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Backgound. Multiple patients with hepatocellular carcinoma (HCC) received sorafenib therapy presented primary or secondary chemoresistance. The underlying mechanisms of chemoresistance are rather vague, ferroptosis is a new kind of cell death, and its discovery provides new opportunities to overcome sorafenib resistance. Therefore, it is essential to investigate the regulators of ferroptosis and implement a corresponding therapy strategy. Methods and Results. The levels of MCF2L were detected by PCR and western blotting assay. The effect of MCF2L on ferroptosis was confirmed by MTT, colony formation assay, Brdu, in vivo animal experiment, and the content of Iron, GSH, ROS, and MDA. The underlying mechanisms were explored by PCR, western blotting, and affinity precipitation assay. Our findings demonstrated that MCF2L is remarkedly upregulated in HCC tissues, and sorafenib can induce the levels of MCF2L, suggesting that MCF2L might function in sorafenib resistance of HCC. Further analysis showed that downregulation of MCF2L enhances HCC cell death induced by sorafenib, and ferroptosis inhibitor can reverse this process. Subsequent experiments showed that downregulation of MCF2L elevates the content of Iron, ROS, and MDA, which are all indicators of ferroptosis. Finally, mechanism analysis showed that MCF2L regulates the PI3K/AKT pathway in a RhoA/Rac1 dependent manner. Conclusions. Our study showed that targeting MCF2L may be a hopeful method to overcome sorafenib-resistance through inducing ferroptosis in HCC.

Aspergillus oryzae is widely used as an industrial koji mold for soy sauce brewing due to its powerful raw material decomposition capability. Although various proteases in A. oryzae have been identified, it remains a challenge to find essential enzymes involved in soy sauce production.

Aortic regurgitation (AR) is a volume overload disease causing eccentric left ventricular (LV) hypertrophy and eventually heart failure. There is currently no approved drug to treat patients with AR. Endoplasmic reticulum (ER) stress and ER stress-mediated apoptosis is involved in many cardiovascular diseases, but whether they also participate in AR-induced heart failure is still elusive. In this study, we found ER stress activation in myocardial samples from patients with AR. With a unique murine model of AR which induced eccentric cardiac hypertrophy and heart failure, we also found aggravation of cardiac ER stress and apoptosis, as evidenced by a reduction of Bcl-2/Bax ratio and an increase of caspase-3 cleavage. We then examined the signaling effectors involved in ER-initiated apoptosis and found volume overload specifically activated C/EBP homologous protein (CHOP), but not caspase-12 or Jun N-terminal kinase (JNK). Interestingly, tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, improved cardiac function, and suppressed ER stress, apoptosis, and CHOP. Furthermore, genetic knockdown of CHOP inhibited cardiac Bcl-2/Bax ratio reduction and caspase-3 activation and rescued cardiac dysfunction. In summary, our findings suggest that ER stress-CHOP signaling is involved in the development of volume overload cardiac hypertrophy induced by AR through promoting cardiomyocytes apoptosis and provide a previously unrecognized target in heart failure induced by volume overload.

Volume overload (VO) and pressure overload (PO) are two common pathophysiological conditions associated with cardiac disease. VO, in particular, often occurs in a number of diseases, and no clinically meaningful molecular marker has yet been established. We intend to find the main differential gene expression using bioinformatics analysis. GSE97363 and GSE52796 are the two gene expression array datasets related with VO and PO, respectively. The LIMMA algorithm was used to identify differentially expressed genes (DEGs) of VO and PO. The DEGs were divided into three groups and subjected to functional enrichment analysis, which comprised GO analysis, KEGG analysis, and the protein–protein interaction (PPI) network. To validate the sequencing data, cardiomyocytes from AR and TAC mouse models were used to extract RNA for qRT-PCR. The three genes with random absolute values of LogFC and indicators of heart failure (natriuretic peptide B, NPPB) were detected: carboxylesterase 1D (CES1D), whirlin (WHRN), and WNK lysine deficient protein kinase 2 (WNK2). The DEGs in VO and PO were determined to be 2761 and 1093, respectively, in this study. Following the intersection, 305 genes were obtained, 255 of which expressed the opposing regulation and 50 of which expressed the same regulation. According to the GO and pathway enrichment studies, DEGs with opposing regulation are mostly common in fatty acid degradation, propanoate metabolism, and other signaling pathways. Finally, we used Cytoscape’s three techniques to identify six hub genes by intersecting 255 with the opposite expression and constructing a PPI network. Peroxisome proliferator-activated receptor (PPARα), acyl-CoA dehydrogenase medium chain (ACADM), patatin-like phospholipase domain containing 2 (PNPLA2), isocitrate dehydrogenase 3 (IDH3), heat shock protein family D member 1 (HSPD1), and dihydrolipoamide S-acetyltransferase (DLAT) were identified as six potential genes. Furthermore, we predict that the hub genes PPARα, ACADM, and PNPLA2 regulate VO myocardial changes via fatty acid metabolism and acyl-Coa dehydrogenase activity, and that these genes could be employed as basic biomarkers for VO diagnosis and treatment.

