extfieldsofstudy: Medicine Chemistry

text:
Reducing tectorial membrane viscoelasticity enhances spontaneous otoacoustic emissions and compromises the detection of low level sound

The mammalian cochlea is able to detect faint sounds due to the presence of an active nonlinear feedback mechanism that boosts cochlear vibrations of low amplitude. Because of this feedback, self-sustained oscillations called spontaneous otoacoustic emissions (SOAEs) can often be measured in the ear canal. Recent experiments in genetically modified mice have demonstrated that mutations of the genes expressed in the tectorial membrane (TM), an extracellular matrix located in the cochlea, can significantly enhance the generation of SOAEs. Multiple untested mechanisms have been proposed to explain these unexpected results. In this work, a physiologically motivated computational model of a mammalian species commonly studied in auditory research, the gerbil, is used to demonstrate that altering the viscoelastic properties of the TM tends to affect the linear stability of the cochlea, SOAE generation and the cochlear response to low amplitude stimuli. These results suggest that changes in TM properties might be the underlying cause for SOAE enhancement in some mutant mice. Furthermore, these theoretical findings imply that the TM contributes to keeping the mammalian cochlea near an oscillatory instability, which promotes high sensitivity and the detection of low level stimuli.

1 Supporting materials and methods 1

.1 Cochlear model Parameters
The addition of longitudinal coupling to the TM bending mode required a slight modification to the model parameters compared to our previous work [1]. The numerical values of the present cochlear model parameters are given in Tables S1 and S2. Some of these parameters appear in Eqs. 4-13 of the manuscript. The other equations of the model are given in previous papers [22,14,17,1].

Electrical Parameters and Model Formulation
The saturating mechanoelectrical transduction current, I max hb , appears in Eq. 4 of the manuscript. I max hb , is related to the parameters given in Table S2 by the following equation: where G max hb is the saturating hair bundle mechanoelectrical conductance and ∆V 0 hb is the resting value of the difference between the scala media potential and intracellular OHC potential.
As discussed in [15], cochlear amplification depends on the slope of the mechanoelectrical conductance vs HB deflection function; as in our previous work [1], the parameters of the mechanoelectrical transduction channel were chosen such that the sensitivity of the BM response is similar to experimental measurements. Because experimental measurements have shown that the mutations have very limited effect on the cochlear microphonic in T ecta Y 1870C/+ and T ectb KO mice [11,26] and on cubic and quadratic distortion product otoacoustic emissions in the Ceacam16 KO mice [2], the operating point of the transduction channel was assumed to be the same for all models.

Middle ear model and parameters
As in our previous work [1], the cochlear model is coupled to a one DOF model of the middle ear with mass, damping and stiffness coefficients. While sound transmission through the middle ear can be represented by more realistic multi-DOF models [19,12], the advantage of a simpler model is that it makes it possible to easily vary the reflection of reverse waves at the stapes to study the influence of the middle ear properties on SOAE generation (see for example Fig. S7). In the frequency domain, the reverse middle ear impedance (i.e, the impedance looking out from the stapes in the reverse direction [21]) is given by: where M s , C s , and K s are the mass, damping coefficient, and stiffness coefficient of the stapes, respectively; ω is the radian frequency and A s is the area of the stapes footplate.
In the theory of coherent reflection [27], the reflection of the reverse traveling wave by the stapes plays a critical role in SOAE generation: SOAEs are expected to be generated only when the amplitude of the stapes reflection coefficient is sufficiently close to 1. This hypothesis was tested in our model. The stapes reflection coefficient, R st , can be calculated using the equation [28,1]: where Z c is the input impedance of the cochlear model, and * denotes the complex conjugate. Two parameter sets for the middle ear model, given in Table S3, were considered: a baseline parameter set (used in all of the numerical results except for some results in Fig. S7), in which |R st | is approximately equal to 0.7 from 10 to 25 kHz; a low |R st | parameter set (used only for Fig. S7) in which |R st | is below 0.2 from 10 to 25 kHz (see Fig. S2 in [1]). At a longitudinal position, x, and frequency, ω, the gain of the BM velocity relative to the stapes velocity is defined as where v bm and v s are the velocities of the BM and stapes, respectively. The pure tone response of the smooth model is compared to experimental data from Refs. [20] and [23] in Fig. S1. As mentioned earlier, model parameters were adjusted so that the gain of the model matched that reported from experimental measurements. At the 34.6 kHz peak position (CF=34.6 kHz, Fig. S1a), the model results are similar to the experimental data in terms of the sharpness of the peaks and relative levels of the gain (both show a ≈ 21 dB difference between low and high SPL). At CF=13.3 kHz (Fig. S1b), the both model and experiment show a ≈27 dB difference between low and high SPL and the peaks are similarly broad. However, there is a noticeable difference of 11 dB between the model and experiment that is possibly due to a mismatch in the structural properties or assumptions of the model. As is the case with many cochlear models (e.g. [30,1,18]), the phase rolls-off or decreases somewhat more rapidly than what is observed experimentally (Figs. S1c and S1d) despite effort during calibration to mitigate this. The mismatch between the model and the experimental data is, however, noticeably smaller than that of the previous version of the model (Fig. 3 in [1]). The pure tone response of the smooth model at all longitudinal positions with the place-frequency map, ratio of the BM gain at CF, and quality factor, Q 10dB are shown in Fig. S2. The model results in Fig. S2 were obtained using a linear formulation of the model; the active and passive models represent the model responses at low and high stimulus levels, respectively. The passive model is obtained by setting G max hb = 0 in Eq. S1. As shown in the figures, the model predictions agree well with the measurements from multiple longitudinal locations.  2 Supporting results

Stability of organ of Corti model
To determine whether the spontaneous oscillations in the model were generated by local instabilities or a global phenomenon, the linear stability of an isolated longitudinal crosssection of the organ of Corti was analyzed (Fig. S3). This organ of Corti model neglects longitudinal coupling and fluid loading. Both the fully active model and a passive model (obtained by setting the mechanoelectrical transduction current to 0) were analyzed. As seen in Fig. S3b, both the passive and active organ of Corti models are linearly stable. The parameters of the cochlear model vary spatially (Tables S1 and S2

Influence of mesh on linear stability
Throughout the results shown in the manuscript, a finite element mesh with elements of longitudinal length ∆x = 25 µm long was used (total of 448 elements along the BM). Furthermore, the roughness function, r(x), is assumed to be a piecewise linear function with breakpoints that are separated by a distance ∆x r . For the results shown in the manuscript, ∆x r was chosen to be also equal to 25 µm. The influence of ∆x and ∆x r on the results was examined. Two different cases were considered: (1) ∆x r was fixed at 25 µm and the element size, ∆x, was varied, shown in Fig.  S4 and (2) the roughness lengthscale, ∆x r , was varied for a fixed element size (∆x = 25 µm) shown in Figs. S5.
The results for the first case (Fig. S4), indicate a slight decrease in stability for models with ∆x = 12.5 µm long elements. When the element size is increased to ∆x = 50 µm, there is a noticeable decrease in the number of linearly unstable models (Fig. S4d). The same roughness variations were used to allow for a meaningful comparison ( Fig. S4a and S4b); using different roughness variations would result in slightly different numbers of linearly unstable modes. For many of the models considered, using the finer mesh results in at most one additional linearly unstable mode than using the mesh used in the manuscript. Shown in Fig. S4c is a less common case, in which two nearly unstable modes for ∆x = 25 µm (indicated by the arrows) become unstable when the element size is reduced to ∆x = 12.5 µm. Such a small decrease in stability (increase in σ) for the finer mesh also affects n inst when N = 20 models are considered, as shown in Fig. S4d. Despite the slight decrease in stability for the finer mesh, the results in Fig. S4d are consistent with results shown in Figs. 2-5 and confirm that the effects of altering viscoelastic coupling on cochlear stability do not change if the size of the element is doubled or divided by two.
Varying the length scale of the roughness, ∆x r , results in different roughness functions, r(x) (Fig. S5). Using a roughness length scale of 10-25 µm is reasonable given the value of the OHCs diameter (8-10 µm [3]) and the variability in OHC organization [13]. While identifying the related individual poles for the three meshes was straightforward in Fig. S4c, there is no such correlation between poles for the results shown in Fig. S5c since different r(x) are used. This difference is due to the roughness having very different spatial variations ( Fig. S5a and S5b). More importantly, the average numbers of linearly unstable modes is not affected much by the changes in ∆x r (Fig. S5d). The results of Fig. S5d show that reducing TM viscoelastic coupling results in a reduction in cochlear stability and an increase in the average number of linearly unstable modes at all ∆x r values that were considered. (c) Linear stability diagram for the "Reduced Both" model for RS=2. (d) Average number of linearly unstable models, n inst , for N=20 RS for the four compling cases, "Baseline" (B), "Reduced Elastic" (RE), "Reduced Viscous" (RV), and "Reduced Both" (RB). The error bar corresponds to ± one standard deviation.

