CRISPR/Cas9-mediated ebony knockout causes melanin pigmentation and prevents moth Eclosion in Ectropis grisescens.

Ectropis grisescens (Lepidoptera: Geometridae) is a destructive tea pest in China. Mimesis, characterized by changing body color, is an important trait of E. grisescens larvae. Hence, identifying melanin pathway-related genes may contribute to developing new pest control strategies. In the present study, we cloned Egebony, a gene potentially involved in melanin pigmentation in E. grisescens, and subsequently conducted CRISPR/Cas9-mediated targeted mutagenesis of Egebony to analyze its role in pigmentation and development. At the larvae, prepupae, and pupae stages, Egebony-knockout individuals exhibited darker pigmentation than the wild-type. However, Egebony knockout did not impact the colors of sclerotized appendants, including ocelli, setae, and claws. While mutant pupae could successfully develop into moths, they were unable to emerge from the puparium. Notably, embryo hatchability and larval survival of mutants remained normal. Further investigation indicated that mutant pupae exhibited significantly stronger shearing force than the wild-type, with the pigmented layer of mutant pupae appearing darker and thicker. Collectively, these results suggest that the loss of Egebony might increase the rigidity of the puparium and prevent moth eclosion. This study provides new insights into understanding the function and diversification of ebony in insect development and identifies a lethal gene that can be manipulated for developing effective pest control strategies.

Microbial community structure in a constructed wetland based on a recirculating aquaculture system: Exploring spatio-temporal variations and assembly mechanisms.

The diversity, composition and performance of microbial communities within constructed wetlands (CW) were markedly influenced by spatio-temporal variations. A pilot-scale integrated vertical-flow constructed wetland (IVCW) as the biological purification unit within a recirculating aquaculture system (RAS) was established and monitored in this study. The investigation aimed to elucidate the responses of community structure, co-occurrence networks, and assembly mechanisms of the microbial community to spatial and temporal changes. Spatially, all a-diversity indices and microbial networks complexity were significantly higher in the upstream pool of the IVCW than in the downstream pool. Temporally, the richness increased over time, while the evenness showed a decreasing trend. The number of nodes and edges of microbial networks increased over time. Notably, the stable pollutant removal efficiencies were observed during IVCW operations, despite a-diversity and bacterial community networks exhibited significant variations across time. Functional redundancy emerged as a likely mechanism contributing to the stability of microbial ecosystem functions. Null model and neutral model analyses revealed the dominance of deterministic processes shaping microbial communities over time, with deterministic influences being more pronounced at lower a-diversity levels. DO and inorganic nitrogen emerged as the principal environmental factor influencing microbial community dynamics. This study provides a theoretical foundation for the regulation of microbial communities and environmental factors within the context of IVCW.

Discovery of novel tumor-targeted near-infrared probes with 6-substituted pyrrolo[2,3-d]pyrimidines as targeting ligands.

Since the overexpression of folate receptors (FRs) in certain types of cancers, a variety of FR-targeted fluorescent probes for tumor detection have been developed. However, the reported probes almost all have the same targeting ligand of folic acid with various fluorophores and/or linkers. In the present study, a series of novel tumor-targeted near-infrared (NIR) molecular fluorescent probes were designed and synthesized based on previously reported 6-substituted pyrrolo[2,3-d]pyrimidine antifolates. All newly synthesized probes showed specific FR binding in vitro, whereas GT-NIR-4 and GT-NIR-5 with a benzene and a thiophene ring, respectively, on the side chain of pyrrolo[2,3-d]pyrimidine exhibited better FR binding affinity than that of GT-NIR-6 with folic acid as targeting ligand. GT-NIR-4 also showed high tumor uptake in KB tumor-bearing mice with good pharmacokinetic properties and biological safety. This work demonstrates the first attempt to replace folic acid with antifolates as targeting ligands for tumor-targeted NIR probes.

Saccharomyces cerevisiae Δ9-desaturase Ole1 forms a supercomplex with Slc1 and Dga1.

