Mmp-induced fat body cell dissociation promotes pupal development and moderately averts pupal diapause by activating lipid metabolism.

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.

Cyp6g2 is the major P450 epoxidase responsible for juvenile hormone biosynthesis in Drosophila melanogaster.

BACKGROUND: Juvenile hormones (JH) play crucial role in regulating development and reproduction in insects. The most common form of JH is JH III, derived from MF through epoxidation by CYP15 enzymes. However, in the higher dipterans, such as the fruitfly, Drosophila melanogaster, a bis-epoxide form of JHB3, accounted most of the JH detected. Moreover, these higher dipterans have lost the CYP15 gene from their genomes. As a result, the identity of the P450 epoxidase in the JH biosynthesis pathway in higher dipterans remains unknown. RESULTS: In this study, we show that Cyp6g2 serves as the major JH epoxidase responsible for the biosynthesis of JHB3 and JH III in D. melanogaster. The Cyp6g2 is predominantly expressed in the corpus allatum (CA), concurring with the expression pattern of jhamt, another well-studied gene that is crucial in the last steps of JH biosynthesis. Mutation in Cyp6g2 leads to severe disruptions in larval-pupal metamorphosis and exhibits reproductive deficiencies, exceeding those seen in jhamt mutants. Notably, Cyp6g2-/-::jhamt2 double mutants all died at the pupal stage but could be rescued through the topical application of JH analogs. JH titer analyses revealed that both Cyp6g2-/- mutant and jhamt2 mutant lacking JHB3 and JH III, while overexpression of Cyp6g2 or jhamt caused a significant increase in JHB3 and JH III titer. CONCLUSIONS: These findings collectively established that Cyp6g2 as the major JH epoxidase in the higher dipterans and laid the groundwork for the further understanding of JH biosynthesis. Moreover, these findings pave the way for developing specific Cyp6g2 inhibitors as insect growth regulators or insecticides.

Experimental Observation of the Fully Ferroelectric-Fully Ferroelastic Effect in Multiferroic Hybrid Perovskites.

Since the concept of "multiferroic" was first proposed in 1968, the coupling effect between different ferroic orders has attracted great interest in energy, information, and biomedical fields. However, the fully ferroelectric-fully ferroelastic effect has never been experimentally observed in hybrid perovskites, even though this effect was predicted to exist half a century ago. Realizing such cross-linking effects of polarization vectors and strain tensors has always been a huge challenge because of the complex difference in these two ferroic origins. Here, we report a multiferroic with full ferroelectricity and full ferroelasticity in two-dimensional (2D) hybrid perovskites based on ferroelectrochemistry. The dynamic molecular reorientations endow (cyclohexanemethylaminium)2PbCl4 with a desired symmetry change of 4̅2mFmm2 at a Curie temperature of 411.8 K. More strikingly, the switchable evolution of ferroelastic domains was directly observed under the control of either electric or mechanical fields, which is the first experimental observation of a fully ferroelectric-fully ferroelastic effect in hybrid perovskites. This work would provide new insights into understanding the intrinsic cross-linking mechanism between ferroelectricity and ferroelasticity toward the development of multichannel interactive microelectronic devices.

Metal-Free Perovskite Ferroelectrics with the Most Equivalent Polarization Axes.