Influence of large roughness variations on linear stability
In Fig. 3c, the influence of varying the amplitude of the random perturbations, ∆R, on the average number of linearly unstable modes was examined for a relatively narrow range of ∆R values (∆R ≤ 1%). For larger random perturbations (1% ≤ ∆R ≤ 40%), shown in Fig.  S6, the average number of unstable modes continues to increases as ∆R is increased, albeit at a slightly lower rate once ∆R exceeeds 5%. In all cases, for a given random perturbation amplitude, reducing TM viscoelastic coupling results in a reduction in cochlear stability and an increase in the average number of linearly unstable modes. The results for the linear stability of the models, shown in Fig. S7, demonstrate that unstable modes are significantly reduced when the magnitude of the stapes reflection coefficient, |R st |, is low. Since SOAEs are only generated when linear unstable modes are present, this implies that SOAE generation requires the stapes reflection coefficient to have a sufficiently high magnitude. This is a consistent with the theory that are SOAEs are a global phenomenon rather than locally generated. Baseline TM Reduced Both Figure S8: Modeling the effect of holes in the TM on linear stability. The random seed (RS) of the electromechanical coupling coefficient is fixed to RS=13 for these results. N = 20 random seeds were used for the roughness in the TM properties. The error bar corresponds to ± one standard deviation.
Because the presence of holes has been reported in the TM of Ceacam16 KO and T ecta Y 1870C/+ mice [2,11], random inhomogeneities in the TM properties were considered. These inhomogeneities could potentially enhance the generation of reflection-type otoacoustic emissions, including SOAEs. These inhomogeneities were introduced by using the following equations of the longitudinal coupling stiffness and viscosity of the TM: where ∆R tm is the amplitude of the random perturbation (which was set to 5%, which is somewhat similar to the percentage of the TM area occupied by holes in the TM of Ceacam16 KO mice [2]), r tm (x) is a number generated by a random number generator based on a normal distribution of average value 0 and standard deviation 1. Since roughness due to holes in the TM or to inhomogeneities in the OHC properties arises from different mechanisms, different random seed numbers were used for r tm (x) in Eq. S5 and r(x) in Eq. 12. The results shown in Fig. S8 show that adding roughness to the TM properties has a limited effect on the linear stability of the models: the N = 20 different models with roughness in the TM properties with the baseline value of the TM parameters have n inst. = 2.5 unstable modes, while the model without roughness in the TM parameters has no unstable modes; for the model with reduced viscosity, the average number of instabilities is slightly higher in the models with roughness in the TM parameters (average=20.4) than in the model without TM roughness (which has 19 unstable modes). The change corresponds to 50% of the baseline value for C LC tm and C tms , 50% of the baseline value for K LC tm and K tms ; 50% and 90% of the baseline value for C f .

Influence of other TM parameters on cochlear stability and SOAEs
Because TM mutations might affect other properties of the TM, the influence of other TM model parameters on linear stability was also analyzed (Fig. S9). For example, given the effect of the mutations on the intrinsic properties of the TM, TM mutations might also cause a reduction in the stiffness or viscosity of the attachment of the TM to the spiral limbus. Reducing the stiffness of the attachment to the spiral limbus, K tms , reduces the average number of linearly unstable modes; this effect is opposite to the increase in linearly unstable modes observed when the TM longitudinal coupling elastic stiffness is reduced. Reducing the viscosity of the attachment of the TM to the spiral limbus slightly increases the average number of linearly unstable modes; however, reducing the TM viscosity has a much more significant effect. It is possible that due to the loss of the Hensen's stripe or broadening of the subtectorial space, TM mutations tend to reduce power dissipation in the subtectorial space, which would affect the effective damping coefficient due to fluid viscosity in the subtectorial space, C f , and would slightly increase the number of instabilities.


extfieldsofstudy: Biology Medicine

text:
Comparative mitogenomics of the Decapoda reveals evolutionary heterogeneity in architecture and composition

The emergence of cost-effective and rapid sequencing approaches has resulted in an exponential rise in the number of mitogenomes on public databases in recent years, providing greater opportunity for undertaking large-scale comparative genomic and systematic research. Nonetheless, current datasets predominately come from small and disconnected studies on a limited number of related species, introducing sampling biases and impeding research of broad taxonomic relevance. This study contributes 21 crustacean mitogenomes from several under-represented decapod infraorders including Polychelida and Stenopodidea, which are used in combination with 225 mitogenomes available on NCBI to investigate decapod mitogenome diversity and phylogeny. An overview of mitochondrial gene orders (MGOs) reveals a high level of genomic variability within the Decapoda, with a large number of MGOs deviating from the ancestral arthropod ground pattern and unevenly distributed among infraorders. Despite the substantial morphological and ecological variation among decapods, there was limited evidence for correlations between gene rearrangement events and species ecology or lineage specific nucleotide substitution rates. Within a phylogenetic context, predicted scenarios of rearrangements show some MGOs to be informative synapomorphies for some taxonomic groups providing strong independent support for phylogenetic relationships. Additional comparisons for a range of mitogenomic features including nucleotide composition, strand asymmetry, unassigned regions and codon usage indicate several clade-specific trends that are of evolutionary and ecological interest.

Traditional molecular systematic research has been largely dependent on phylogenetic reconstructions based on few molecular markers obtained via individual targeted polymerase chain reaction (PCR) and Sanger sequencing approaches [1][2][3][4][5][6][7] . More recently, the advent of second-generation sequencing (SGS), coupled with the development of simplified and streamlined laboratory and bioinformatic pipelines [8][9][10][11][12] , has seen a proliferation of eukaryotic and prokaryotic genomic resources. Notably, advances in genomic approaches have greatly increased the efficiency of recovering complete and near-complete mitochondrial genomes from species across the animal and plant kingdoms, making it a popular candidate for phylogenetic and comparative genomic studies, in addition to its role as a useful barcoding locus for species identification 13 . Despite the rapid growth in mitogenome resources, current datasets predominately come from small and disconnected studies on single or a limited number of related species, introducing sampling biases and impeding research of broad taxonomic relevance [14][15][16] . While there is a general consensus that new mitogenome resources remain valuable for comparative genomic and systematic research, improved representation of mitogenome resources for poorly-represented biological lineages is still required for studies aimed at addressing fundamental evolutionary, genomic and systematic questions 14 . Table 1. Specimen sources and mitogenome accession numbers for 21 decapod species (from 7 infraorders) contributed in this study.
www.nature.com/scientificreports www.nature.com/scientificreports/ Established as a gene order common to insects and crustaceans 67 , the pancrustacean ground pattern (coloured orange and labelled Gr) is predominantly observed for basal decapod groups such as the suborder Dendrobranchiata and infraorders Caridea, Achelata and Glypheidea, appears in a limited number of species within the Astacidea and Gebiidea, and is absent in all species sequenced so far in the other infraorders. Within the context of the inferred phylogeny, gene rearrangement events shared by members of specific clades provide examples of unifying evolutionary signatures (synapomorphies) from higher levels such as the infraorder-specific St1 pattern shared by the four analysed Stenopodidea species, through the superfamily-specific As1 for species in the Astacoidea, and family-specific rearrangements at the base of the Upogebiidae clade illustrated by the Ge1 pattern.