Biosynthesis of the various lipid species that compose cellular membranes and lipid droplets depends on the activity of multiple enzymes functioning in coordinated pathways. The flux of intermediates through lipid biosynthetic pathways is regulated to respond to nutritional and environmental demands placed on the cell necessitating that there be flexibility in pathway activity and organization. This flexibility can in part be achieved through the organization of enzymes into metabolon supercomplexes. However, the composition and organization of such supercomplexes remain unclear. Here, we identified protein-protein interactions between acyltransferases Sct1, Gpt2, Slc1, Dga1, and the Δ9 acyl-CoA desaturase Ole1 in Saccharomyces cerevisiae. We further determined that a subset of these acyltransferases interact with each other independent of Ole1. We show that truncated versions of Dga1 lacking the carboxyl-terminal 20 amino acid residues are nonfunctional and unable to bind Ole1. Furthermore, charged-to-alanine scanning mutagenesis revealed that a cluster of charged residues near the carboxyl terminus was required for the interaction with Ole1. Mutation of these charged residues disrupted the interaction between Dga1 and Ole1 but allowed Dga1 to retain catalytic activity and to induce lipid droplet formation. These data support the formation of a complex of acyltransferases involved in lipid biosynthesis that interacts with Ole1, the sole acyl-CoA desaturase in S. cerevisiae, that can channel unsaturated acyl chains toward phospholipid or triacylglycerol synthesis. This desaturasome complex may provide the architecture that allows for the necessary flux of de novo-synthesized unsaturated acyl-CoA to phospholipid or triacylglycerol synthesis as demanded by cellular requirements.

Design, synthesis and biological evaluation of pyrrolopyrimidine derivatives as novel and selective positive modulator of the small conductance Ca2+-activated K+ channels.

The type 2 small conductance Ca2+-activated K+ channels (SK2) have been considered as one of the most promising therapeutic targets for spinocerebellar ataxias type 2 (SCA2) by playing a critical role in the control of normal purkinje cells (PCs) pacemaking. Herein, a novel series of pyrrolopyrimidine derivatives were designed and synthesized from the lead compound NS13001 as subtype-selective modulators of SK channels. Among them, the halogen-substituted compound 12b (EC50 = 0.34 ± 0.044 µM) was identified with a ∼5.4-fold higher potency on potentiating SK2-a channels at submicromolar concentrations as compared to NS13001 (EC50 = 1.83 ± 0.50 µM). Furthermore, compound 12b exhibited selectivity on SK2-a/SK3 subtype by displaying 93.33 ± 3.26% efficacies on SK2-a channels, and 84.54% ± 7.49% on SK3 channels. In addition, compound 12b demonstrated the potential to cross the blood-brain barrier (BBB) with suitable pharmacokinetic properties and low cytotoxicity. Molecular docking study also unveiled the binding interactions of compound 12b with SK2-CaM protein complex. Overall, the novel pyrrolopyrimidines provide an insightful guidance for future structural optimization of SK channel agonists.

Olfactory Gene Families in Scopula subpunctaria and Candidates for Type-II Sex Pheromone Detection.

Scopula subpunctaria, an abundant pest in tea gardens, produce type-II sex pheromone components, which are critical for its communicative and reproductive abilities; however, genes encoding the proteins involved in the detection of type-II sex pheromone components have rarely been documented in moths. In the present study, we sequenced the transcriptomes of the male and female S. subpunctaria antennae. A total of 150 candidate olfaction genes, comprising 58 odorant receptors (SsubORs), 26 ionotropic receptors (SsubIRs), 24 chemosensory proteins (SsubCSPs), 40 odorant-binding proteins (SsubOBPs), and 2 sensory neuron membrane proteins (SsubSNMPs) were identified in S. subpunctaria. Phylogenetic analysis, qPCR, and mRNA abundance analysis results suggested that SsubOR46 may be the Orco (non-traditional odorant receptor, a subfamily of ORs) of S. subpunctaria. SsubOR9, SsubOR53, and SsubOR55 belonged to the pheromone receptor (PR) clades which have a higher expression in male antennae. Interestingly, SsubOR44 was uniquely expressed in the antennae, with a higher expression in males than in females. SsubOBP25, SsubOBP27, and SsubOBP28 were clustered into the moth pheromone-binding protein (PBP) sub-family, and they were uniquely expressed in the antennae, with a higher expression in males than in females. SsubOBP19, a member of the GOBP2 group, was the most abundant OBP in the antennae. These findings indicate that these olfactory genes, comprising five candidate PRs, three candidate PBPs, and one candidate GOBP2, may be involved in type II sex pheromone detection. As well as these genes, most of the remaining SsubORs, and all of the SsubIRs, showed a considerably higher expression in the female antennae than in the male antennae. Many of these, including SsubOR40, SsubOR42, SsubOR43, and SsubIR26, were more abundant in female antennae. These olfactory and ionotropic receptors may be related to the detection of host plant volatiles. The results of this present study provide a basis for exploring the olfaction mechanisms in S. subpunctaria, with a focus on the genes involved in type II sex pheromones. The evolutionary analyses in our study provide new insights into the differentiation and evolution of lepidopteran PRs.

