Single-cell RNA-sequencing of zebrafish hair cells reveals novel genes potentially involved in hearing loss.

Hair cells play key roles in hearing and balance, and hair cell loss would result in hearing loss or vestibular dysfunction. Cellular and molecular research in hair cell biology provides us a better understanding of hearing and deafness. Zebrafish, owing to their hair cell-enriched organs, have been widely applied in hair cell-related research worldwide. Similar to mammals, zebrafish have inner ear hair cells. In addition, they also have lateral line neuromast hair cells. These different types of hair cells vary in morphology and function. However, systematic analysis of their molecular characteristics remains lacking. In this study, we analyzed the GFP+ cells isolated from Tg(Brn3c:mGFP) larvae with GFP expression in all hair cells using single-cell RNA-sequencing (scRNA-seq). Three subtypes of hair cells, namely macula hair cell (MHC), crista hair cell (CHC), and neuromast hair cell (NHC), were characterized and validated by whole-mount in situ hybridization analysis of marker genes. The hair cell scRNA-seq data revealed hair cell-specific genes, including hearing loss genes that have been identified in humans and novel genes potentially involved in hair cell formation and function. Two novel genes were discovered to specifically function in NHCs and MHCs, corresponding to their specific expression in NHCs and MHCs. This study allows us to understand the specific genes in hair cell subpopulations of zebrafish, which will shed light on the genetics of both human vestibular and cochlear hair cell function.

THOC1 deficiency leads to late-onset nonsyndromic hearing loss through p53-mediated hair cell apoptosis.

Apoptosis of cochlear hair cells is a key step towards age-related hearing loss. Although numerous genes have been implicated in the genetic causes of late-onset, progressive hearing loss, few show direct links to the proapoptotic process. By genome-wide linkage analysis and whole exome sequencing, we identified a heterozygous p.L183V variant in THOC1 as the probable cause of the late-onset, progressive, non-syndromic hearing loss in a large family with autosomal dominant inheritance. Thoc1, a member of the conserved multisubunit THO/TREX ribonucleoprotein complex, is highly expressed in mouse and zebrafish hair cells. The thoc1 knockout (thoc1 mutant) zebrafish generated by gRNA-Cas9 system lacks the C-startle response, indicative of the hearing dysfunction. Both Thoc1 mutant and knockdown zebrafish have greatly reduced hair cell numbers, while the latter can be rescued by embryonic microinjection of human wild-type THOC1 mRNA but to significantly lesser degree by the c.547C>G mutant mRNA. The Thoc1 deficiency resulted in marked apoptosis in zebrafish hair cells. Consistently, transcriptome sequencing of the mutants showed significantly increased gene expression in the p53-associated signaling pathway. Depletion of p53 or applying the p53 inhibitor Pifithrin-α significantly rescued the hair cell loss in the Thoc1 knockdown zebrafish. Our results suggested that THOC1 deficiency lead to late-onset, progressive hearing loss through p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss.

Metabolic differences of two constructive species in saline-alkali grassland in China.

BACKGROUND: Salinization of soil is an urgent problem that restricts agroforestry production and environmental protection. Substantial accumulation of metal ions or highly alkaline soil alters plant metabolites and may even cause plant death. To explore the differences in the response strategies between Suaeda salsa (S. salsa) and Puccinellia tenuiflora (P. tenuiflora), two main constructive species that survive in saline-alkali soil, their metabolic differences were characterized. RESULT: Metabolomics was conducted to study the role of metabolic differences between S. salsa and P. tenuiflora under saline-alkali stress. A total of 68 significantly different metabolites were identified by GC-MS, including 9 sugars, 13 amino acids, 8 alcohols, and 34 acids. A more detailed analysis indicated that P. tenuiflora utilizes sugars more effectively and may be saline-alkali tolerant via sugar consumption, while S. salsa utilizes mainly amino acids, alcohols, and acids to resist saline-alkali stress. Measurement of phenolic compounds showed that more C6C3C6-compounds accumulated in P. tenuiflora, while more C6C1-compounds, phenolic compounds that can be used as signalling molecules to defend against stress, accumulated in S. salsa. CONCLUSIONS: Our observations suggest that S. salsa resists the toxicity of saline-alkali stress using aboveground organs and that P. tenuiflora eliminates this toxicity via roots. S. salsa has a stronger habitat transformation ability and can provide better habitat for other plants.