Lack of correlation between MGO variation and substitution rates.
Overall, evidence of episodic positive selection is observed in 10 out of 13 mitochondrial PCGs (ATP6, COX1, COX2, COX3, CYTB, ND1, ND2, ND4, ND5, ND6) and for main branches of most infraorders/suborders, with the exception of Dendrobranchiata, Glypheidea and Axiidea (Supplementary Data S4). Notably, a higher frequency of statistically significant selection is reported in Caridea mostly detected along branches leading to the genera Alpheus, Palaemon and Macrobrachium. On the other hand, accelerated evolutionary rates are observed for multiple decapod lineages across all infraorders (Supplementary Data S4). Based on observation, while there appears to be a link between substitution rates and heightened gene rearrangements in some taxonomic clades, for example in Parastacoidea (southern hemisphere crayfish) or Caridea, there are also instances where accelerated rates are  Decapod phylogenetic tree. This cladogram was inferred using the maximum-likelihood method based on Dataset I (13 mitochondrial PCGs, 10 359 nucleotide alignment). Clades are coloured according to the different infraorders. The outer colour strip in the phylogenetic tree represents the distribution of mitochondrial gene orders (MGO) in various infraorders and summarises a total of 59 different MGOs across the 246 different decapod species analysed, labelled for each infraorder in the panels below. Orange-coloured MGO labelled with 'Gr' refers to the pancrustacean ground pattern; other derived MGOs are numerically labelled and attached with a 2-letter infraorder prefix. MGOs that differ from the ground pattern are a result of a series of CREx-predicted gene rearrangement events: transposition (T), reversal (r), reverse transposition (rT), duplication (d), deletion (x) and tandem duplication-random loss (tdrl). Yellow-or red-coloured circles on some nodes reflect the level of uncertainty for the TreeREx reconstruction of each ancestral MGO, with red exhibiting highest level of uncertainty, yellow for mid-level and no circle for consistent reconstruction (see Babbucci,et al. 42 for details). Subsequent outer rings indicate, to the best of our knowledge, the possible environments (terrestrial, freshwater, marine, vents/seeps) inhabited by each decapod species.
www.nature.com/scientificreports www.nature.com/scientificreports/ observed for lineages with highly conserved MGOs as well. However, across Decapoda as a whole, the Spearman correlation analysis does not suggest a link between variable MGOs and nucleotide/amino acid substitution rates (Supplementary Data S4).

Mitogenome characteristics vary within and between decapod infraorders. Applying the
Hyper-Empirical Relative Mitochondrial Evolutionary Speed (HERMES) index developed by Plazzi, et al. 41 , the amount of mitochondrial evolution was estimated based on several genomic features found to be phylogenetically congruent in their tested datasets. These genomic features include the root-to-tip distance for each species (RtoTdist), ML distance from the E. pacifica outgroup (MLdist), percentage of Unassigned Regions (URs), amount of mitochondrial identical gene arrangements (AMIGA) for PCGs and strand usage skew (SUskew). The HERMES index appears to be informative for our dataset with reasonable goodness-of-fit statistics: Tucker-Lewis Index (TLI) 70 = 0.939; root mean square of the residuals (SRMR) = 0.053; root mean squared error of approximation (RMSEA) = 0.058, all close to boundaries suggested by Hu and Bentler 71 . The highest communality was for AMIGA at 85.5% whereas other variables scored at only 10 to 20%, resulting in a mean of 29.1% (i.e. the HERMES index accounts for 29.1% of the total variability of the source matrix). Since gene order is the key parameter in this system, the HERMES factor analysis in Fig. 3 separates individuals into two distinct groups. The first group exhibits low HERMES index and consists of individuals with MGO that is identical to or highly similar to the pancrustacean ground pattern (Gr) while the second group comprises mostly of individuals that have undergone tdrl events resulting in large rearrangements of mitochondrial PCGs (Fig. 2).
Several notable observations can be made on the clustering of data points based on various mitogenomic features, as observed in principal component analysis (PCA) plots in Fig. 4. The plot in Fig. 4a is based on the same five variables in the previous HERMES analysis. While most individuals with MGOs identical or similar to the pancrustacean ground pattern tend to form a tight cluster, some of these are distinctly separated from others based on the proportion of their unassigned regions, most of them either containing more than one control region (e.g. Metanephrops, Munida) or long intergenic regions (e.g. Geothelphusa, Longpotamon) (Fig. 4a). Additionally, Fig. 4b-d summarise nucleotide composition, asymmetry (skew) information and amino acid composition respectively. The AT content separates most of the brachyuran crabs from caridean shrimps, whereas GC-skew distinguishes the two Astacidea superfamilies (Astacoidea, Parastacoidea) (Fig. 4b). The two burrowing mud shrimp infraorders, Axiidea and Gebiidea, are often clearly split into different quadrants for plots based on nucleotide features, whereas data points representing anomuran species are generally widely scattered across all PCA plots.