Animal feeding, which directly affects growth and metabolism, is an important physiological process. However, the contribution of PIWI proteins and PIWI-interacting RNAs (piRNAs) to the regulatory mechanism of animal feeding is unknown. Here, we report a novel function of Piwi and piRNAs in regulating food intake in locusts. Our study shows that the locust can serve as a representative species for determining PIWI function in insects. Knockdown of Piwi1 expression suppresses anabolic processes and reduces food consumption and body weight. The reduction in food intake by knockdown of Piwi1 expression results from decreased expression of neuropeptide NPF1 in a piRNA-dependent manner. Mechanistically, intronic piRNAs might enhance RNA splicing of NPF1 by preventing hairpin formation at the branch point sites. These results suggest a novel nuclear PIWI/piRNA-mediated mechanism that controls food intake in the locust nervous system.

Background Alternative splicing is the process of selecting different combinations of splice sites to produce variably spliced mRNAs. However, the relationships between alternative splicing prevalence and level (ASP/L) and variations of intron size and organism complexity (OC) remain vague. Here, we developed a robust protocol to analyze the relationships between ASP/L and variations of intron size and OC. Approximately 8 Tb raw RNA-Seq data from 37 eumetazoan species were divided into three sets of species based on variations in intron size and OC. Results We found a strong positive correlation between ASP/L and OC, but no correlation between ASP/L and intron size across species. Surprisingly, ASP/L displayed a positive correlation with mean intron size of genes within individual genomes. Moreover, our results revealed that four ASP/L-related pathways contributed to the differences in ASP/L that were associated with OC. In particular, the spliceosome pathway displayed distinct genomic features, such as the highest gene expression level, conservation level, and fraction of disordered regions. Interestingly, lower or no obvious correlations were observed among these genomic features. Conclusions The positive correlation between ASP/L and OC ubiquitously exists in eukaryotes, and this correlation is not affected by the mean intron size of these species. ASP/L-related splicing factors may play an important role in the evolution of OC.

The genus Weissella is attracting an increasing amount of attention because of its multiple functions and probiotic potential. In particular, the species Weissella confusa is known to have great potential in industrial applications and exhibits numerous biological functions. However, the knowledge on this bacterium in insects is not investigated. Here, we isolated and identified W. confusa as the dominant lactic acid bacteria in the gut of the migratory locust. We named this strain W. confusa LM1, which is the first genome of an insect-derived W. confusa strain with one complete chromosome and one complete plasmid. Among all W. confusa strains, W. confusa LM1 had the largest genome. Its genome was the closest to that of W. confusa 1001271B_151109_G12, a strain from human feces. Our results provided accurate evolutionary relationships of known Weissella species and W. confusa strains. Based on genomic analysis, the pan-genome of W. confusa is in an open state. Most strains of W. confusa had the unique genes, indicating that these strains can adapt to different ecological niches and organisms. However, the variation of strain-specific genes did represent significant correlations with their hosts and ecological niches. These strains were predicted to have low potential to produce secondary metabolites. Furthermore, no antibiotic resistance genes were identified. At the same time, virulence factors associated with toxin production and secretion system were not found, indicating that W. confusa strains were not sufficient to perform virulence. Our study facilitated the discovery of the functions of W. confusa LM1 in locust biology and their potential application to locust management.