Metformin and an insulin/IGF-1 receptor inhibitor are synergistic in blocking growth of triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor survival outcomes. Metformin has been shown to have antitumor effects by lowering serum levels of the mitogen insulin and having pleiotropic effects on cancer cell signaling pathways. BMS-754807 is a potent and reversible inhibitor of both insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR). Both drugs have been reported to have some efficacy in TNBC. However, it is unclear whether the combination of the two drugs is more effective than single drug treatment in TNBC. METHODS: We treated a panel of TNBC cell lines with metformin and BMS-754807 alone and in combination and tested cell viability using MTS assays. We used the CompuSyn software to analyze for additivity, synergism, or antagonism. We also examined the molecular mechanism by performing reverse phase protein assay (RPPA) to detect the candidate pathways altered by single drugs and the drug combination and used Western blotting to verify and expand the findings. RESULTS: The combination of metformin and BMS-754807 showed synergy in 11 out of 13 TNBC cell lines tested (85%). RPPA analysis detected significant alterations by the drug combination of multiple proteins known to regulate cell cycle and tumor growth. In particular, the drug combination significantly increased levels of total and phosphorylated forms of the cell cycle inhibitor p27Kip1 and decreased the level of the p27Kip1 E3 ligase SCFSkp2. CONCLUSIONS: We conclude that the combination of metformin and BMS-754807 is more effective than either drug alone in inhibiting cell proliferation in the majority of TNBC cell lines, and that one important mechanism may be suppression of SCFSkp2 and subsequent stabilization of the cell cycle inhibitor p27Kip1. This combination treatment may represent an effective targeted therapy for a significant subset of TNBC cases and should be further evaluated.

Systematic profiling of early regulators during tissue regeneration using zebrafish model.

Great progresses have been made in comprehension of tissue regeneration process. However, one of the central questions in regeneration research remains to be deciphered is what factors initiate regenerative process. In present study, we focused on systematic profiling of early regulators in tissue regeneration via high-throughput screening on zebrafish caudal fin model. Firstly, 53 GO-annotated regeneration-related genes, which were specifically activated upon fin amputation, were identified according to the transcriptomic analysis. Moreover, qRT-PCR analysis of a couple of randomly selected genes from the aforementioned gene list validated our sequencing results. These studies confirmed the reliability of transcriptome sequencing analysis. Fibroblast growth factor 20a (fgf20a) is a key initial factor in the regeneration of zebrafish. Through a gene expression correlation analysis, we discovered a collection of 70 genes correlating with fgf20a, whose expression increased promptly at 2 days post amputation (dpa) and went down to the basal level until the completion of fin regeneration. In addition, two genes, socs3b and nppc, were chosen to investigate their functions during the fin regeneration. Inhibition of either of those genes significantly delayed the regenerative process. Taken together, we provided a simple and effective time-saving strategy that may serve as a tool for identifying early regulators in regeneration and identified 71 genes as early regulators of fin regeneration.

Transcriptional repressor GATA binding 1-mediated repression of SRY-box 2 expression suppresses cancer stem cell functions and tumor initiation.

Cancer stem cells (CSCs) have been reported in a variety of cancers. SRY-box 2 (SOX2) is a member of the SOX family of transcription factors and has been shown to play a critical role in maintaining the functions of CSCs and promoting tumor initiation. However, the underlying mechanisms for the transcriptional regulation of the SOX2 gene in CSCs are unclear. In this study, using in silico and experimental approaches, we identified transcriptional repressor GATA binding 1 (TRPS1), an atypical GATA-type transcription factor, as a critical transcriptional regulator that represses SOX2 expression and thereby suppresses cancer stemness and tumorigenesis. Mechanistically, TRPS1 repressed SOX2 expression by directly targeting the consensus GATA-binding element in the SOX2 promoter as elucidated by ChIP and luciferase reporter assays. Of note, in vitro mammosphere formation assays in culture and in vivo xenograft tumor initiation experiments in mouse models revealed that TRPS1-mediated repression of SOX2 expression suppresses CSC functions and tumor initiation. Taken together, our study provides detailed mechanistic insights into CSC functions and tumor initiation by the TRPS1-SOX2 axis.