Ferroelectricity in metal-free perovskites (MFPs) has emerged as an academic hotspot for their lightweight, eco-friendly processability, flexibility, and degradability, with considerable progress including large spontaneous polarization, high Curie temperature, large piezoelectric response, and tailoring coercive field. However, their equivalent polarization axes as a key indicator are far from enough, although multiaxial ferroelectrics are highly preferred for performance output and application flexibility that profit from as many equivalent polarization directions as possible with easier reorientation. Here, by implementing the synergistic overlap of regulating anionic geometries (from spherical I- to octahedral [PF6]- and to tetrahedral [ClO4]- or [BF4]-) and cationic asymmetric modification, we successfully designed multiaxial MFP ferroelectrics CMDABCO-NH4-X3 (CMDABCO = N-chloromethyl-N'-diazabicyclo[2.2.2]octonium; X = [ClO4]- or [BF4]-) with the lowest P1 symmetry. More impressively, systemic characterizations indicate that they possess 24 equivalent polarization axes (Aizu notations of 432F1 and m3̅mF1, respectively)─the maximum number achievable for ferroelectrics. Benefiting from the multiaxial feature, CMDABCO-NH4-[ClO4]3 has been demonstrated to have excellent piezoelectric sensing performance in its polycrystalline sample and prepared composite device. Our study provides a feasible strategy for designing multiaxial MFP ferroelectrics and highlights their great promise for use in microelectromechanical, sensing, and body-compatible devices.

Organic-Inorganic Rare-Earth Double Perovskite Ferroelectric with Large Piezoelectric Response and Ferroelasticity for Flexible Composite Energy Harvesters.

Hybrid organic-inorganic perovskite (HOIP) ferroelectric materials have great potential for developing self-powered electronic transducers owing to their impressive piezoelectric performance, structural tunability and low processing temperatures. Nevertheless, their inherent brittle and low elastic moduli limit their application in electromechanical conversion. Integration of HOIP ferroelectrics and soft polymers is a promising solution. In this work, a hybrid organic-inorganic rare-earth double perovskite ferroelectric, [RM3HQ]2RbPr(NO3)6 (RM3HQ = (R)-N-methyl-3-hydroxylquinuclidinium) is presented, which possesses multiaxial nature, ferroelasticity and satisfactory piezoelectric properties, including piezoelectric charge coefficient (d33) of 102.3 pC N-1 and piezoelectric voltage coefficient (g33) of 680 × 10-3 V m N-1. The piezoelectric generators (PEG) based on composite films of [RM3HQ]2RbPr(NO3)6@polyurethane (PU) can generate an open-circuit voltage (Voc) of 30 V and short-circuit current (Isc) of 18 µA, representing one of the state-of-the-art PEGs to date. This work has promoted the exploration of new HOIP ferroelectrics and their development of applications in electromechanical conversion devices.

Unusual Thermal Quenching of Photoluminescence from an Organic-Inorganic Hybrid [MnBr4 ]2- -based Halide Mediated by Crystalline-Crystalline Phase Transition.

The ability to generate and manipulate photoluminescence (PL) behavior has been of primary importance for applications in information security. Excavating novel optical effects to create more possibilities for information encoding has become a continuous challenge. Herein, we present an unprecedented PL temporary quenching that highly couples with thermodynamic phase transition in a hybrid crystal (DMML)2 MnBr4 (DMML=N,N-dimethylmorpholinium). Such unusual PL behavior originates from the anomalous variation of [MnBr4 ]2- tetrahedrons that leads to non-radiation recombination near the phase transition temperature of 340 K. Remarkably, the suitable detectable temperature, narrow response window, high sensitivity, and good cyclability of this PL temporary quenching will endow encryption applications with high concealment, operational flexibility, durability, and commercial popularization. Profited from these attributes, a fire-new optical encryption model is devised to demonstrate high confidential information security. This unprecedented optical effect would provide new insights and paradigms for the development of luminescent materials to enlighten future information encryption.

Organic-Inorganic Hybrid Ferroelectric and Antiferroelectric with Afterglow Emission.

Luminescent ferroelectrics are holding exciting prospect for integrated photoelectronic devices due to potential light-polarization interactions at electron scale. Integrating ferroelectricity and long-lived afterglow emission in a single material would offer new possibilities for fundamental research and applications, however, related reports have been a blank to date. For the first time, we here achieved the combination of notable ferroelectricity and afterglow emission in an organic-inorganic hybrid material. Remarkably, the presented (4-methylpiperidium)CdCl3 also shows noticeable antiferroelectric behavior. The implementation of cationic customization and halogen engineering not only enables a dramatic enhancement of Curie temperature of 114.4 K but also brings a record longest emission lifetime up to 117.11 ms under ambient conditions, realizing a leapfrog improvement of at least two orders of magnitude compared to reported hybrid ferroelectrics so far. This finding would herald the emergence of novel application potential, such as multi-level density data storage or multifunctional sensors, towards the future integrated optoelectronic devices with multitasking capabilities.