Discussion
This study adds to the growing list that has benefited from the use of museum-preserved specimens for mitogenomic studies 37,72-77 , with mitogenomes for 21 species strategically sampled to fill important taxonomic gaps, particularly in under-represented decapod infraorders. The higher level topologies of all three Maximum likelihood trees reconstructed in this study are congruent at the base and top of the trees (Fig. 1), consistently recovering: [1] The suborder Dendrobranchiata as sister group to the rest of egg-brooding Pleocyemata, [2] Caridea as basal to Pleocyemata, [3] Stenopodidea as sister to all of Reptantia, [4] Axiidea and Gebiidea as separate www.nature.com/scientificreports www.nature.com/scientificreports/ infraorders, and [5] Anomura and Brachyura placed as sister taxa high in the tree, generally in agreement with previous reports 1,11,36,37 though with points of distinction with others 2,3,6,78,79 . While the higher level topology of the Bayesian-inferred tree generally exhibits similar observations, there are deviations with respect to the relationship between Caridea and Stenopodidea (recovered as sister clades) and the shuffle in positions of Axiidea, Anomura and Brachyura. Generally, incongruent topologies show that inter-relationships among lobster and crayfish infraorders still remain unresolved 1,2,6,7,78,80,81 , requiring further taxonomic sampling and/or additional nuclear gene information. It is also noteworthy that the taxonomically-impoverished infraorder Procarididea 5,82 is absent from our study, but is considered by most to likely be one of the basal lineages within the Decapoda 5 .
The phylogenetic analysis presented in this study represents one of the most comprehensive samplings of decapod species, based on mitochondrial data. While genome-based phylogenies are still rare for the Decapoda due to the generally large sizes of its genomes (1 to 40 Gbp), the increasingly lower costs of sequencing are enabling studies to target more genomic loci through genome skimming or anchored hybrid enrichment methods [83][84][85] . However, these still lack the fine-grained resolution obtained in this study. Two noteworthy studies that have been recently published have attempted to elucidate decapod inter-relationships based on genomic (Wolfe,et al. 84 ; 94 decapod species, 410 loci, greater than 86 000 bp) or transcriptomic data (Schwentner,et al. 86 ; 16 decapod species, 81 to 684 orthogroups, 17 690 to 242 530 amino acid positions). Mitogenome-based maximum likelihood phylogenies in this study share similarities in parts of their topologies with that obtained from these studies, specifically in the recovery of shrimp and prawn groups as basal clades and the traditional Meiura (Brachyura + Anomura) as more derived lineages, increasing the confidence of these nodes based on support from different underlying genetic data. Nevertheless, several nodes are still in contention. This study recovers a paraphyletic Caridea and Stenopodidea clade whereas both Wolfe, et al. 84 and Schwentner, et al. 86 recovered these infraorders as sister clades. The position of Achelata also remains unresolved in most trees as well as the interrelationships among crayfish and lobster groups, as well as the positions of the traditional thalassinids (Axiidea, Gebiidea). Overall, topologies inferred in this study are generally more similar to that obtained by Wolfe, et al. 84 .
The contrasting MGO patterns across all available decapod mitogenomes have revealed some interesting findings. Most notably, a number of highly-rearranged gene orders are observed, occurring in unequal frequencies across various decapod infraorders or at lower taxonomic levels, with a level of diversity higher than or at least comparable to the levels observed in other metazoan groups 42,47,49,63,87,88 . We see the prevalence of the pancrustacean ground pattern (Gr) 67 , and/or other highly-similar MGOs in infraorders at both the basal and more derived positions, indicating that this highly-conserved ground pattern is likely to be ancestral for decapods, with modified MGOs within each infraorder being more derived traits 35 . Hence, instances of clade-specific MGO patterns or MGO "hot spots" suggest that the utility of MGOs as synapomorphies is amplified for certain decapod groups, acting as unifying evolutionary signatures that can be used to either support or question existing classifications at a range of taxonomic levels 31,32,34,35,62,66,67,69 . For example, brachyuran species associated with the Varunidae (superfamily Grapsoidea) and Macrophthalmidae (superfamily Ocypodoidea) that form sister clades 31,66,89,90 also share the Br2 MGO pattern, which provides complementary support to other phylogenetic studies that call for a re-evaluation of classifications for the two large paraphyletic superfamilies 33,[89][90][91] . Nonetheless, it is equally important that we continue to be cautious when evaluating MGO information within a phylogenetic context 42,66,92 , keeping in mind the caveats related to potential homoplastic or convergent arrangements 42,87,93,94 , a saturation of signals from reversible rearrangement events 95,96 or possibly inaccurate inferences from unresolved phylogenies 66 .
In contrast to findings that found a positive correlation between gene rearrangements and elevated nucleotide substitution rates in insect and bivalve mitogenomes 41,45 , our correlation analysis indicates no such association for the Decapoda (Spearman correlation in Supplementary Data S4). Although the exact drivers of MGO rearrangements remain unclear, other studies have noted broad associations with adaptations to extreme ecological niches and lifestyles 34,62,66,68,97,98 . Similarly, accelerated rates of MGO rearrangements occur in various decapod lineages such as burrowing crayfish 34 , freshwater crabs 66 , and other species adapted to extreme deep-sea environments or temperatures 66,98 . However, again, our study indicates no such correlation between MGOs and ecologically-similar decapod groups. For example, freshwater crabs and crayfish (southern hemisphere parastacids) display highly variable MGOs, while other freshwater groups such as the shrimps and northern hemisphere crayfish maintain the same gene order among all sampled species (although the members of northern hemisphere crayfish group have a large distinctive rearrangement that acts as a synapomorphy for the superfamily). The same observation applies to other crab and shrimp species inhabiting deep sea vent niches, or burrowing axiid mud shrimps, which exhibit only a few rearranged MGOs.
However, ecological transitions and the evolution of a distinct Bauplan or the adoption of different lifestyles (e.g. parasitism or adaptation to freshwater) may be achieved via different evolutionary pathways 99-101 and the consequences for energy demands for an organism may be just one of a plethora of factors including thermal tolerance 102 , aerial exposure 103 , sensitivity to salinity 104 , light 105 or metal concentrations 106 . In addition to the regulation of their transcription and translation 107,108 , mitochondria are very dynamic structures often undergoing continuous fusion, fission and motility, events that can potentially affect their bioenergetic capacities 109 . However, the extent of these rearrangements and the cues that trigger them are still poorly understood. Thus, we do not rule out an association of heightened MGO rearrangements and profound changes at deep taxonomic levels (e.g. the two groups of crayfish) to clade-specific adaptations and ecological/life history transitions, but these cannot be confidently determined until we achieve a better understanding of the stressors involved at the cellular and physiological level and the associated adaptive responses.
Previous studies have compared decapod MGO patterns at lower taxonomic levels across the Decapoda 31,32,34,35,60,66,110,111 . However, this study is one of the few to investigate the extent of MGO diversity at the ordinal level and a large number of species (246 species), the other being a phylomitogenomic study on 86 malacostracan mitogenomes 38 . Other large-scale comparative MGO analyses for various metazoan groups have also reported on the distribution of MGOs, some showing relatively high rates of MGO evolution from www.nature.com/scientificreports www.nature.com/scientificreports/ across major taxonomic groups such as beetles, echinoids, batoids and elasmobranchs 64,65,119,120 . While decapod mitogenomes clearly showcase a high diversity of MGO patterns as a whole (59 MGOs across 246 species), it is premature to claim that this degree of diversity is unusual or comparable to that of other metazoan groups since results are influenced by the number of mitogenomes compared (i.e. scale of comparison), the taxonomic level of interest or as a result of unbalanced or biased sampling. We therefore highly recommend interpreting the findings from any comparative analyses with caution especially those still lacking representation for major groups and again, urge for more consideration to be given to better sampling strategies.
Comprehensive studies on the evolutionary trends of mitogenomes (both small and large scale) are also emerging for various animal groups, following the increased availability of mitogenomic resources 41,119,[121][122][123][124][125][126] . To determine if there are other aspects of the decapod mitogenome that may show clade-specific associations, aside from the arrangement of genes, we compared additional aspects of the molecular architecture and composition of mitogenomes across the major decapod groups. Mitogenomes analysed in this study are generally around 16 kbp in length, though some outliers were identified (17-20 kbp) with lengths inflated by a higher proportion of unassigned regions, i.e. containing more than one control region (e.g. Metanephrops species 61 ) or relatively long stretches of intergenic spaces such as in most Potamoidea species 127,128 . Though proportions of unassigned regions may potentially be the discerning feature for some taxonomic groups 129 , this notion should also be treated with caution as the sizes of these regions may be influenced by the assembly or annotation methods used to predict genes, exemplified by the identification of three trnL genes by Segawa and Aotsuka 127 as opposed to only two copies reported following re-annotations by MITOS in this study 10 . But generally, decapod mitogenome sizes are relatively stable compared to those of other metazoan groups such as bivalves, nematodes and sponges that exhibit strong heterogeneity in size 88 , with some larger molecules spanning lengths of over 20 kbp and even up to 48 kbp 130 .
Strand and nucleotide composition asymmetries are other mitochondrial characteristics that have been noted to vary across animal groups 49,50,[131][132][133] . In this study, we point out observed trends from PCA plots that suggest molecular signatures for certain decapod groups. Similar to insects, annelids and arachnids, decapod mitogenomes are generally AT-rich, as opposed to lower compositional AT bias often observed in chordates (e.g. fishes, birds, reptiles) 134 . Within the Decapoda itself, recognizable clusters were observed for Caridea (average: 65%, range: 59% to 70%) and Brachyura (average: 71%, range: 65% to 77%) with minor overlaps. Other noteworthy features include positive GC-skew values that appear to be a unifying characteristic for all northern hemisphere freshwater crayfish (Astacoidea) as well as for the four Coenobita anomuran species included in this study, as opposed to negative GC-skew values for most other decapods. Further, species from infraorders Axiidea and Gebiidea are well separated due to substantial differences in compositional bias and asymmetry, which, in addition to previous reports from phylogenetic and MGO analyses 6,35,135,136 , is consistent with their status as separate infraorders instead of united in what was once the Thalassinidea 1,78,137,138 . On the other hand, data points for anomuran species are dispersed across all plots, highlighting their higher plasticity and mitogenomic variability within this infraorder notorious for its morphological and ecological diversity, including the convergent evolution of the "crab" form 66,69,101,139,140 and taxonomic controversies [141][142][143][144] .
For all discussed groups with their own unique signatures, it is of interest to see if these patterns still hold when more mitogenomes are included in future analyses. Though this study is limited in scope to comparisons across whole mitogenomes, we recognise that this only skims the surface of the range of possible compositional comparisons (e.g. at the gene level) and that there are a myriad of other factors that can be included in further detailed investigations, e.g. testing different codon sites 43,44 or the effects of these asymmetries on nucleotide versus amino acid compositions 50,145 .
All Maximum-likelihood trees reconstructed from the three mitochondrial-based datasets (Datasets I to III) were rooted with E. pacifica (Euphausiacea), which is often treated as sister clade to the Decapoda 159 . Support at each node is evaluated with 1000 ultrafast bootstrap replicates (UFBoot) 160 and 1000 SH-aLRT replicates (SH) 161 .