Phylogeny, Divergent Evolution, and Speciation of Sulfur-Oxidizing Acidithiobacillus Populations.

BACKGROUND: Habitats colonized by acidophiles as an ideal physical barrier may induce genetic exchange of microbial members within the common communities, but little is known about how species in extremely acidic environments diverge and evolve. RESULTS: Using the acidophilic sulfur-oxidizer Acidithiobacillus as a case study, taxonomic reclassifications of many isolates provides novel insights into their phylogenetic lineage. Whole-genome-based comparisons were attempted to investigate the intra- and inter-species divergence. Recent studies clarified that functional and structural specificities of bacterial strains might provide opportunities for adaptive evolution responding to local environmental conditions. Acidophilic microorganisms play a key role in the acidification of natural waters and thus the formation of extremely acidic environments, and the feedbacks of the latter might confer the distinct evolutionary patterns of Acidithiobacillus spp. Varied horizontal gene transfer events occurred in different bacterial strains, probably resulting in the expansion of Acidithiobacillus genomes. Gene loss as another evolutionary force might cause the adaptive phenotypic diversity. A conceptual model for potential community-dependent evolutionary adaptation was thus proposed to illustrate the observed genome differentiation. CONCLUSIONS: Collectively, the findings shed light on the phylogeny and divergent evolution of Acidithiobacillus strains, and provided a useful reference for evolutionary studies of other extremophiles.

A ribosomal DNA-hosted microRNA regulates zebrafish embryonic angiogenesis.

MicroRNAs (miRNAs) are single-stranded small non-coding RNAs, generally 18-25 nucleotides in length, that act as repressors of gene expression. miRNAs are encoded by independent genes or processed from a variety of different RNA species. So far, there is no evidence showing that the ribosomal DNA-hosted microRNA is implicated in vertebrate development. Currently, we found a highly expressed small RNA hosted in ribosomal DNA was predicted as a novel miRNA, named miR-ntu1, in zebrafish endothelial cells by deep sequencing analysis. The miRNA was validated by custom-designed Taqman PCR, Northern Blot, and in silico analysis. Furthermore, we demonstrated that miR-ntu1 played a crucial role in zebrafish angiogenesis via modulation of Notch signaling. Our findings provide a notable case that a miRNA hosted in ribosomal DNA is involved in vertebrate development.

Tricho-rhino-phalangeal syndrome 1 protein functions as a scaffold required for ubiquitin-specific protease 4-directed histone deacetylase 2 de-ubiquitination and tumor growth.

BACKGROUND: Although numerous studies have reported that tricho-rhino-phalangeal syndrome type I (TRPS1) protein, the only reported atypical GATA transcription factor, is overexpressed in various carcinomas, the underlying mechanism(s) by which it contributes to cancer remain unknown. METHODS: Both overexpression and knockdown of TRPS1 assays were performed to examine the effect of TRPS1 on histone deacetylase 2 (HDAC2) protein level and luminal breast cancer cell proliferation. Also, RT-qRCR, luciferase reporter assay and RNA-sequencing were used for transcription detection. Chromatin immunoprecipitation (ChIP) using H4K16ac antibody in conjunction with qPCR was used for determining H4K16ac levels in targeted genes. Furthermore, in vitro cell proliferation assay and in vivo tumor xenografts were used to detect the effect of TRPS1 on tumor growth. RESULTS: We found that TRPS1 scaffolding recruits and enhances interaction between USP4 and HDAC2 leading to HDAC2 de-ubiquitination and H4K16 deacetylation. We detected repression of a set of cellular growth-related genes by the TRPS1-USP4-HDAC2 axis indicating it is essential in tumor growth. In vitro and in vivo experiments confirmed that silencing TRPS1 reduced tumor growth, whereas overexpression of HDAC2 restored tumor growth. CONCLUSION: Our study deciphered the TRPS1-USP4-HDAC2 axis as a novel mechanism that contributes to tumor growth. Significantly, our results revealed the scaffolding function of TPRS1 in USP4-directed HDAC2 de-ubiquitination and provided new mechanistic insights into the crosstalk between TRPS1, ubiquitin, and histone modification systems leading to tumor growth.

