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A series of novel phenothiazine derivatives with N–H have been synthesized, and those materials were suitable for colourimetric detection of fluoride ions and electrochromism.

The isomerism of glucaric acids and the complexity of the composition of Leonurus japonicus Houtt. increased the difficulty of the separation of glucaric acids from the herb. In the present study, three glucaric acids were isolated from Leonurus japonicus Houtt. by using high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography. Cation exchange resin chromatography was applied to remove the alkaloids and enrich the glucaric acid fractions. Preliminary separation of the glucaric acid extract by high-speed countercurrent chromatography was carried out at 45℃ by using an optimized solvent system of ethyl acetate/n-butanol/formic acid/water (1:1:0.01:2, v/v/v/v) with satisfied stationary phase retention and separation factor. The semi-preparative high-performance liquid chromatography was used for further separation and purification of the target fractions, and three monomeric compounds were obtained with purities of 90.0, 91.0, and 95.3%. UV spectroscopy, NMR spectroscopy, and mass spectrometry were employed to identify their structures, which were assigned as 2-syringyl glucaric acid, 2,4-disyringyl glucaric acid, and 3,4-disyringyl glucaric acid, respectively, and 2,4-disyringyl glucaric acid was reported for the first time.

A novel switchable molecular dielectric material [3-3-difluorocyclobutylammonium]2CdCl4 was synthesized. It shows a reversible phase transition at 353.95 K and rapid switching and reversibility between high and low dielectric states for several cycles.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant (Met) and germ-cell expressed (Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases. Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C (PKC) which phosphorylated ultraspiracle (USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20E through its nuclear receptor complex EcR-USP. The uspS35A mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20E signaling that delayed developmental timing. The uspS35A mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.

In Lepidoptera and Diptera, the fat body dissociates into single cells in nondiapause pupae, but it does not dissociate in diapause pupae until diapause termination. Using the cotton bollworm, Helicoverpa armigera , as a model of pupal diapause insects, we illustrated the catalytic mechanism and physiological importance of fat body cell dissociation in regulating pupal development and diapause. In nondiapause pupae, cathepsin L (CatL) activates matrix metalloproteinases (Mmps) that degrade extracellular matrix proteins and cause fat body cell dissociation. Mmp-induced fat body cell dissociation activates lipid metabolism through transcriptional regulation, and the resulting energetic supplies increase brain metabolic activity (i.e., mitochondria respiration and insulin signaling) and thus promote pupal development. In diapause pupae, low activities of CatL and Mmps prevent fat body cell dissociation and lipid metabolism from occurring, maintaining pupal diapause. Importantly, as demonstrated by chemical inhibitor treatments and CRISPR-mediated gene knockouts, Mmp inhibition delayed pupal development and moderately increased the incidence of pupal diapause, while Mmp stimulation promoted pupal development and moderately averted pupal diapause. This study advances our recent understanding of fat body biology and insect diapause regulation.

Callicarpa nudiflora, belonging to the family Verbenaceaae, is wildly used as a traditional Chinese herbal medicine (Luo-hua-zi-zhu) for hemostasis, antibiosis and antiphlogosis in clinic. However, the underlying chemical basis of C. nudiflora for the significant effects remains obscure. Hence, an ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry method was established for the characterization of multi-constituents in C. nudiflora. As a result, 57 chemical compounds were identified based on their retention times, accurate masses and MS/MS data, and 20 of them were uncovered for the first time in C. nudiflora. In addition, an optimized UHPLC fingerprint analysis, combined with chemometrics including similarity analysis, principal component analysis and partial least squares-discriminant analysis was developed for quality assessment and origin discrimination of C. nudiflora. Multivariate data analysis revealed the resemblances and differences of C. nudiflora related to regions, while partial least squares-discriminant analysis screened nine characteristic markers including luteoloside, acteoside, luteolin-4'-O-β-D-glucopyranoside, pachypodol, isoquercitrin, nudifloside, 5,7,3',4'-tetrahydroxy-8-methoxy-6-C-β-D-glucopyranosylflavone, 7α-acetoxysandaracopimaric acid and sandaracopimaric acid which contributed the most to the classification. This was the first report on the comprehensive profiling of chemical components in C. nudiflora, which helped to uncover the material basis of C. nudiflora and possess potential value for quality evaluation and clinical application purpose.