Mitochondrial gene order (MGO) analysis.
Prior to gene order analyses, public mitogenome entries downloaded from GenBank were manually inspected for inaccuracies via cross-checks against results from the MITOS webserver 10 . Any discrepancies found (e.g. incorrect strands specified, missing genes, mislabelling, etc) were corrected and new GenBank files were generated (Supplementary Data S5). Mitogenomes were then processed through the MitoPhAST v3.0 pipeline, which compares and clusters MGOs into groups according to gene order patterns 66 . Similar to Tan, et al. 66 , the output Fasta format file containing MGO for every mitogenome was pre-processed to remove missing genes in one or more species and also to retain only a single copy of duplicated genes. Using each of the generated Maximum-likelihood trees (nucleotide-and amino acid-based) as a phylogenetic guide, gene re-arrangement pathways and putative ancestral MGOs were reconstructed with TreeREx v.1.85 54 . To obtain more accurate inferences, TreeREx results were further used to guide pairwise comparisons using web-based CREx 55 to enable the inclusion of fuller sets of genes that would have been excluded in the TreeREx analysis. The interactive Tree of Life (iTOL) online phylogenetic tool 162 was used to illustrate the distribution of MGOs across various infraorders in the phylogenetic tree.

Comparison analyses of other mitochondrial features.
Excluding individuals with partial mitogenomes, Hyper-Empirical Relative Mitochondrial Evolutionary Speed (HERMES) index is generated for each of 239 individuals using HERMES v.1.0 41 to estimate the amount of mitochondrial evolution. Given a maximum likelihood tree, the program computes the root-to-tip distance for each species (RtoTdist) and ML distance from the E. pacifica outgroup (MLdist) then merges these with other mitochondrial characteristics including the percentage of Unassigned Regions (URs), amount of mitochondrial identical gene arrangements (AMIGA) for PCGs and strand usage skew (SUskew). Also, codon usage counts in each individual were obtained with EMBOSS v.6.6.0 163 with minor adjustments for the Invertebrate Mitochondrial Code, followed by the calculation of relative synonymous codon usage (RSCU) values by taking the ratio of the actual number of times a codon appears to the expected frequency of the codon if all synonymous codons for the same amino acid are used equally 164 . Various mitogenome characteristics were then summarised by Principal Component Analysis (PCA) using Pearson correlation carried out with XLSTAT v.2018.5.52460 165 . Episodic selection, evolutionary rates and spearman correlation test. Without making an a priori assumption on the likelihood of specific lineages undergoing episodic positive selection, the aBSREL method 166 implemented in the command-line version of the HyPhy v.2.3.11 package 167 was used on the codon alignment of each mitochondrial protein-coding gene to test each branch in the maximum -likelihood phylogeny inferred from Dataset I, to inspect whether a proportion of sites have evolved under positive selection (ω > 1). Further, evolutionary rates were estimated from the same codon alignments using BEAST v.2.5.0 168 . An uncorrelated, lognormal relaxed clock model was applied to each partition and the Yule Model was used as the tree prior since taxa consist of individuals from different species, with Euphausia pacifica set as outgroup. Two BEAST MCMC runs of 6 × 10 9 were performed and convergence was checked with Tracer v.1.7.1 169 , applying a burn-in of 20% and checking for sufficient Effective Sample Size (ESS) (>200). Finally, trees from both MCMC runs were combined and a maximum clade credibility tree was constructed. Rates were visualised with Figtree v.1.4.3 170 . Correlations among different MGOs, habitat and substitution rates were measured by the Spearman correlation coefficient carried out with XLSTAT v.2018.5.52460 165 , details in Supplementary Data S4.

Conclusion
Moving forward, further research is needed to contrast the composition and architecture of mitogenomes across other metazoan groups to determine if our observed trends are common or specific to the Decapoda. As more mitogenomic data is made available, coupled with bioinformatics tools such as the MGO analysis feature in the MitoPhAST pipeline, it becomes increasingly feasible to conduct more complete and larger-scale comparisons for any animal group of interest in the future, the bottleneck now being to ensure that annotations in public database entries are accurate, which in this study was still a manual process. We have been fortunate to benefit from mitogenomes generated by various research groups that have contributed to enriching these resources for the order Decapoda but also realise that there is still need for new mitogenomes, carefully sampled to achieve a more balanced representation of infraorders. More importantly, by demonstrating what is possible for large-scale comparative MGO analyses in this study, it is our hope to inspire the undertaking of future research equivalent to or surpassing the extent of this study, contributing as a collective to the eventual reformation of the field of mitochondrial genomics.

Data Availability
Assembled mitogenomes are available on NCBI repository (See Table 1 for GenBank SRA accession numbers). Raw datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry Medicine

text:
Catalpol attenuates lipopolysaccharide-induced inflammatory responses in BV2 microglia through inhibiting the TLR4-mediated NF-κB pathway.

Catalpol, an iridoid glucoside mainly found in the root of Rehmannia glutinosa Libosch, is known to possess various pharmacological effects. Here, we investigated its inhibitory potential against inflammatory responses in lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results showed that catalpol significantly suppressed LPS-induced secretion of pro-inflammatory mediators, including nitric oxide (NO) and prostaglandin E2. Consistent with these results, catalpol downregulated LPS-stimulated expression of their regulatory enzymes, such as inducible NO synthase and cyclooxygenase-2. Catalpol also inhibited LPS-induced production and expression of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β. Additionally, catalpol suppressed the nuclear factor-kappa B (NF-κB) signaling pathway by disrupting the phosphorylation and degradation of inhibitor of κB-α and blocking the nuclear translocation of NF-κB p65. Moreover, catalpol inhibited LPS-induced expression of toll-like receptor 4 (TLR4) and myeloid differentiation factor 88, which was related to suppression of the binding of LPS with TLR4 on the cell surface. Furthermore, catalpol markedly reduced LPS-induced generation of reactive oxygen species (ROS). Collectively, these results suggest that catalpol can repress LPS-mediated inflammatory action in BV2 microglia through inactivating NF-κB signaling by antagonizing TLR4 and eliminating ROS, indicating that catalpol can have potential benefits by inhibiting the onset and/or treatment of inflammatory diseases.

Introduction
Glial cells, including microglia and astrocytes, are the central nervous system (CNS) immune cells that play a critical role in the development and maintenance of the brain. However, these cells dysfunction due to hyperactivity in response to inflammatory signals is closely related to the onset and progression of various neurodegenerative diseases by damaging brain nerve cells (Tremblay et al. 2011;Gomez-Nicola and Perry 2015). Toll-like receptors (TLRs) among the various receptors that stimulate inflammatory signaling belong to the family of Toll homologous proteins, which functions as a receptor for recognition of pathogen-associated immune responses. Among bacterial endotoxins, lipopolysaccharides (LPS), present in the outer membrane of gram-negative bacteria, can induce excessive activation of glial cells through binding to TLR4, which was coupled with increased expression of TLR4 (Glass et al. 2010;Cherry et al. 2014). TLR4 ultimately activates a variety of downstream signaling pathways, including the nuclear factor-kappa B (NF-κB) signaling pathway, leading to elevated secretion of pro-inflammatory mediators and cytokines (Li and Verma 2002;Kopitar-Jerala, 2015;Lee et al. 2017). Therefore, antagonists to TLR4 can counteract the pro-inflammatory downstream signaling pathway by inhibiting different target gene expression and cellular responses. In addition, over-activated microglial cells stimulated by LPS can induce oxidative stress by increasing the generation of reactive oxygen species (ROS); this further aggravates the inflammatory response (von Bernhardi et al. 2015;Daulatzai 2016). These observations suggest that blocking excessive activation of microglia is an important tool to delay the induction and progression of several brain diseases.
Rehmanniae Radix, the root of Rehmannia glutinosa Libosch, belongs to the Scrophulariaceae family, which has long been used for the prevention and treatment of various diseases in Korea and other Asian countries Matsumoto and Sekimizu 2016;Liu et al. 2017a). Catalpol is one of the iridoid glycoside compounds purified from Rehmanniae Radix. It is known to have several remarkable pharmacological effects, such as anti-asthma , anti-diabetic (Bao et al. 2016;Zhu et al. 2016), hypoglycemic (Li et al. 2014), and antitumor (Jin et al. 2015;Liu et al. 2017b;Wang and Zhan-Sheng 2018) properties. It has also been reported that catalpol acts as a neuroprotective agent for ischemic injury and neurodegenerative diseases (Tian et al. 2006;Zhang et al. 2007;Zheng et al. 2017). In addition, its antioxidant effect can be achieved by blocking ROS production Bi et al. 2013;Cai et al. 2016). As previously reported, catalpol has also shown potent anti-inflammatory effects in a variety of experimental models using LPS and inflammatory cytokines (Tian et al. 2006;Zhang et al. 2009Zhang et al. , 2016Bi et al. 2013;Fu et al. 2014;Zhou et al. 2015), which were found to be related to inhibition of NF-κB signaling activity (Zhang et al. 2009Choi et al. 2013;Xiao et al. 2014;Zhou et al. 2015). Although it has been suggested that TLR4 may be involved in the antiinflammatory effects of catalpol (Bi et al. 2013;Fu et al. 2014;Zhang et al. 2016), the precise role of TLR4 in relation to the protective effects of catalpol has not yet been established. Moreover, studies on the microglial cell model have not been performed properly. Therefore, the objective of this study was to examine the anti-inflammatory potency of catalpol and determine the effect of catalpol on activation of the TLR4 signaling pathway in BV2 microglia stimulated by LPS.

