assay demonstrates the relative level of ␣1(XII) collagen mRNA in sternal cartilage, maxillary dento-alveolar tissue, eye, skin, and tail of adult mice. The relative amounts of mRNA encoding the type XII collagen alternative isoforms containing long and short NC1 domains were separately analyzed against a housekeeping control, -actin. tion. The newly discovered variations in the NC1 domain of type XII collagen can provide a clue as to the puzzling tissue distribution of type XII collagen in structurally and functionally diverse adult tissues. It is tempting to speculate that type XII collagen may contribute to different extracellular matrix organizations in these adult tissues, potentially through altering the fibril association schemes through its NC1 domain. | 2018-04-03T00:17:01.159Z | 1999-07-30T00:00:00.000 | {
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237597210 | pes2o/s2orc | v3-fos-license | Adverse events reported by Iranian patients with multiple sclerosis after the first dose of Sinopharm BBIBP-CorV
MS patients were one of the first populations vaccinated in Iran. To date, the most used vaccine brand on Iranian MS patients is Sinopharm COVID-19 vaccine. Here is the first study on the adverse events after the first dose of Sinopharm vaccine on 583 Iranian MS patients. A Google form link was sent to MS patients through social networks, between May 1, 2021 and May 22, 2021. No serious adverse event was reported. At least one complaint (mostly transient) was reported by 350 (60%) of vaccine recipients. Constitutional symptoms (malaise, fatigue, fever, shivering, & generalized body pain) (51%) and headache (9%) were the most reported complaints. We found a relation between gender and prior infection with COVID-19 and reported symptoms (p value less than 0.05). Only five recipients (0.9%) reported MS relapse after vaccination. MS worsening was a minor incident related to fever.
Introduction
Evidence suggests that Multiple Sclerosis (MS) prevalence has been rising in Iran in the recent years [1]. Immune system modulation or suppression is the key approach in MS treatment modalities. However, this could predispose the patients to an increased risk of infection [2]. This is the reason that experts in this field have been trying to develop adapted diagnostic, therapeutic, and followup protocols through the pandemic [3]. As the vaccines brought rays of hope to end this dark era, MS patients were one of the first populations vaccinated in Iran. To date, the most used vaccine brand on Iranian MS patients is Sinopharm COVID-19 vaccine. As world health organization (WHO) reported: ''Sinopharm BBIBP-CorV, is a 2-dose b-propiolactone-inactivated, aluminium hydroxide-adjuvanted vaccine administered on a 0/21-28-day schedule". It was authorized by the National Medical Products Administration of China on December 31, 2020, and by about 45 countries/jurisdictions to be used for adults 18 years [4]. It seems a safe choice for patients with MS, regarding safety of previous inactivated vaccines on MS patients and also the lack of severe adverse effects of the Sinopharm vaccine in the primary trial phases [5,6]. To our knowledge, there is only one report of vaccination results of MS patients [7], with none on Sinopharm. Here is the first study on the adverse events after the first dose of Sinopharm vaccine on 583 Iranian MS patients.
Methods: A pilot questionnaire was designed. Following initial revision by two MS patients, the questionnaire was checked by three MS experts. The final Google form link was sent to MS patients through social networks (mainly Telegram Ò ), between May 1, 2021 and May 22, 2021. Each participant was previously registered in the National MS Registry by a unique code. To ensure both the patients' privacy and validity of the data, the responders were asked to enter their code (Supplementary 1). Data on the expanded disability status scale (EDSS), MS type, COVID-19 diagnosis, and the attack were checked by the principal investigator. No other individual access was possible. COVID-19 diagnosis was confirmed by an internist based on either clinical criteria or PCR test results. To differentiate between transient worsening of MS symptoms and a true relapse, full neurologic investigations (history taking, physical exam, imaging) were performed by an MS fellowship.
As the main vaccine for this population was Sinopharm, those patients who got other vaccine types (most of whom were medical staffs) were excluded. The excluded cases have received one of Sputnik V, AstraZeneca, or Bharat Biotech COVAXIN.
Statistical analysis:
Descriptive statistics was used to evaluate the mean and its standard deviation (SD), or frequency of different basic demographic characteristics of the patients (
Contents lists available at ScienceDirect
Vaccine j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e younger than 50), employment status, body mass index (BMI) (those with BMI more or less than 25), education level (illiterate, without or with academic degree)). Same approach was used to study prior COVID-19 infection, MS types (relapsing-remitting (RR) versus progressive), EDSS (those with EDSS higher or lower than 3), MS treatment (receiving anti CD20s like rituximab or ocrelizumab versus other drugs), and also different reported side effects. Side effects were grouped into constitutional (malaise, fatigue, fever, shivering, & generalized body pain), headache, injection site reactions, gastrointestinal (GI) (nausea, vomiting, abdominal pain, diarrhea), and respiratory (coughs, dyspnea). Regarding BMI, patients were asked to enter their weight and height, and the final calculation was made by the software. Binary logistic regression methods were adopted to assess the associations between adverse events and basic patients' characteristics. IBM Ò SPSS Ò version 26 was employed for analyses.