Fructus Ligustri Lucidi (FLL) has been preclinically and clinically used to treat musculoskeletal diseases. However, whether and how FLL affect the canonical Wnt/β-catenin signaling in the management of osteoporosis remains largely unknown. To this end, ovariectomized (OVX) rats and primary osteoblasts were administrated with FLL aqueous extract and medicated serum, respectively. Supplement of FLL to OVX rats maintains bone quality by attenuating the reduction in bone mineral density, strength and microstructure. The maintenance may be associated with upregulating the expression of insulin-like growth factor-1, osteoprotegerin, phospho (p)-low-density lipoprotein receptor-related protein 6, p-glycogen synthase kinase 3 beta (GSK3β), β-catenin, Runx2 and c-Myc, and downregulating the expressions of sclerostin (SOST), dickkopf-related protein 1 (DKK1), GSK3β and p-β-catenin in rat femurs and tibias. In addition, the medicated serum promotes osteoblastic bone formation through activation of Wnt/β-catenin signaling via inhibition of DKK1 and SOST overexpression. Salidroside may be one of the active ingredients in FLL that are beneficial for bone homeostasis. In summary, our results suggest that FLL may preserve bone quality through induction of canonical Wnt/β-catenin signaling via inhibition of DKK1 and SOST overexpression. And FLL may offer a new source of the DKK1 or SOST inhibitors in protection against osteoporosis.

Nitraria tangutorum B. (NT), Hippophae rhamnoides L. (HR), Lycium ruthenicum M. (LR), Lycii fructus (LF), Rosa xanthina L. (RX), and Rubuscor chorifolius L. f. (RC) are six berries from Tibetan Plateau. They have been used in traditional folk medicine with a long history, which are rich in anthocyanins. However, detailed study of their anthocyanins remains scarce. Therefore, a method for rapid simultaneous identification and quantification of 12 anthocyanins from berries using UPLC-Quadruple-Orbitrap MS system (UPLC-Q-Orbitrap MS) was established in this work. It was verified with limit of detection (3.86–11.61 µg/L), limit of quantification (3.86–11.61 µg/L), precision (0.95–2.38%), repeatability (0.96–2.08%), stability (0.86–2.31%), mean recovery (95.8–103.1%), recovery range (93.1–107.2%) and RSD less than 5.21%. It was then used in the analysis of anthocyanins in six berries species; 8, 7, 7, 7, 6 and 9 species of anthocyanins have been identified in NT, LF, LR, HR, RC and RX, respectively based on their own retention time and exact mass in positive mode, and for the first time quantified successfully in each berry (31.11 ± 0.42–2978 ± 25.67 μg.g−1). Finally, 2, 2-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging activity (0.92 ± 0.12–5.61 ± 0.23 mM TE/100 g), ferric reducing antioxidant power (FRAP) (1.23 ± 0.15–7.42 ± 0.28 mM TE/100 g) and total antioxidant activity (T-AOC) assays were used to evaluate the antioxidant activities of the six berries.

A stable porous sorbent M1 was achieved through the specific transformation of flexible thioalkyl groups and metal cluster sites in a zirconium MOF (metal–organic framework; Zr-L) template. The target polymer combines sulfoxide/sulfone and phosphoric acid in a single framework, which was fully characterized by 1H-NMR, PXRD, IR, and elemental analysis. When employed as the heavy metal adsorbent, M1 exhibit a remarkable Eu­(III) sorption behavior, achieving both high chemical affinity (Kd = 105) and sorption capacity (the maximum Eu­(III) sorption capacity reached 220 mg g–1 at pH = 4.0 and T = 298 K calculated from the Langmuir model). Recyclability and selectivity test of M1 further prove that the sorbent is highly stable and effective for europium enrichment in the aqueous solution. This work takes focus on the introduction of multifunctional groups into a single polymeric framework in a feasible and environmentally friendly way and highlights the sorption efficiency for europium removal from the aqueous solution.