Cell culture and LPS stimulation
BV2 microglia were maintained in DMEM containing 10% FBS, L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere with 5% CO 2 and 95% air. Catalpol was dissolved in DMSO and adjusted to the final concentration using complete culture medium. To stimulate BV2 cells, the medium was replaced with fresh DMEM and 100 ng/ml LPS was added in the presence or absence of catalpol for the indicated time periods.

Assessment of cell viability
To determine cell viability, BV2 cells were seeded into 96well plates at a density of 1×10 4 cells per well. After 24 h of incubation, cells were treated with various concentrations of catalpol or pretreated with different concentrations of catalpol for 1 h before LPS (100 ng/ml) treatment for 24 h. Afterward, the medium was removed and 0.5 mg/ml MTT solution was added to each well. After incubation at 37°C for 3 h, the supernatant was then replaced with DMSO to dissolve blue formazan crystals in each well. After 10 min of incubation at 37°C, the optical density was measured at a wavelength of 540 nm on an ELISA microplate reader (Dynatech Laboratories, Chantilly, VA, USA). The optical density of formazan formed in control (untreated) cells was taken as 100% viability.

Measurement of pro-inflammatory mediators and cytokines production
Levels of NO production were indirectly determined by measuring stable NO catabolite nitrite in a medium by Griess reaction. Briefly, the conditioned medium (100 µl) was mixed with the same volume of Griess reagent and incubated at room temperature for 10 min. The optical density at 540 nm was then measured on an ELISA microplate reader and the concentration of nitrite was calculated according to a standard curve generated from known concentrations of sodium nitrite. Levels of PGE 2 , TNF-α and IL-1β in the culture medium were measured using commercial ELISA kits, according to the manufacturer's instructions, as described previously (Choi et al. 2017).

RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR)
BV2 cells were pretreated with various concentrations of catalpol for 1 h, followed by treatment with 100 ng/ml LPS for 24 h. Total RNA from isolated cells was prepared using a TRIzol reagent, following the manufacturer's instructions, and quantified. For mRNA expression analysis, cDNA was synthesized from 1 μg of total RNA using AccuPower® RT PreMix (Bioneer, Daejeon, Korea) containing moloney murine leukemia virus reverse transcriptase. The RT-generated cDNA was amplified using selected primers, under the following conditions: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. GAPDH was used as an internal control.

Protein isolation and Western blot analysis
Cells were collected, and cellular proteins were prepared, as described previously . Cytosolic and nuclear proteins were separated using a commercial kit according to the manufacturer's protocol. For Western blotting, equal amounts of protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. Subsequently, the membranes were blocked with 5% non-fat dry milk/Trisbuffered saline containing 0.1% Triton X-100 (TBST) for 1 h and incubated with the appropriate antibodies at 4°C overnight. After washing membranes with TBST, they were incubated with the appropriate HRP-conjugated secondary antibodies for 2 h at room temperature. Protein bands were visualized using an ECL kit according to the manufacturer's instructions.

Immunofluorescence staining for nuclear translocation of NF-κB and formation of LPS/TLR4 complexes
After cells were pretreated with or without 500 µM catalpol for 1 h, they were treated with 100 ng/ml LPS. The cells were then fixed in 3.7% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 15 min, and blocked with PBS containing 5% BSA. The cells were then stained with primary antibody against NF-κB p65 at 4°C overnight followed by incubation with a fluorescein-conjugated anti-rat IgG in the dark at 37°C for 40 min. In addition, these cells were treated with AF 488-conjugated LPS (100 ng/ml) for 30 min in the presence or absence of catalpol to analyze the formation of LPS/TLR4 complexes. Fixed cells were also stained with anti-TLR4 antibody at 4°C for 90 min and then incubated with secondary antibody conjugated with Alexa Fluor 594 at room temperature for 1 h. Nuclei were sequentially stained with DAPI solution (2.5 µg/ml). Slides were mounted and fluorescence images were captured by fluorescence microscopy (Carl Zeiss, Oberkochen, Germany).

Measurement of TLR4 expression on cell surface
To investigate the effect of catalpol on TLR4 expression on the cell surface, AF-LPS was used to treat BV2 cells pretreated with or without catalpol for 1 h. After treatment, cells were washed twice with PBS, harvested with 0.005% ethylenediaminetetraacetic acid, and then analyzed by flow cytometry. Alexa 488 was excited using 488 argon ion laser lines and detected on channel FL1 using a 530 nm emission filter. Fluorescence emission of samples was recorded by flow cytometry (Becton Dickinson, San Jose, CA, USA) as previously described (Lee et al. 2012).

Detection of intracellular ROS levels
Production of intracellular ROS was monitored using DCF-DA. In short, cells were treated with 500 µM catalpol for 1 h or pretreated with 500 µM catalpol for 1 h followed by culture for 1 h in the presence or absence of 100 ng/ml LPS. These cells were harvested and stained with 10 µM DCF-DA in the dark at 37°C for 15 min. After rinsing twice with PBS, cells were immediately analyzed by flow cytometry with an excitation wavelength of 480 nm and an emission wavelength of 525 nm. To observe the degree of ROS production by fluorescence microscopic observation, cells attached to glass coverslips were stimulated with or without 100 ng/ml LPS after 500 µM catalpol treatment for 1 h. These cells were stained with DCF-DA, washed twice with PBS, and fixed with 4% paraformaldehyde (pH 7.4) for 20 min, after which the fixed cells were analyzed by fluorescence microscopy.

Statistical analysis
Results are presented as the mean ± the standard deviation (SD). Statistical significance was assessed using Statistical Package for the Social Sciences (SPSS) software (version 18.0) to identify significant differences based on one-way analysis of variance (ANOVA) followed by Dunn's post-hoc tests. Statistical significance was considered at p value < 0.05.

Catalpol suppresses LPS-induced NO and PGE 2 production in BV2 microglial cells
To examine the inhibitory effect of catalpol on LPS-induced production of pro-inflammatory mediators, including NO and PGE 2 , BV2 cells were pretreated with different concentrations of catalpol (0, 100, 250 or 500 µM) for 1 h and then stimulated with 100 ng/ml LPS for another 24 h. Levels of NO and PGE 2 in culture supernatants were determined by Griess reaction assay and ELISA, respectively. As shown in Figures 1A and B, stimulation with LPS alone markedly increased NO and PGE 2 production in comparison with no stimulation with LPS (p < 0.05 vs. control; about 12-fold and 15-fold, respectively). Conversely, catalpol significantly suppressed LPS-induced secretion of NO and PGE 2 in BV2 cells in a concentration-dependent manner. Significant repression was observed with 100 µM of catalpol (p < 0.05 vs. LPS).