Introducing Ferroelasticity into 1D Hybrid Lead Halide Semiconductor by Halogen Substitution Strategy.

Organic-inorganic hybrid lead halide perovskites (OLHPs), represented by (CH3 NH3 )PbI3 , are one of the research focus due to their exceptional performance in optoelectronic applications, and ferroelastic domain walls are benign to their charge carrier transport that is confirmed recently. Among them, the 1D OLHPs feature better stability against desorption and moisture, but related 1D ones possessing ferroelasticity are rarely investigated and reported so far. In this work, the 1D ferroelastic semiconductor (N-iodomethyl-N-methyl-morpholinium)PbI3 ((IDMML)PbI3 ) is prepared successfully by introducing successively halogenate atoms from Cl, Br to I into the organic cation of the prototype (N,N-dimethylmorpholinium)PbI3 ((DMML)PbI3 ). Notably, (IDMML)PbI3 shows the narrow bandgap energy (≈2.34 eV) according to the ultraviolet-visible absorption spectrum and the theoretical calculation, and possesses the evident photoconductive characteristic with the on/off ratio of current of ≈50 under the 405 nm light irradiation. This work provides a new case for the ferroelastic OLHPs and will inspire intriguing research in the field of optoelectronic.

Fluorescence Emission Is Highly Structure-Dependent in Hybrid Lead Halides.

Hybrid lead halide perovskites have received extensive scientific attention owing to their great potential in the field of fluorescent displays and light-emitting diodes. Currently, most luminescent materials contain functional molecular and rare-earth metal ion parts. However, the mechanism of photoluminescence property in two-dimensional hybrid lead halide perovskites with different layered inorganic skeletons has been reported rarely. To better understand the effect of an inorganic skeleton on the fluorescence property, here, we report three organic-inorganic hybrid materials with different layered inorganic frameworks: (MACH)2·PbBr4 (Prv-1, MACH = cyclohexylmethylammonium), (2-MPQ)·PbBr4 (Prv-2, 2-MPQ = 2-methylpiperazinium), and (TMBA)4·Pb3Br10 (Prv-3, TMBA = N'N'N'-trimethylbenzylammonium). Among them, Prv-1 is a (100)-oriented perovskite, Prv-2 belongs to the (110)-oriented perovskite, and the inorganic framework of Prv-3 possesses [Pb3Br10] units. Interestingly, Prv-1 has a strong blue-violet fluorescence emission, while the luminescence effect of Prv-2 is very weak; notably, Prv-3 emits a charming bright-orange light. Meanwhile, results of theoretical computational studies also reveal that the electronic structure of all three compounds is highly dependent on structurally distorted [PbBr6] octahedra, and the frontier molecular orbital (FMO) analysis further suggests that HOMO and LUMO of Prv-3 are contributed by inorganic and organic components, respectively. In addition, all three materials belong to direct band gap semiconductors, and the band gaps are 2.79, 2.97, and 2.76 eV, respectively. Significantly, there are obvious differences in conduction bands. Based on the above analysis, the photoluminescence mechanism of three hybrid materials is explained from the electronic levels. Consequentially, this work might provide practical strategies and perspectives for exploring novel structure-related properties.

Precise Design of Molecular Ferroelectrics with High TC and Tunable Band Gap by Molecular Modification.