Cordyceps sinensis (CS) is an herbal tonic in traditional Chinese medicine and is used to treat a wide range of disorders, including immune, kidney, respiratory, lung and cardiovascular diseases, in China. Most studies are focused mainly on nucleotides and polysaccharides from CS and consider them to be the main active ingredients, while other ingredients are often disregarded. Hundreds of sphingolipids have been identified from CS and showed inhibitory effects on mouse splenic lymphocytes.This study aimed to establish a method for preparing a fraction of sphingolipids from the mycelial powder of CS and evaluate its immunosuppressive activity.Fraction of sphingolipids (Fr-SPLs) were prepared by silica gel chromatography and reversed-phase chromatography. Its components were identified and quantified by Quadrupole-Orbitrap UHPLC-MS/MS. PBMCs were prepared from human blood, and splenic lymphocytes, B cells, and T cells were prepared from mouse spleens. The inhibitory effect of Fr-SPLs on cell viability was evaluated by CCK-8 assay. PBMC apoptosis and the ratio of CD4A fraction containing 84.83% of sphingolipids (SPLs) was prepared from the mycelia of CS and named Fr-SPLs. 15 SPLs were identified from the Fr-SPLs. Fr-SPLs significantly inhibited the viability of human peripheral blood mononuclear cells (PBMCs) with an ICFr-SPLs show immunosuppressive activity, and this study will be useful for preparing immunosuppressive components from CS and its mycelia for hyperimmune disease.

As metabolites of the gut microbiome, short-chain fatty acids (SCFAs) played an important role in the diagnosis of the metabolic diseases. Because of the high polarity, high volatility and complex matrix of biological samples, the highly sensitive, selective and accurate method to determine SCFAs remains a major challenge. Herein, a new method for simultaneous quantification of eleven SCFAs by derivatization combined with solid phase microextraction (SPME) and gas chromatography tandem mass spectrometry (GC-MS/MS) was developed. Isobutyl chloroformate coupled with isobutanol was used as the reaction reagent to derivatize SCFAs. The method validation data showed a satisfactory linearity with the linear regression coefficients (R) ranging from 0.9964 to 0.9996. The limit of detection (LOD) of all SCFAs ranges from 0.01 ng•mL−1 to 0.72 ng•mL−1 and the limit of quantification (LOQ) ranges from 0.04 ng•mL−1 to 2.41 ng•mL−1. The intra-day and inter-day precision (RSDs) ranged from 0.65% to 8.92% and 1.62% to 15.61%, respectively. The recovery ranged from 88.10% to 108.71%. Finally, the developed method was successfully used to determine SCFAs in mice fecal sample, and ten of the SCFAs were found in feces of mice, including formic acid.

A method based on liquid critical condition chromatography (LCCC) was established for the quantification of hydroxy-terminated polybutadiene (HTPB) in polymer blends. The critical conditions for HTPB were investigated using a C18 column as the stationary phase, and tetrahydrofuran (THF)-acetonitrile (ACN) or THF-water as the mobile phase. Irrespective of the relative molecular mass, HTPB was eluted under the critical conditions of THF-ACN (70.7:29.3, v/v) or THF-water (92:8, v/v). Under the optimal conditions, the HTPB exhibited satisfactory linearity in the range of 46.7-216.4 mg/L, with a correlation coefficient (

The total alkaloids extracted from the seeds of Sophora moorcroftiana (TAs-SM) have the potential to treat alveolar echinococcosis, a disease included by the WHO in a list of 17 key neglected diseases world-wide. The aims of the current study were first to develop a supercritical fluid extraction (SFE) method for optimizing TAs-SM extraction, and second, to develop an optimized method for evaluating TAs-SM pharmacokinetics in vivo. The Box-Behnken response surface method was used to optimize the extraction process, and ultra-high liquid chromatography coupled with high resolution electrospray mass spectrometry (UPLC-HR-ESI-MS) was used to determine the pharmacokinetics of TAs-SM in SD rats. The results indicated the following optimal SFE extraction conditions: pressure = 31 MPa, temperature = 70 °C, time = 162.18 min. With these parameters, total alkaloids could be extracted from each gram of S. moorcroftiana, with the total content being 68.88 μg. The linear range of UPLC-HR-ESI-MS is 0.78-200.00 ng/ml, R