Transport of trans-tiliroside (kaempferol-3-ß-D-(6"-p-coumaroyl-glucopyranoside) and related flavonoids across Caco-2 cells, as a model of absorption and metabolism in the small intestine.

1. Absorption and metabolism of tiliroside (kaempferol 3-ß-D-(6"-p-coumaroyl)-glucopyranoside) and its related compounds kaempferol, kaempferol-3-glucoside and p-coumaric acid were investigated in the small intestinal Caco-2 cell model. Apparent permeation (Papp) was determined as 0.62 × 10(-6) cm/s, 3.1 × 10(-6) cm/s, 0 and 22.8 × 10(-6) cm/s, respectively. 2. Mechanistic study showed that the transportation of tiliroside, kaempferol-3-glucoside and p-coumaric acid in Caco-2 model were transporter(s) involved, while transportation of kaempferol was solely by passive diffusion mechanism. 3. Efflux transporters, multi-drug-resistance-associated protein-2 (MRP2), were shown to play a role in limiting the uptake of tiliroside. Inhibitors of MRP2, (MK571 and rifampicin) and co-incubation with kaempferol (10 µM), increased transfer from the apical to the basolateral side by three to five fold. 4. Metabolites of kaempferol-3-glucoside and p-coumaric acid were not detected in the current Caco-2 model, while tiliroside was metabolised to a limited extent, with two tiliroside mono-glucuronides identified; and kaempferol was metabolised to a higher extent, with three mono-glucuronides and two mono-sulfates identified. 5. In conclusion, tiliroside was metabolised and transported across Caco-2 cell membrane to a limited extent. Transportation could be increased by applying MRP2 inhibitors or co-incubation with kaempferol. It is proposed that tiliroside can be absorbed by human; future pharmacokinetics studies are warranted in order to determine the usefulness of tiliroside as a bioactive agent.

Cupid: simultaneous reconstruction of microRNA-target and ceRNA networks.

We introduce a method for simultaneous prediction of microRNA-target interactions and their mediated competitive endogenous RNA (ceRNA) interactions. Using high-throughput validation assays in breast cancer cell lines, we show that our integrative approach significantly improves on microRNA-target prediction accuracy as assessed by both mRNA and protein level measurements. Our biochemical assays support nearly 500 microRNA-target interactions with evidence for regulation in breast cancer tumors. Moreover, these assays constitute the most extensive validation platform for computationally inferred networks of microRNA-target interactions in breast cancer tumors, providing a useful benchmark to ascertain future improvements.

Genome-wide perturbations by miRNAs map onto functional cellular pathways, identifying regulators of chromatin modifiers.

BACKGROUND: Regulation of gene expression by microRNAs (miRNAs) is critical for determining cellular fate and function. Dysregulation of miRNA expression contributes to the development and progression of multiple diseases. miRNA can target multiple mRNAs, making deconvolution of the effects of miRNA challenging and the complexity of regulation of cellular pathways by miRNAs at the functional protein level remains to be elucidated. Therefore, we sought to determine the effects of expression of miRNAs in breast and ovarian cancer cells on cellular pathways by measuring systems-wide miRNA perturbations to protein and phosphoproteins. METHODS: We measure protein level changes by reverse-phase protein array (RPPA) in MDA-MB-231, SKOV3.ip1 and HEYA8 cancer cell lines transfected by a library of 879 human miRNA mimics. RESULTS: The effects of multiple miRNAs-protein networks converged in five broad functional clusters of miRNA, suggesting a broad overlap of miRNA action on cellular pathways. Detailed analysis of miRNA clusters revealed novel miRNA/cell cycle protein networks, which we functionally validated. De novo phosphoprotein network estimation using Gaussian graphical modeling, using no priors, revealed known and novel protein interplay, which we also observed in patient ovarian tumor proteomic data. We identified several miRNAs that have pluripotent activities across multiple cellular pathways. In particular we studied miR-365a whose expression is associated with poor survival across several cancer types and demonstrated that anti-miR-365 significantly reduced tumor formation in animal models. CONCLUSIONS: Mapping of miRNA-induced protein and phosphoprotein changes onto pathways revealed new miRNA-cellular pathway connectivity, paving the way for targeting of dysregulated pathways with potential miRNA-based therapeutics.