Molecular ferroelectric materials are widely applied in piezoelectric converters, non-volatile memorizers, and photovoltaic devices due to their advantages of adjustable structure, lightweight, easy processing, and environmental friendliness. However, designing multifunctional molecular ferroelectrics with excellent properties has always been a great challenge. Herein, a multiaxial molecular ferroelectric is successfully designed by modifying the quasi-spherical cation dabco with CuBr2 to obtain halogenated [Bretdabco]CuBr4 (Bretdabco = N-bromoethyl-N'-diazabicyclo [2.2.2]octane), which crystallizes in polar point groups (C6). Typical ferroelectric behaviors featured by the P-E hysteresis loop and switched ferroelectric domain are exhibited. Notably, the molecular ferroelectric shows a high TC of 460 K, which is rare in the field and could greatly expand the application range of this material. In addition, the band gap is adjustable through the regulation of halogen. Both the UV absorption spectra and theoretical calculations indicate that the molecular ferroelectrics belong to a direct band gap (2.14 eV) semiconductor. This tunable and narrow band gap semiconductor molecular ferroelectric material with high TC can be utilized more effectively in the study of optoelectronics and sensors, including piezoelectric energy harvesters. This research may provide a promising approach for the development of multiaxial molecular ferroelectrics with a tiny band gap and high TC.

A liquid chromatography-tandem mass spectrometry method for comprehensive determination of metabolites in the purine pathway of rat plasma and its application in anti-gout effects of Lycium ruthenicum Murr.

It has been proved that purine metabolites are implicated in various biological syndromes and disorders. Therefore, the realization of panoramic detection of purine metabolites will be of great significance to the pathogenesis of purine metabolic disorders. In the present study, an ultra-high performance liquid chromatography with tandem mass spectrometry method was developed for the comprehensive quantification of purine metabolites in rat plasma. The 17 purine metabolites were separated and quantified in the short running time of 15 min. The proposed method was strictly validated by applying SeraSub solution as a matrix and proved to be linear (R2 ≥ 0.9944), accurate (the recoveries of all analytes ranged from 85.3% to 103.0%, with relative standard deviation values ≤ 9.3%), and precise (the intra- and inter-day precisions were less than 10.8% and 12.4%, respectively). The method was then successfully applied to the qualification of the endogenous purine metabolites in acute gouty arthritis rats, as well as colchicine and anthocyanin-intervened rats. Results showed that uric acid, xanthine, hypoxanthine, and xanthine were considered the key factors of acute gouty arthritis. The established method and measurement of purines in rat plasma might help the investigation of the action mechanisms between purine disorders and related diseases.

Chemical Profiling of Nitraria roborowskii Kom. by UPLC-Q-Orbitrap-MS and Their Hypolipidemic Effects in Vivo.

The Nitraria roborowskii Kom. (NRK) berries, as fruits of the genus Nitraria of the Zygophyllceae family, have been widely used as folk medicine. Modern pharmacological research has demonstrated that Nitraria berries had hypolipidemic, hypoglycemic, and immunomodulatory effects. However, more research needs to be reported on the chemical composition and biological activity of NRK. Hence, the phenolic compounds in the NRK berries were comprehensively analyzed and characterized by Ultra Performance Liquid Chromatography-Quadruple-Orbitrap MS system (UPLC-Q-Orbitrap MS) in this study. In total, 52 phenolics were identified, and all were reported for the first time. In addition, the hypolipidemic efficacy of NRK berries extract was studied in the hyperlipidemic mouse model. After treatment, the high dose group of NRK substantially reversed total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol levels. Through lipidomics technology, 27 potential biomarkers were characterized. And there was a significant callback at 25 of them after NRK treatment by using statistical analysis methods. Pathway analysis results demonstrated that NRK might exert therapeutic effects by regulating glycerophospholipid and glycerolipid metabolism pathways. This study could provide firsthand information on NRK berries for their phenolic compounds and potential application in preventing and treating hyperlipidemia.

Two-Step Dielectric Responsive Organic-Inorganic Hybrid Material with Mid-Band Light Emission.