Dme-Hsa Disease Database (DHDD): Conserved Human Disease-Related miRNA and Their Targeting Genes in Drosophila melanogaster.

Abnormal expressions of microRNA (miRNA) can result in human diseases such as cancer and neurodegenerative diseases. MiRNA mainly exert their biological functions via repressing the expression of their target genes. Drosophila melanogaster (D. melanogaster) is an ideal model for studying the molecular mechanisms behind biological phenotypes, including human diseases. In this study, we collected human and D. melanogaster miRNA as well as known human disease-related genes. In total, we identified 136 human disease-related miRNA that are orthologous to 83 D. melanogaster miRNA by mapping "seed sequence", and 677 human disease-related genes that are orthologous to 734 D. melanogaster genes using the DRSC Integrative Ortholog Prediction Tool Furthermore, we revealed the target relationship between genes and miRNA using miRTarBase database and target prediction software, including miRanda and TargetScan. In addition, we visualized interaction networks and signalling pathways for these filtered miRNA and target genes. Finally, we compiled all the above data and information to generate a database designated DHDD This is the first comprehensive collection of human disease-related miRNA and their targeting genes conserved in a D. melanogaster database. The DHDD provides a resource for easily searching human disease-related miRNA and their disease-related target genes as well as their orthologs in D. melanogaster, and conveniently identifying the regulatory relationships among them in the form of a visual network.

Comprehensive data mining reveals RTK/RAS signaling pathway as a promoter of prostate cancer lineage plasticity through transcription factors and CNV.

Prostate cancer lineage plasticity is a key driver in the transition to neuroendocrine prostate cancer (NEPC), and the RTK/RAS signaling pathway is a well-established cancer pathway. Nevertheless, the comprehensive link between the RTK/RAS signaling pathway and lineage plasticity has received limited investigation. In particular, the intricate regulatory network governing the interplay between RTK/RAS and lineage plasticity remains largely unexplored. The multi-omics data were clustered with the coefficient of argument and neighbor joining algorithm. Subsequently, the clustered results were analyzed utilizing the GSEA, gene sets related to stemness, multi-lineage state datasets, and canonical cancer pathway gene sets. Finally, a comprehensive exploration of the data based on the ssGSEA, WGCNA, GSEA, VIPER, prostate cancer scRNA-seq data, and the GPSAdb database was conducted. Among the six modules in the clustering results, there are 300 overlapping genes, including 3 previously unreported prostate cancer genes that were validated to be upregulated in prostate cancer through RT-qPCR. Function Module 6 shows a positive correlation with prostate cancer cell stemness, multi-lineage states, and the RTK/RAS signaling pathway. Additionally, the 19 leading-edge genes of the RTK/RAS signaling pathway promote prostate cancer lineage plasticity through a complex network of transcriptional regulation and copy number variations. In the transcriptional regulation network, TP63 and FOXO1 act as suppressors of prostate cancer lineage plasticity, whereas RORC exerts a promoting effect. This study provides a comprehensive perspective on the role of the RTK/RAS pathway in prostate cancer lineage plasticity and offers new clues for the treatment of NEPC.

Metabolomic responses to the mechanical wounding of Catharanthus roseus' upper leaves.