Catalpol attenuates LPS-induced iNOS and COX-2 expression in BV2 microglial cells
We next investigated whether the inhibitory effects of catalpol on NO and PGE 2 production were related to regulation of the expression of their synthesis enzymes, such as iNOS and COX-2. The results of RT-PCR and immunoblotting demonstrated that catalpol concentration-dependently Figure 1. Suppression of LPS-induced production NO and PGE 2 and expressions of iNOS and COX-2 by catalpol in BV2 microglial cells. Cells were pretreated with the indicated concentrations of catalpol for 1 h prior to incubation with 100 ng/ml LPS for 24 h. The levels of NO (A) and PGE 2 (B) in culture media were measured by Griess assay and a commercial ELISA kit, respectively. Each value indicates the mean ± SD as a representative result obtained from three independent experiments (* p < 0.05 compared to control; # p < 0.05 compared to cells cultured with 100 ng/ml LPS). The mRNA (C) and protein (D) levels of iNOS and COX-2 were determined by RT-PCR and Western blot analysis, respectively. GAPDH and β-actin were used as internal controls for the RT-PCR and Western blot assay, respectively. E. Bands were quantified using ImageJ and normalized to GAPDH and β-actin, and the ratio was determined. Data are expressed as mean ± SD. All experiments were repeated three times (* p < 0.05 compared with the LPS group).
inhibited expression levels of iNOS and COX-2 mRNA and protein in LPS-stimulated BV2 cells (p < 0.05 vs. LPS). This finding indicates that catalpol can suppress NO and PGE 2 production by reducing the expression of their encoding genes at the transcriptional levels.

Catalpol reduces the production and expression of pro-inflammatory cytokines in LPS-stimulated BV2 microglial cells
The effect of catalpol on the production of pro-inflammatory cytokines, including TNF-α and IL-1β, in LPS-stimulated BV2 cells was determined. According to our ELISA results (Figures 2A and B), the production of the two cytokines was significantly increased in the culture medium of LPSstimulated BV2 cells (p < 0.05 vs. control) but decreased in the presence of catalpol in a concentration-dependent manner (p < 0.05 vs. LPS). In addition, suppression of TNF-α and IL-1β production by catalpol was associated with inhibition of the expression of these genes ( Figures 2C and D), suggesting that catalpol LJGP prevented pro-inflammatory cytokine production through suppression of their gene expression in LPS-stimulated BV2 microglia.

Effect of catalpol on cell viability in BV2 microglial cells
An MTT assay was performed to investigate whether the inhibitory effect of catalpol on production of pro- Figure 2. Inhibition of LPS-induced production and expression of TNF-α and IL-1β by catalpol in BV2 microglial cells. BV2 cells were pretreated with various concentrations of catalpol for 1 h followed by treatment with 100 ng/ml LPS for 24 h. The levels of TNF-α (A) and IL-1β (B) present in the supernatants were measured using the corresponding ELISA kits. Each value indicates the mean ± SD as a representative result obtained from three independent experiments (* p < 0.05 compared to control; # p < 0.05 compared to cells cultured with 100 ng/ml LPS). The mRNA (C) and protein (D) levels of TNF-α and IL-1β were determined by RT-PCR and Western blot analysis, respectively. GAPDH and β-actin were used as internal controls for the RT-PCR and Western blot assay, respectively. E. Bands were quantified using ImageJ and normalized to GAPDH and β-actin, and the ratio was determined. Data are expressed as mean ± SD. All experiments were repeated three times (* p < 0.05 compared with the LPS group). inflammatory mediators and cytokines was due to the cytotoxicity of catalpol. Figure 3 showed that the survival rate of BV2 cells was not significantly affected by treatment with catalpol alone at a concentration of up to 500 µM for 24 h, even in the presence of 100 ng/ml LPS. LPS markedly induced morphological changes of BV2 microglia after treatment with LPS, which was consequently improved by the treatment with catalpol (data not shown). Therefore,  . Inhibition of NF-κB nuclear translocation by catalpol in LPS-stimulated BV2 microglial cells. A. Cells were pre-treated with the indicated concentrations of catalpol for 1 h before treatment with 100 ng/ml LPS for 1 h. Nuclear and cytosolic proteins were prepared for Western blot analysis using anti-NF-κB p65, anti-p-IκBα, and anti-IκBα antibodies with an ECL detection system. Nucleolin and β-actin were used as internal controls for nuclear and cytosolic fractions, respectively. B. Bands were quantified using ImageJ and normalized to nucleolin and β-actin, and the ratio was determined. Data are expressed as mean ± SD. All experiments were repeated three times (* p <0.05 compared to control). C. Cells were pre-treated with 500 µM catalpol for 1 h before treatment with 100 ng/ml LPS for 1 h. The localization of NF-κB p65 was visualized by fluorescence microscopy after immunofluorescence staining with an anti-NF-κB p65 antibody (green). These cells were also stained with DAPI to visualize the nuclei (blue). Results are representative of three independent experiments. Scale bars, 10 μm. the anti-inflammatory effect by catalpol was not due to the cytotoxicity of catalpol, but to catalpol itself.

Catalpol alleviates LPS-induced NF-κB nuclear translocation and inhibitor of κB-α (IκBα) degradation in BV2 microglial cells
Subsequently, we determined whether catalpol could attenuate LPS-induced activation of NF-κB in BV2 cells. Immunoblotting data using cytoplasmic and nuclear extracts showed that catalpol pretreatment inhibited nuclear accumulation of the NF-κB p65 subunit associated with attenuation of phosphorylation and degradation of IκBα in LPS-stimulated BV2 cells in a concentration-dependent manner ( Figure  4A). Consistent with these results, immunocytochemical analysis indicated that the fluorescence intensity of NF-κB p65 in the nucleus was increased in LPS-stimulated cells. However, LPS-mediated nuclear translocation of NF-κB was considerably blocked by pretreatment with 500 µM catalpol ( Figure 4B), implying that catalpol could attenuate Figure 5. Attenuation of LPS-induced expression of TLR4 and Myd88, and interaction between LPS and TLR4 by catalpol in BV2 microglial cells. A, B. Cells were pretreated with the indicated concentrations of catalpol for 1 h prior to 100 ng/ml LPS treatment for 6 h. The mRNA (A) and protein (B) levels of TLR4 and Myd88 were determined by RT-PCR and Western blot analysis, respectively. GAPDH and β-actin were used as internal controls for the RT-PCR and Western blot assay, respectively. C. Bands were quantified using ImageJ and normalized to GAPDH and β-actin, and the ratio was determined. Data are expressed as mean ± SD. All experiments were repeated three times (* p < 0.05 compared with the LPS group). D. Cells were incubated with 100 ng/ml AF-LPS for 1 h in the absence or presence of 500 µM catalpol. After 6 h of incubation, the distribution of AF-LPS and TLR4 was detected by fluorescence microscopy. Experiments were repeated three times with similar results. Scale bars, 10 μm. E. After treatment with 100 ng/ml AF-LPS for 1 h in the absence or presence of 500 µM catalpol, LPS binding on the surface of BV2 cells was measured by flow cytometry. Data are presented as the mean ± SD as representative results obtained from three independent experiments (* p < 0.05 compared to control; # p < 0.05 compared to cells cultured with 100 ng/ml AF-LPS).
LPS-induced activation of NF-κB. Therefore, these results indicate that the inactivation of NF-κB by catalpol may be a mechanism responsible for suppression of NO, PGE 2 and pro-inflammatory cytokines in BV2 cells.

Catalpol inhibits LPS-induced TLR4 and Myd88 expression, and interaction between LPS and TLR4 in BV2 microglial cells
To determine whether the anti-inflammatory effect of catalpol was related to blocking the TLR4 signaling pathway, the effects of LPS and catalpol on expression of TLR4 and myeloid differentiation factor 88 (Myd88) were investigated. Results of immunoblotting revealed that levels of the TLR4 and Myd88 proteins were markedly increased by LPS treatment; however, when catalpol was pretreated, LPS-induced expression of these proteins was markedly inhibited ( Figure  5A). We further assessed whether catalpol could inhibit the interaction between LPS and TLR4 on an LPS-treated BV2 cell surface. As shown in Figures 5B-D, the increased fluorescence of LPS and TLR4 was observed outside the cell membrane in LPS-treated BV2 cells. However, the fluorescence intensity of TLR4 and the binding activity of LPS on the cell surface were significantly attenuated in BV2 cells treated with LPS in the presence of catalpol (p < 0.05 vs. LPS).