The study protocol was approved at the ethics committee of Tehran University of medical sciences by IRB code of ''IR.TUMS.N I.REC.1400.012".
Result
Out of 1217 MS patients vaccinated till May 22, 2021, 583 cases who received Sinopharm vaccine filled in the questionnaire. Basic characteristics of the patients are summarized in Table 1. Among the participants, 449 (78%) were female and 324 (56%) were unemployed. Mean age was 36.3 (SD: 8.2) with only 44 (8%) of cases older than 50. All the participants were literate, 149 (26%) of whom did not have any academic degree. BMI was>25 in 236 (41%) cases.
At least one complaint was reported by 350 (60%) of vaccine recipients. Constitutional complaints were reported by 299 (51%) cases. Headache was the next most-reported symptom (n = 55, 9%). Injection site reactions were seen in 43 (7%) cases. Thirtythree (6%) complained of GI discomfort. Respiratory symptoms were observed in 25 (4%) cases ( Table 2). The duration of the symptoms was between some hours to 15 days with mean of 2 (SD:2) days.
Two (0.3%) patients complained of worsened MS symptoms; both were due to post-vaccination fever and improved by fever resolution. Five patients (0.9%) reported MS attacks. All happened within two weeks from the vaccination. Different management strategies were employed based on the attack severity (Table 3).
Constitutional symptoms were significantly more reported by female patients (OR:1.87, 95% CI: 1.24-2.80) (p value: 0.003) and those with prior history of COVID-19 infection (OR:1.49, 95% CI: 1.01-2.21) (p value: 0.05). GI complaints were more common among those with prior infection with COVID-19 (OR:2.26, 95% CI: 1.09 -4.67) (p value: 0.03) ( Table 4). No other association was seen between reported adverse events and basic characteristics (both demographic or MS related ones) (p value > 0.05). As may be noted, missing data are not presented in the table, so the total count of each category may be less than 583. (3) 10 (2) Headache 55 (9) Injection site reaction 43 (7) 3. Discussion As far as the authors of the present study are concerned, this is the first study of reported adverse events after the first dose of Sinopharm COVID-19 vaccine on MS population. No serious adverse event was found. The prevalence of at least one adverse reaction was found to be more in our study (60%) compared to the phase two trial of the vaccine (29%) [6]. It is also more than adverse events reported after COVID-19 BNT162b2 vaccine (30% after the first dose) [7]. A recent study in United Arab Emirates (UAE) also showed higher prevalence of at least one postvaccination symptom [8]. Similar to the results of previous studies [68], side effects were mostly transient in our cases.
The most notable symptoms were of constitutional type, followed by injection site reactions, which is in line with the previous studies [6][7][8]. In the phase two trial of the vaccine, fever was the most common post inoculation systemic adverse event of the Sinopharm vaccine [6]. Fatigue and malaise were more common among our population. This may be explained by high prevalence of fatigue among MS patients as general [9] and particularly among Iranian MS patients [10]. Saeed et al. also reported fatigue as a more reported symptom after vaccination in UAE participants [8]. However, no such distribution was reported after COVID-19 BNT162b2 vaccine [7]. They found a minor association between adverse events and young age, lower EDSS, and immunomodulatory drugs [7]. We found a relation between gender and prior infection with COVID-19 and reported symptoms. The latter association may be explained by enhanced immune response after the second exposure to the viral antigens [11]. Regarding gender differences, other observational evidences also suggests higher prevalence of symptoms like lethargy, fatigue, and back pain in female participants [8,12]. Previous studies indicate a greater response to the viral vaccine, followed by more reported adverse events due to the greater inflammatory reaction, in female patients [13,14]. Hormonal and environmental factors are considered as underlying factors [15].
No hypothesis could be addressed to the observed relationship between the post-vaccination GI symptoms and previous COVID-19 infection, so further investigation is encouraged.
Only five recipients (0.9%) reported MS relapse after vaccination. This is close to the acute relapse risk reported in relation to COVID-19 BNT162b2 vaccine (2.1% after the first and 1.6% after the second dose) [7]. MS worsening was a minor incident related to fever, similar to what was found in relation to COVID-19 BNT162b2 vaccine [7].
As confirmed MS diagnosis was a necessity in this step of national vaccination protocol, those with clinically isolated syndrome (CIS) or radiologically isolated syndrome (RIS) were not included in our surveillance.
This primary report involved a limited number of patients in a short duration of follow-up. Further studies with more patients and with longer follow up periods are encouraged.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | 2021-09-23T13:08:52.678Z | 2021-09-23T00:00:00.000 | {
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