Accumulating evidence suggests that mitochondrial dysfunction and adipocyte differentiation promote lipid accumulation in the development of obesity and diabetes. Curcumin is an active ingredient extracted from Curcuma longa that has been shown to exhibit antioxidant and anti-inflammatory potency in metabolic disorders. However, the underlying mechanisms of curcumin in adipocytes remain largely unexplored. We studied the effects of curcumin on adipogenic differentiation and mitochondrial oxygen consumption and analysed the possible mechanisms. 3T3-L1 preadipocytes were used to assess the effect of curcumin on differentiation of adipocytes. The Mito Stress Test measured by Seahorse XF Analyzer was applied to investigate the effect of curcumin on mitochondrial oxygen consumption in 3T3-L1 adipocytes. The effect of curcumin on the morphology of both white and brown adipose tissue (WAT and BAT) was evaluated in a high-fat diet-induced obese mice model. We found that curcumin dose-dependently (10, 20 and 35 µM) induced adipogenic differentiation and the intracellular fat droplet accumulation. Additionally, 10 µM curcumin remarkably enhanced mature adipocyte mitochondrial respiratory function, specifically, accelerating basic mitochondrial respiration, ATP production and uncoupling capacity via the regulation of peroxisome proliferator-activated receptor γ (PPARγ) ( p < 0.01). Curcumin administration also attenuated the morphological changes in adipose tissues in high-fat diet-induced obese mice. Moreover, curcumin markedly increased the mRNA and protein expressions of mitochondrial uncoupling protein 1 (UCP1), PPARγ, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and PR domain protein 16 (PRDM16) in vivo and in vitro . Collectively, the results demonstrate that curcumin promotes the adipogenic differentiation of preadipocytes and mitochondrial oxygen consumption in 3T3-L1 mature adipocytes by regulating UCP1, PRDM16, PPARγ and PGC-1α expression.

The diet compositions of MD12031 and MD12033 purchased from Mediscience Co., Ltd.

Matrix metalloproteinases (MMPs) are the major proteinases that process or degrade numerous extracellular matrix (ECM) components and are evolutionarily conserved from nematodes to humans. During molting in insects, the old cuticle is removed and replaced by a new counterpart. Although the regulatory mechanisms of hormones and nutrients in molting have been well studied, very little is known about the roles of ECM-modifying enzymes in this process. Here, we found that MMPs are necessary for imaginal molting of the American cockroach, Periplaneta americana. Inhibition of Mmp activity via inhibitor treatment led to the failure of eclosion and wing expansion. Five Mmps genes were identified from the P. americana genome, and PaMmp2 played the dominant roles during molting. Further microscopic investigations showed that newly formed adult cuticles were attenuated and that then chitin content was reduced upon Mmp inhibition. Transcriptomic analysis of the integument demonstrated that multiple signaling and metabolic pathways were changed. Microscopic investigation of the wings showed that epithelial cells were restrained together because they were incapable of degrading the ECM upon Mmp inhibition. Transcriptomic analysis of the wing identified dozens of possible genes functioned in wing expansion. This is the first study to show the essential roles of Mmps in the nymph-adult transition of hemimetabolous insects.

We report ruthenium(II)-catalyzed ortho-C–H arylation of N,N-dialkylthiobenzamides with boronic acids. The method employs [RuCl2(p-cym)]2 in the presence of Cu(OTf)2 and Ag2O oxidant. The reaction ...