Purpose: Plant secondary metabolites are used to treat various human diseases. However, it is difficult to produce a large number of specific metabolites, which largely limits their medicinal applications. Many methods, such as drought and nutrient application, have been used to induce the biosynthetic production of secondary metabolites. Among these secondary metabolite-inducing methods, mechanical wounding maintains the composition of secondary metabolites with little potential risk. However, the effects of mechanical stress have not been fully investigated, and thus this method remains widely unused. Methods: In this study, we used metabolomics to investigate the metabolites produced in the upper and lower leaves of Catharanthus roseus in response to mechanical wounding. Results: In the upper leaves, 13 different secondary metabolites (three terpenoid indole alkaloids and 10 phenolic compounds) were screened using an orthogonal partial least squares discriminant analysis (OPLS-DA) score plot. The mechanical wounding of different plant parts affected the production of secondary metabolites. Specifically, when lower leaves were mechanically wounded, the upper leaves became a strong source of resources. Conversely, when upper leaves were injured, the upper leaves themselves became a resource sink. Changes in the source-sink relationship reflected a new balance between resource tradeoff and the upregulation or downregulation of certain metabolic pathways. Conclusion: Our findings suggest that mechanical wounding to specific plant parts is a novel approach to increase the biosynthetic production of specific secondary metabolites. These results indicate the need for a reevaluation of production practices for secondary metabolites from select commercial plants.

Expression analysis of nel during zebrafish embryonic development.

Nel is a multimeric extracellular glycoprotein which predominantly expressed in the nervous system and play an important role in neural development and functions. There are three nel paralogues included nell2a, nell2b, and nell3 in zebrafish, while systematic expression analysis of the nel family is still lacking. In this study, we performed a phylogenetic analysis on 7 species, in different species the nell2a are highly conserved, as is nell2b. Then, the expression profiles of nell2a, nell2b and nell3 were detected by in situ hybridization in zebrafish embryo, and the result showed that nel genes highly enriched in the central nervous system, but distributed in different regions of the brain. In addition, nell2a is also expressed in the olfactory pit, spinal cord, otic vesicle and retina (ganglion cell layer), nell2b was detected to express in gill arches, olfactory epithelium, olfactory pit, spinal cord, photoreceptor and retina (ganglion cell layer), it should be noted that the expression of nell3 is special, was only detected at 96 hpf in the brain and spinal cord of zebrafish. Overall, our results indicate that nell2a and nell2b genes are expressed in the nervous system and eyes of zebrafish embryo, while nell3 is expressed in different regions in the nervous system. The phylogenetic analysis also shows that nell3 sequences are significantly different from nell2a and nell2b. This study provides new evidence to better understand the role of nel in zebrafish embryo development.

Dual-Specificity Phosphatase 14 Regulates Zebrafish Hair Cell Formation Through Activation of p38 Signaling Pathway.

Most cases of acquired hearing loss are due to degeneration and subsequent loss of cochlear hair cells. Whereas mammalian hair cells are not replaced when lost, in zebrafish, they constantly renew and regenerate after injury. However, the molecular mechanism among this difference remains unknown. Dual-specificity phosphatase 14 (DUSP14) is an important negative modulator of mitogen-activated protein kinase (MAPK) signaling pathways. Our study was to investigate the effects of DUSP14 on supporting cell development and hair cell regeneration and explore the potential mechanism. Our results showed that dusp14 gene is highly expressed in zebrafish developing neuromasts and otic vesicles. Behavior analysis showed that dusp14 deficiency resulted in hearing defects in zebrafish larvae, which were reversed by dusp14 mRNA treatment. Moreover, knockdown of dusp14 gene caused a significant decrease in the number of neuromasts and hair cells in both neuromast and otic vesicle, mainly due to the inhibition of the proliferation of supporting cells, which results in a decrease in the number of supporting cells and ultimately in the regeneration of hair cells. We further found significant changes in a series of MAPK pathway genes through transcriptome sequencing analysis of dusp14-deficient zebrafish, especially mapk12b gene in p38 signaling. Additionally, inhibiting p38 signaling effectively rescued all phenotypes caused by dusp14 deficiency, including hair cell and supporting cell reduction. These results suggest that DUSP14 might be a key gene to regulate supporting cell development and hair cell regeneration and is a potential target for the treatment of hearing loss.

FGF binding protein 3 is required for spinal cord motor neuron development and regeneration in zebrafish.