Catalpol diminishes LPS-induced ROS generation in BV2 microglial cells
To investigate the antioxidant potential of catalpol, the effect of catalpol on LPS-induced ROS production was examined. Flow cytometry results showed that the level of ROS was gradually increased after treatment with LPS (p < 0.05 vs. control), peaking at 1 h. It was decreased thereafter (data not shown). However, treatment with catalpol alone did not induce ROS generation, and pretreatment with catalpol effectively attenuated the level of ROS released by LPS (p < 0.05 vs. LPS; Figure 6A). The inhibitory effect of catalpol on ROS production was similarly observed in experiments using fluorescence microscopy ( Figure 6B and C). In N-acetyl cysteine (NAC, 10 mM)-pretreated cells used as a positive control, the production of ROS stimulated by LPS was significantly blocked (p < 0.05 vs. LPS), indicating that catalpol had a strong ROS scavenging effect.

Discussion
Expression of iNOS and COX-2 in glial cells is increased in inflammatory conditions leading to the increased synthesis of Figure 6. Inhibition of LPSinduced ROS generation by catalpol in BV2 microglial cells. Cells were pretreated with 500 µM catalpol or 10 mM NAC for 1 h followed by stimulation with or without 100 ng/ml LPS for 1 h. A. After staining with DCF-DA, images were obtained by fluorescence microscopy (original magnification, ×200). Scale bars, 10 μm. These images are representative of at least three independent experiments. B. Cells were incubated with DCF-DA and DCF fluorescence was measured by flow cytometry. These images are representative of at least three independent experiments. C. Each value indicates the mean ± SD as a representative result obtained from three independent experiments (* p < 0.05 compared to control; # p < 0.05 compared to cells cultured with 100 ng/ml LPS). NO and PGE 2 . Such regulation of NO and PGE 2 production during neuroinflammation plays an important role in the regulation of inflammatory balance by glial cells (Tremblay et al. 2011;Gomez-Nicola and Perry 2015). Results of the current study showed that catalpol inhibited LPS-induced inflammatory signaling in BV2 microglia, a brain microglial cell line. Similar to results of previous studies (Tian et al. 2006;Bi et al. 2013), catalpol could significantly inhibit the increased production of NO and PGE 2 by LPS in the absence of cytotoxicity, which was associated with suppression of iNOS and COX-2 mRNA and protein expression. We also found that catalpol reduced the release of inflammatory cytokines such as TNF-α and IL-1β by blocking their expression in LPS-stimulated BV2 cells, very similar to the results of previous studies (Tian et al. 2006;Fu et al. 2014;. These results suggest that catalpol may attenuate inflammatory response by inhibiting the expression of genes that regulate the production of pro-inflammatory factors. NF-κB is a key transcription factor that increases the expression of pro-inflammatory enzymes and cytokines only if it has migrated to the nucleus. In un-activated cells, NF-κB is usually located in the cytoplasm in association with IκBα, which suppresses the activation signal upon translocalization to the nucleus. When IκBα is phosphorylated and degraded, a key step in the activation of NF-κB, NF-κB is isolated and translocated to the nucleus (Li and Verma 2002;Kopitar-Jerala 2015;Lee et al. 2017). Thus, we determined whether catalpol could inhibit LPS-induced degradation of IκBα and nuclear translocation of NF-κB. Our results indicated that catalpol could effectively block nuclear expression of NF-κB (p65), and the phosphorylation and degradation of IκBα in LPS-treated BV2 microglial cells. These results suggest that catalpol can reduce the expression and production of pro-inflammatory mediators and cytokines by inhibiting the NF-κB pathway in LPS-stimulated BV2 microglia. This is in line with previous findings that catalpol inhibited the activation of NF-κB in LPS-induced acute lung injury and acute systemic inflammation, and LPS-mediated inflammatory responses in astrocytic primary cultures (Zhang et al. 2009;Bi et al. 2013;Fu et al. 2014).
Immune cells, including microglia, can recognize pathogen-associated molecular patterns through TLR pattern recognition receptors expressed on the cell surface. Among the various TLRs, TLR4 is known to recruit adapter molecules, including MyD88, LPS-binding protein (LPB), myeloid differentiation protein 2 (MD2), and differentiation cluster co-receptor when immune cells are activated by LPS (Tremblay et al. 2011;Gomez-Nicola and Perry 2015). Upon activation of TLR4 by LPS, a TLR4-MyD88-mediated signal can induce the activation of mitogen-activated protein kinases (MAPKs), which eventually promotes the activation of NF-κB signaling and ultimately produces proinflammatory mediators and cytokines (Glass et al. 2010;Cherry et al. 2014). In this study, we found that catalpol suppressed LPS-induced expression of TLR4 and MyD88. It also reduced the binding of TLR4 to LPS on the cell surface. This indicates that catalpol can inhibit the expression of pro-inflammatory enzymes and cytokines by blocking the TLR4 signaling pathway, the early stage of intracellular signaling in LPS-stimulated cells. This finding demonstrates that catalpol can attenuate the onset of the LPS-mediated intracellular signaling pathway by suppressing activation of NF-κB and inhibiting the binding of LPS to TLR4 in BV2 cells. According to the results of Fu et al. (2014), catalpol significantly inhibited expression of TLR4 and activation of NF-κB, and MAPKs in LPS-mediated acute lung injury. Bi et al. (2013) also reported that suppressive action of catalpol on inflammation was mediated via inhibiting TLR expression and NF-κB activation by LPS plus interferon (INF)-γ stimulation in primary cultured astrocytes. Thus, catalpol might be able to inhibit the NF-κB and MAPK signaling pathways by showing antagonistic effects on the binding of LPS to TLR4.
Oxidative stress (along with inflammatory stimuli) is another major cause of CNS damage. Low levels of ROS play an important role as signaling molecules that regulate the immune response to pathogens, but the overproduction of ROS contributes to neurotoxicity (von Bernhardi et al. 2015;Ohl et al. 2016;Fetisova et al. 2017). In many previous studies, inflammatory responses by LPS in microglia were directly related to increased ROS production, and inhibition of inflammatory response was reported to be associated with blocking ROS production (Fan et al. 2015;Iizumi et al. 2016;Garcia et al. 2017;). TLR4 signaling-mediated generation of ROS by LPS accelerates inflammatory response by activating downstream signaling cascades containing NF-κB (Zeng et al. 2012;Slusarczyk et al. 2016). Therefore, inhibiting ROS production is an important strategy to suppress inflammatory responses and oxidative stress. From the perspective of inhibiting the production of ROS, our results are consistent with several previous studies. For example, the beneficial effects of catalpol on LPS-induced neurotoxicity in mesencephalic neuron-glia cultures and LPS plus IFN-γ-induced inflammatory response in astrocytes primary cultures are directly related to its antioxidant effects (Tian et al. 2006;Bi et al. 2013). In addition, the protective effect of catalpol on oxidative stress-and ischemia-induced damage in astrocytes and oligodendrocytes is associated with its blocking of ROS production (Bi et al. 2008(Bi et al. , 2013Li et al. 2008;Cai et al. 2016). These results are in line with results showing its antioxidant efficacy, observed in the present study, showing that catalpol can effectively block the production of excessive ROS induced by LPS. Although the inhibitory effect of catalpol on ROS production in the microglia model is reported in this study for the first time, additional studies in primary cultures of microglia and astrocytes are needed to determine the direct linkage between ROS production blockade and anti-inflammatory efficacy.
In conclusion, the results obtained in this study demonstrated that catalpol had a potent anti-inflammatory effect on BV2 microglial cells. In LPS-stimulated BV2 cells, catalpol was able to reduce pro-inflammatory mediators and cytokine production, which was associated with decreased expression of their regulatory genes by suppressing NF-κB activity and ROS accumulation. Moreover, catalpol could block the aggregation of LPS to TLR4 on the cell surface, and interfering TLR4 signaling increased the anti-inflammatory potential of catalpol in BV2 microglia, suggesting the antagonistic effect of catalpol against TLR4, suggesting the antagonistic effect of catalpol against TLR4 (Figure 7). Although the results of the current study may provide a partial understanding of the mechanism involved in the anti-inflammatory effect of catalpol, further studies are needed to assess the mechanical role of catalpol in a variety of oxidative stresses and inflammatory mediated diseases.

Conflict of interest.
The author has no conflict of interest to declare.