Zhishi-Xiebai-Guizhi Decoction (ZSXBGZD), a traditional Chinese medicine (TCM) formula, has been used for treatment of coronary heart disease and myocardial infarction for nearly two thousand years. However, the chemical composition of ZSXBGZD is still unclear. In order to obtain the chemical profile of ZSXBGZD, an ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) method was utilized for the identification of its multi-constituents. As a result, a total of 148 compounds were identified based on their retention times, accurate masses and MS/MS data. In addition, an optimized UPLC fingerprint analysis, combined with chemometrics such as similarity analysis (SA), hierarchical cluster analysis (HCA), principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) was developed for quality assessment of ZSXBGZD. Multivariate data analysis revealed that samples could be classified correctly according to their geographic origins, and four compounds neohesperidin, naringin, guanosine and adenosine contributed the most to classification. The established UPLC method with multi-wavelength detection was further validated and implemented for simultaneous quantification of 12 representative ingredients in the prescription, including guanosine, adenosine, 2'-deoxyadenoside, syringin, magnoloside A, forsythoside A, naringin, hesperidin, cinnamaldehyde, neohesperidin, honokiol and magnolol. This is the first report on the comprehensive profiling of major chemical components in ZSXBGZD. The results of the study could help to uncover the chemical basis of ZSXBGZD and possess potential value for quality evaluation purpose.

Five traditional medicinal food from the Tibetan plateau including Nitraria tangutorum Bobrov (NT), Hippophae rhamnoides L. (HR), Lycium ruthenicum Murray (LR), Lycium barbarum L. (LB) and Rubus corchorifolius L.f. (RC) are rich in phenolic compounds. However, the detailed studies about the phenolic compounds remain scarce. Therefore, we established a rapid method for the simultaneous identification and quantification of the phenolic compounds from berries via Ultra Performance Liquid Chromatography-Quadruple-Orbitrap MS system (UPLC-Q-Orbitrap MS). This method was verified from many aspects including detection limit, quantification limit, precision, repeatability, stability, average recovery rate and recovery range, and then was used to analyze the phenolic compounds in these five species of berries. Finally, a total of 21 phenolic compounds were directly identified by comparing the retention time and exact mass, of which 14 compounds were identified by us for the first time in berries from the Tibetan plateau, including one flavonoid aglycone (myricetin), 11 phenolic acids (gallic acid, protocatechuate, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, 2-hydroxybenzeneacetic acid and ellagic acid), one flavanol (catechin) and one dihydrochalcone flavonoid (phloretin). Quantitative results showed that rutin, myricetin, quercetin and kaempferol were the main flavonoids. Moreover, a variety of phenolic acid compounds were also detected in most of the berries from the Tibetan plateau. Among these compounds, the contents of protocatechuate and chlorogenic acid were high, and high levels of catechin and phloretin were also detected in these plateau berries.

Although radix Salviae miltiorrhizae (RSM) is reported to exhibit the antiosteoporotic effect in preclinical study, the underlying mechanism is unclear. To this end, ovariectomized (OVX) rats were employed with administration of RSM (5 g/kg) for 14 weeks. The disturbed serum levels of alkaline phosphatase (ALP), osteoprotegerin (OPG), tartrate-resistant acid phosphatase, and receptor activator of nuclear factor-κB ligand (RANKL) in OVX rats were improved by RSM treatment. Furthermore, supplement of RSM to OVX rats resulted in an increase in femoral bone mineral density and bone strength as well as an improvement in bone microstructures. Moreover, the decreased expression of phosphor (p)-LRP6, insulin-like growth factor-1(IGF-1), ALP, and OPG, as well as increased expression of RANKL and cathepsin K in the tibias and femurs of OVX rats were shifted by RSM treatment. Additionally, RSM reversed the decreased ratio of p-glycogen synthase kinase 3β (GSK3β) to GSK3β and increased ratio of p-β-catenin to β-catenin in OVX rats. Altogether, it is suggestive that RSM improves bone quantity and quality by favoring Wnt/β-catenin and OPG/RANKL/cathepsin K signaling pathways in OVX rats thereby suggesting the potential of this herb to be a novel source of antiosteoporosis drugs.