Impact of miRNA sequence on miRNA expression and correlation between miRNA expression and cell cycle regulation in breast cancer cells.

The miRNAs regulate cell functions by inhibiting expression of proteins. Research on miRNAs had usually focused on identifying targets by base pairing between miRNAs and their targets. Instead of identifying targets, this paper proposed an innovative approach, namely impact significance analysis, to study the correlation between mature sequence, expression across patient samples or time and global function on cell cycle signaling of miRNAs. With three distinct types of data: The Cancer Genome Atlas miRNA expression data for 354 human breast cancer specimens, microarray of 266 miRNAs in mouse Embryonic Stem cells (ESCs), and Reverse Phase Protein Array (RPPA) transfected by 776 miRNAs in MDA-MB-231 cell line, we linked the expression and function of miRNAs by their mature sequence and discovered systematically that the similarity of miRNA expression enhances the similarity of miRNA function, which indicates the miRNA expression can be used as a supplementary factor to predict miRNA function. The results also show that both seed region and 3' portion are associated with miRNA expression levels across human breast cancer specimens and in ESCs; miRNAs with similar seed tend to have similar 3' portion. And we discussed that the impact of 3' portion, including nucleotides 13-16, is not significant for miRNA function. These results provide novel insights to understand the correlation between miRNA sequence, expression and function. They can be applied to improve the prediction algorithm and the impact significance analysis can also be implemented to similar analysis for other small RNAs such as siRNAs.

Modeling miRNA-mRNA interactions: fitting chemical kinetics equations to microarray data.

BACKGROUND: The miRNAs are small non-coding RNAs of roughly 22 nucleotides in length, which can bind with and inhibit protein coding mRNAs through complementary base pairing. By degrading mRNAs and repressing proteins, miRNAs regulate the cell signaling and cell functions. This paper focuses on innovative mathematical techniques to model gene interactions by algorithmic analysis of microarray data. Our goal was to elucidate which mRNAs were actually degraded or had their translation inhibited by miRNAs belonging to a very large pool of potential miRNAs. RESULTS: We proposed two chemical kinetics equations (CKEs) to model the interactions between miRNAs, mRNAs and the associated proteins. In order to reduce computational cost, we used a non linear profile clustering method named minimal net clustering and efficiently condensed the large set of expression profiles observed in our microarray data sets. We determined unknown parameters of the CKE models by minimizing the discrepancy between model prediction and data, using our own fast non linear optimization algorithm. We then retained only the CKE models for which the optimized fit to microarray data is of high quality and validated multiple miRNA-mRNA pairs. CONCLUSION: The implementation of CKE modeling and minimal net clustering reduces drastically the potential set of miRNA-mRNA pairs, with a high gain for further experimental validations. The minimal net clustering also provides good miRNA candidates that have similar regulatory roles.

The complete mitochondrial genome of bean goose (Anser fabalis) and implications for anseriformes taxonomy.