Hybrid organic-inorganic perovskite (HOIP) have received tremendous scientific attention because of the phase transition and photovoltaic properties. However, achieving the special perovskite structure with both two-step dielectric response and luminescence characteristics is rarely reported. Herein, we report an organic-inorganic hybrid perovskite, [(BA)2 ⋅ PbI4 ] (Compound 1, BA=n-butylamine) by introducing flexible organic cations (HBA+ ), with direct mid-band gap as 2.28 eV. Interestingly, this material exhibits two-step reversible dielectric response at 350 K and 460 K (in heating process), respectively. Besides, the photoluminescence was found: it emits charming green light under 365 nm lamp (Photoluminescence quantum yield is 9.52 %). The outstanding two-step dielectric response and luminescence characteristics of this compound might pave the way for the application of dielectric and ferroelectric functional materials in temperature sensors and mechanical switches.

Ferroelasticity in Organic-Inorganic Hybrid Perovskites.

Molecular ferroelastics have received particular attention for potential applications in mechanical switches, shape memory, energy conversion, information processing, and solar cells, by taking advantages of their low-cost, light-weight, easy preparation, and mechanical flexibility. The unique structures of organic-inorganic hybrid perovskites have been considered to be a design platform for symmetry-breaking-associated order-disorder in lattice, thereby possessing great potential for ferroelastic phase transition. Herein, we review the research progress of organic-inorganic hybrid perovskite ferroelastics in recent years, focusing on the crystal structures, dimensions, phase transitions and ferroelastic properties. In view of the few reports on molecular-based hybrid ferroelastics, we look forward to the structural design strategies of molecular ferroelastic materials, as well as the opportunities and challenges faced by molecular-based hybrid ferroelastic materials in the future. This review will have positive guiding significance for the synthesis and future exploration of organic-inorganic hybrid molecular ferroelastics.

Integrated Reversible Thermochromism, High Tc , Dielectric Switch and Narrow Band Gap in One Multifunctional Ferroic.

Organic-inorganic Hybrid (OIH) materials for multifunctional switchable applications have attracted enormous attention in recent years due to their excellent optoelectronic properties and good structural tunability. However, it still remains challenging to fabricate one simple OIH compound with multi-functionals properties, such as dielectric switching, thermochromic properties, semiconductor characteristics and ferroelasticity. Under this context, we successfully synthesized [2-(2-fluorophenyl)ethan-1- ammonium]2 SnBr6 (compound 1), which has a higher phase transition temperature of 427.7 K. Additionally, it exhibits a semiconducting property with an indirect band gap of 2.36 eV. Combining ferroelastic, narrow band gap, thermochromic, and dielectric properties, compound 1 can be considered as a rarely reported multi-functional ferroelastic material, which is expected to give inspiration for broadening the applications in the smart devices field.

Isolation of three glucaric acids from Leonurus japonicus Houtt. by using high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography.

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.

Chemical Profiling and Quality Evaluation of Callicarpa Nudiflora from Different Regions in China by UPLC-QTOF-MS Fingerprint.

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-ß-D-glucopyranoside, pachypodol, isoquercitrin, nudifloside, 5,7,3',4'-tetrahydroxy-8-methoxy-6-C-ß-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 preserves bone quality through induction of canonical Wnt/ß-catenin signaling pathway in ovariectomized rats.

Fructus Ligustri Lucidi (FLL) has been preclinically and clinically used to treat musculoskeletal diseases. However, whether and how FLL affect the canonical Wnt/ß-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 (GSK3ß), ß-catenin, Runx2 and c-Myc, and downregulating the expressions of sclerostin (SOST), dickkopf-related protein 1 (DKK1), GSK3ß and p-ß-catenin in rat femurs and tibias. In addition, the medicated serum promotes osteoblastic bone formation through activation of Wnt/ß-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/ß-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.

The expression, purification, and substrate analysis of matrix metalloproteinases in Drosophila melanogaster.

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.

Correction: Methyl Farnesoate Plays a Dual Role in Regulating Drosophila Metamorphosis.

[This corrects the article DOI: 10.1371/journal.pgen.1005038.].