Matrix metalloproteinases (MMPs) are evolutionarily conserved extracellular matrix proteinases. Genetic analysis of the Drosophila MMPs, Mmp1 and Mmp2, in vivo reveal that they play vital roles in tissue remodeling. Although the catalytic domain (CD) undertakes most MMP functions, few studies have sought to demonstrate the biochemical properties of the CDs of fly MMPs. Here, we identified the overexpression, purification, and refolding of the CDs of Drosophila Mmp1 and Mmp2 for biochemical studies. Zymography assays and substrate degradation analysis showed that both Mmp1-CD and Mmp2-CD were able to digest casein, gelatin, fibronectin, collagen (types I, IV, and V), while Mmp2-CD showed much higher degradation activity compared with Mmp1-CD. Moreover, human collagen III could be degraded by Mmp1-CD but not Mmp2-CD, and rat collagen I and laminin could be degraded by Mmp2-CD but not Mmp1-CD, suggesting that Drosophila Mmp1 and Mmp2 might have overlapping yet distinct substrate specificity. Using synthetic fluorescent substrates, we further demonstrated that the enzymatic activity of Mmp1-CD and Mmp2-CD could be inhibited by human tissue inhibitors of metalloproteinases (TIMPs). These results reveal the context of the cooperative yet distinct roles of Mmp1 and Mmp2 in tissue remodeling.

A sensitive and selective fluorescent sensor for Pb2+ ion based on phenothiazine-polyamide was built (named sensor PP). Due to introducing of four diethanolamine groups to polyamide, this sensor was totally water soluble. PP could detect Pb2+ ion within 1 min in the presence of other metal ions in aqueous solution, the detect limit was 9.11 × 10−8 M.

Since it was first postulated by Wigglesworth in 1934, juvenile hormone (JH) is considered a status quo hormone in insects because it prevents metamorphosis that is initiated by the molting hormone 20-hydroxyecdysone (20E). During the last decade, significant advances have been made regarding JH signaling. First, the bHLH-PAS transcription factor Met/Gce was identified as the JH intracellular receptor. In the presence of JH, with the assistance of Hsp83, and through physical association with a bHLH-PAS transcriptional co-activator, Met/Gce enters the nucleus and binds to E-box-like motifs in promoter regions of JH primary-response genes for inducing gene expression. Second, the zinc finger transcription factor Kr-h1 was identified as the anti-metamorphic factor which transduces JH signaling. Via Kr-h1 binding sites, Kr-h1 represses expression of 20E primary-response genes (i.e. Br, E93 and E75) to prevent 20E-induced metamorphosis. Third, through the intracellular signaling, JH promotes different aspects of female reproduction. Nevertheless, this action varies greatly from species to species. Last, a hypothetical JH membrane receptor has been predicted to be either a GPCR or a tyrosine kinase receptor. In future, it will be a great challenge to understand how the JH intracellular receptor Met/Gce and the yet unidentified JH membrane receptor coordinate to regulate metamorphosis and reproduction in insects.

Many cockroach species have adapted to urban environments, and some have been serious pests of public health in the tropics and subtropics. Here, we present the 3.38-Gb genome and a consensus gene set of the American cockroach, Periplaneta americana. We report insights from both genomic and functional investigations into the underlying basis of its adaptation to urban environments and developmental plasticity. In comparison with other insects, expansions of gene families in P. americana exist for most core gene families likely associated with environmental adaptation, such as chemoreception and detoxification. Multiple pathways regulating metamorphic development are well conserved, and RNAi experiments inform on key roles of 20-hydroxyecdysone, juvenile hormone, insulin, and decapentaplegic signals in regulating plasticity. Our analyses reveal a high level of sequence identity in genes between the American cockroach and two termite species, advancing it as a valuable model to study the evolutionary relationships between cockroaches and termites., The American cockroach (Periplaneta americana) is an hemimetabolous insect with rapid growth, high fecundity, and remarkable tissue-regeneration capability. Here Li et al sequence its 3.38-Gb genome and perform the functional studies, yielding insights into its environmental adaptation and developmental plasticity.