Fibroblast growth factor binding protein 3 (Fgfbp3) have been known to be crucial for the process of neural proliferation, differentiation, migration, and adhesion. However, the specific role and the molecular mechanisms of fgfbp3 in regulating the development of motor neurons remain unclear. In this study, we have investigated the function of fgfbp3 in morphogenesis and regeneration of motor neuron in zebrafish. Firstly, we found that fgfbp3 was localized in the motor neurons and loss of fgfbp3 caused the significant decrease of the length and branching number of the motor neuron axons, which could be partially rescued by fgfbp3 mRNA injection. Moreover, the fgfbp3 knockdown (KD) embryos demonstrated similar defects of motor neurons as identified in fgfbp3 knockout (KO) embryos. Furthermore, we revealed that the locomotion and startle response of fgfbp3 KO embryos were significantly restricted, which were partially rescued by the fgfbp3 overexpression. In addition, fgfbp3 KO remarkably compromised axonal regeneration of motor neurons after injury. Lastly, the malformation of motor neurons in fgfbp3 KO embryos was rescued by overexpressing drd1b or neurod6a, respectively, which were screened by transcriptome sequencing. Taken together, our results provide strong cellular and molecular evidence that fgfbp3 is crucial for the axonal morphogenesis and regeneration of motor neurons in zebrafish.

Ionomic and Metabolomic Analyses Reveal Different Response Mechanisms to Saline-Alkali Stress Between Suaeda salsa Community and Puccinellia tenuiflora Community.

Soil salinization imposes severe stress to plants, inhibits plant growth, and severely limits agricultural productivity and land utilization. The response of a single plant to saline-alkali stress has been well investigated. However, the plant community that usually works as a group to defend against saline-alkali stress was neglected. To determine the functions of plant community, in our current work, Suaeda salsa (S. salsa) community and Puccinellia tenuiflora (P. tenuiflora) community, two communities that are widely distributed in Hulun Buir Grassland in Northeastern China, were selected as research objects. Ionomic and metabolomic were applied to compare the differences between S. salsa community and P. tenuiflora community from the aspects of ion transport and phenolic compound accumulation, respectively. Ionomic studies demonstrated that many macroelements, including potassium (K) and calcium (Ca), were highly accumulated in S. salsa community whereas microelement manganese (Mn) was highly accumulated in P. tenuiflora community. In S. salsa community, transportation of K to aboveground parts of plants helps to maintain high K+ and low Na+ concentrations whereas the accumulation of Ca triggers the salt overly sensitive (SOS)-Na+ system to efflux Na+. In P. tenuiflora community, enrichment of Mn in roots elevates the level of Mn-superoxide dismutase (SOD) and increases the resistance to saline-alkali stress. Metabolomic studies revealed the high levels of C6C1-compounds and C6C3C6-compounds in S. salsa community and also the high levels of C6C3-compounds in P. tenuiflora community. C6C1-compounds function as signaling molecules to defend against stress and may stimulate the accumulation of C6C3C6-compounds. C6C3-compounds contribute to the elimination of free radicals and the maintenance of cell morphology. Collectively, our findings determine the abundance of phenolic compounds and various elements in S. salsa community and P. tenuiflora community in Hulun Buir Grassland and we explored different responses of S. salsa community and P. tenuiflora community to cope with saline-alkali stress. Understanding of plant response strategies from the perspective of community teamwork may provide a feasible and novel way to transform salinization land.

Claudin h Is Essential for Hair Cell Morphogenesis and Auditory Function in Zebrafish.

Hereditary hearing loss caused by defective hair cells is one of the most common congenital diseases, whose nosogenesis is still unclear because many of the causative genes remain unidentified. Claudins are one kind of transmembrane proteins that constitute the most important components of the tight junctions and paracellular barrier and play important roles in neurodevelopment. In this study, we investigated the function of claudin h in morphogenesis and auditory function of the hair cell in zebrafish. The results of in situ hybridization showed that claudin h was specifically localized in the otic vesicle and neuromasts in zebrafish embryos. The deficiency of claudin h caused significant reduction of otic vesicle size and loss of utricle otolith. Moreover, the startle response and vestibulo-ocular reflex experiments revealed that loss of claudin h led to serious hearing loss and vestibular dysfunction. Importantly, the confocal microscopy observation found that compared to the control zebrafish, the claudin h morphants and mutants displayed significantly reduced the number of cristae hair cells and shortened kinocilia. Besides, the deficiency of claudin h also caused the loss of hair cells in neuromasts which could be rescued by injecting claudin h mRNA into the mutant embryos at one cell stage. Furthermore, the immunohistochemistry experiments demonstrated remarkable apoptosis of hair cells in the neuromasts, which might contribute to the loss of hair cells number. Overall, these data indicated that claudin h is indispensable for the development of hair cells, vestibular function, and hearing ability of zebrafish.