Mitochondrial DNA plays an important role in living organisms, and has been used as a powerful molecular marker in a variety of evolutionary studies. In this study, we determined the complete mtDNA of Bean goose (Anser fabalis), which is 16,688 bp long and contains 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a control region. The arrangement is similar to that of typical Anseriform species. All protein-coding genes, except for Cyt b, ND5, COI, and COII, start with an ATG codon. The ATG start codon is also generally observed in the 12 other Anseriform species, including 2 Anser species, with sequenced mitochondrial genomes. TAA is the most frequent stop codon, one of three-TAA, TAG, and T- -commonly observed in Anseriformes. All tRNAs could be folded into canonical cloverleaf secondary structures except for tRNA(Ser)(AGY) and tRNA(Leu)(CUN), which are missing the dihydrouridine (DHU) arm. The control region of Bean goose mtDNA, with some conserved sequence boxes, such as F, E, D, and C, identified in its central domain. Phylogenetic analysis of complete mtDNA data for 13 Anseriform species supports the classification of them into four major branches: Anatinae, Anserinae, Dendrocygninae and Anseranatidae. Phylogenetic analyses were also conducted on 36 Anseriform birds using combined Cyt b, ND2, and COI sequences. The results clearly support the genus Somateria as an independent lineage classified in its own tribe, the Somaterini. Recovered topologies from both complete mtDNA and combined DNA sequences strongly indicate that Dendrocygninae is an independent subfamily within the family Anatidae and Anseranatidae represents an independent family. Based on the results of this study, we conclude that combining ND2, Cyt b, and COI sequence data is a workable solution at present for resolving phylogenetic relationships among Anseriform species in the absence of sufficient complete mtDNA data.

Effects of pH on the ability of flavonoids to act as Pickering emulsion stabilizers.

The flavonoids tiliroside, rutin and naringin have been investigated as stabilizers of Pickering oil-in-water (O/W) emulsions. The mean droplet size of tetradecane emulsions was considerably smaller at higher pH, especially for rutin. The solubility of flavonoids in the aqueous phase was 4-6 times higher at pH 8 compared to pH 2 for tiliroside and rutin, although all absolute solubilities remained low (<1 mM). This agreed with a slight increase in surface activity of tiliroside and rutin at the O-W interface at pH 8 compared to pH 2. However, improved emulsion stabilization at higher pH is better explained by the significant increase in ζ-potential of the flavonoid particles to more negative values at pH 8, which will improve particle dispersion and increase the charge on the droplets stabilized by them. A buckwheat tea extract, rich in rutin, was also shown to be an effective stabilizer of sunflower O/W emulsions.

miRNA regulatory circuits in ES cells differentiation: a chemical kinetics modeling approach.

MicroRNAs (miRNAs) play an important role in gene regulation for Embryonic Stem cells (ES cells), where they either down-regulate target mRNA genes by degradation or repress protein expression of these mRNA genes by inhibiting translation. Well known tables TargetScan and miRanda may predict quite long lists of potential miRNAs inhibitors for each mRNA gene, and one of our goals was to strongly narrow down the list of mRNA targets potentially repressed by a known large list of 400 miRNAs. Our paper focuses on algorithmic analysis of ES cells microarray data to reliably detect repressive interactions between miRNAs and mRNAs. We model, by chemical kinetics equations, the interaction architectures implementing the two basic silencing processes of miRNAs, namely "direct degradation" or "translation inhibition" of targeted mRNAs. For each pair (M,G) of potentially interacting miRMA gene M and mRNA gene G, we parameterize our associated kinetic equations by optimizing their fit with microarray data. When this fit is high enough, we validate the pair (M,G) as a highly probable repressive interaction. This approach leads to the computation of a highly selective and drastically reduced list of repressive pairs (M,G) involved in ES cells differentiation.

Particle-stabilizing effects of flavonoids at the oil-water interface.

It has been shown that some common food flavonoids can act as excellent stabilizers of oil-in-water emulsions through their adsorption as water-insoluble particles to the surface of the oil droplets, i.e., Pickering emulsions are formed. Flavonoids covering a wide range of octanol-water partition coefficients (P) were screened for emulsification behavior by low shear mixing of flavonoid+n-tetradecane in a vortex mixer. Most flavonoids with very high or very low P values were not good emulsifiers, although there were exceptions, such as tiliroside, which is very insoluble in water. When a high shear jet homogenizer was used with 20 vol% oil in the presence of 1 mM tiliroside, rutin, or naringin, much finer emulsions were produced: the average droplet sizes (d32) were 16, 6, and 5 µm, respectively. These results may be highly significant with respect to the delivery of such insoluble compounds to the gut, as well as their digestion and absorption.