Biomass-derived cellulose nanoparticles display considerable neurotoxicity in zebrafish.

The widespread use of nanomaterials poses a great threat to human living environments. Among them, biomass-derived cellulose nanoparticle (CN) is one of the widely used nanomaterials. To date, the toxicity of CNs during embryonic development remains undetermined. In this study, we exposed zebrafish embryos to cellulose nanofibrils (CNFs) and cellulose nanocrystals (CNCs) to evaluate the toxicity of these CNs. Exposure to CNFs or CNCs below 30 mg/ml exhibited no dose-dependent increases in malformation and mortality in zebrafish embryos. Then we demonstrated that CNs were highly enriched in zebrafish embryo via imaging analyses of embryos treated with FITC-coupled CNCs. In addition, we found that CNF or CNC exposure resulted in compromised motor ability of zebrafish larva. Furthermore, it was revealed that the differentiation and the morphogenesis of motor neurons were significantly interrupted. While, blood vessels were normally patterned, suggesting the specific neurotoxicity of these nanomaterials. Transcriptome sequencing assay showed that the neurotoxicity of CNs in the motor neurons might be attributed to the expression alteration of neural genes. In summary, we discovered the neurotoxicity of CNs for the first time.

Insecticidal Activity of Artemisia vulgaris Essential Oil and Transcriptome Analysis of Tribolium castaneum in Response to Oil Exposure.

Red flour beetle (Tribolium castaneum) is one of the most destructive pests of stored cereals worldwide. The essential oil (EO) of Artemisia vulgaris (mugwort) is known to be a strong toxicant that inhibits the growth, development, and reproduction of T. castaneum. However, the molecular mechanisms underlying the toxic effects of A. vulgaris EO on T. castaneum remain unclear. Here, two detoxifying enzymes, carboxylesterase (CarEs) and cytochrome oxidase P450 (CYPs), were dramatically increased in red flour beetle larvae when they were exposed to A. vulgaris EO. Further, 758 genes were differentially expressed between EO treated and control samples. Based on Gene Ontology (GO) analysis, numerous differentially expressed genes (DEGs) were enriched for terms related to the regulation of biological processes, response to stimulus, and antigen processing and presentation. Our results indicated that A. vulgaris EO disturbed the antioxidant activity in larvae and partially inhibited serine protease (SP), cathepsin (CAT), and lipase signaling pathways, thus disrupting larval development and reproduction as well as down-regulating the stress response. Moreover, these DEGs showed that A. vulgaris indirectly affected the development and reproduction of beetles by inducing the expression of genes encoding copper-zinc-superoxide dismutase (CuZnSOD), heme peroxidase (HPX), antioxidant enzymes, and transcription factors. Moreover, the majority of DEGs were mapped to the drug metabolism pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Notably, the following genes were detected: 6 odorant binding proteins (OBPs), 5 chemosensory proteins (CSPs), 14 CYPs, 3 esterases (ESTs), 5 glutathione S-transferases (GSTs), 6 UDP-glucuronosyltransferases (UGTs), and 2 multidrug resistance proteins (MRPs), of which 8 CYPs, 2 ESTs, 2 GSTs, and 3 UGTs were up-regulated dramatically after exposure to A. vulgaris EO. The residual DEGs were significantly down-regulated in EO exposed larvae, implying that partial compensation of metabolism detoxification existed in treated beetles. Furthermore, A. vulgaris EO induced overexpression of OBP/CYP, and RNAi against these genes significantly increased mortality of larvae exposed to EO, providing further evidence for the involvement of OBP/CYP in EO metabolic detoxification in T. castaneum. Our results provide an overview of the transcriptomic changes in T. castaneum in response to A. vulgaris EO.