Lung and breast cancer are the two most common causes of malignant pleural effusion (MPE). MPE diagnosis plays a crucial role in determining staging and therapeutic interventions in these cancers. However, our understanding of the pathogenesis and progression of MPE at the molecular level is limited. Extracellular Vesicles (EVs) and their contents, including microRNAs (miRNAs), can be isolated from all bodily fluids, including pleural fluid. This study aims to compare EV-miRNA patterns of expression in MPE caused by breast (BA-MPE) and lung (LA-MPE) adenocarcinomas compared to the control group of heart-failure-induced effusions (HF-PE). We conducted an analysis of 24 pleural fluid samples (8 LA-MPE, 8 BA-MPE, and 8 HF-PE). Using NanoString technology, we profiled miRNAs within EVs isolated from 12 cases. Bioinformatic analysis demonstrated differential expression of miR-1246 in the MPE group vs. HF-PE group and miR-150-5p and miR-1246 in the BA-MPE vs. LA-MPE group, respectively. This difference was demonstrated and validated in an independent cohort using real-time PCR (RT-PCR). miRNA-1246 demonstrated 4-fold increased expression (OR: 3.87, 95% CI: 0.43, 35) in the MPE vs. HF-PE group, resulting in an area under the curve of 0.80 (95% CI: 0.60, 0.99). The highest accuracy for differentiating MPE vs. HF-PE was seen with a combination of miRNAs compared to each miRNA alone. Consistent with prior studies, this study demonstrates dysregulation of specific EV-based miRNAs in breast and lung cancer; pleural fluid provides direct access for the analysis of these EV-miRNAs as biomarkers and potential targets and may provide insight into the underlying pathogenesis of tumor progression. These findings should be explored in large prospective studies.

Background Non-Drosophila insects provide diverse aging types and important complementary systems for studies of aging biology. However, little attention has been paid to the special roles of non-Drosophila insects in aging research. Here, the aging-related features of the migratory locust, Locusta migratoria, were determined at the physiological, cellular, and transcriptional levels. Results In physiological assessments, the flight performance and sperm state of locusts displayed clear aging-related decline in male adults. Transcriptional analyses demonstrated locusts have similar aging-related genes with model species. However, different from those of Drosophila and mammals, the organ-specific aging transcriptional features of locusts were characterized by intensive expression changes in flight muscle and fat body and little transcriptional changes in brain. The predominant transcriptional characteristics of flight muscle and fat body aging were changes in expression of mitochondrion-related genes and detoxification and phagocytosis genes, respectively. Cellular assessments revealed the incidence of mitochondrial abnormalities significantly increased in aged flight muscle, and apoptotic signals and nuclear abnormalities were enhanced in aged fat body but not in brain. In addition, some well-known aging genes and locust aging-related genes (i.e., IAP1, PGRP-SA, and LIPT1), whose roles in aging regulation were rarely reported, were demonstrated to affect lifespan, metabolism, and flight ability of locusts after RNAi. Conclusion This study revealed multi-level aging signatures of locust, thus laying a foundation for further investigation of aging mechanisms in this famous insect in the future.

Maintenance of a balance between the levels of viral replication and selective pressure from the immune systems of insect vectors is one of the prerequisites for efficient transmission of insect-borne propagative phytoviruses. The mechanism regulating the adaptation of RNA viruses to insect vectors by genomic variation remains unknown. Our previous study demonstrated an extension of the 3’-untranslated terminal region (UTR) of two genomic segments of rice stripe virus (RSV). In the present study, a reverse genetic system for RSV in human cells and an insect vector, the small brown planthopper Laodelphax striatellus, was used to demonstrate that the 3’-terminal extensions suppressed viral replication in vector insects by inhibiting promoter activity due to structural interference with the panhandle structure formed by viral 3’- and 5’-UTRs. The extension sequence in the viral RNA1 segment was targeted by an endogenous insect microRNA, miR-263a, which decreased the inhibitory effect of the extension sequence on viral promoter activity. Surprisingly, the expression of miR-263a was negatively regulated by RSV infection. This elaborate coordination between terminal variation of the viral genome and endogenous insect microRNAs controls RSV replication in planthopper, thus reflecting a distinct strategy of adaptation of phytoviruses to insect vectors., Author summary Mutations frequently happen when insect-transmitted RNA viruses circulate between insect vectors and plant or mammalian hosts. However, the significance of these mutations for viral fitness in the two distinct organisms is poorly understood. We discovered that a high proportion of rice stripe virus (RSV) had terminally extended genomes when the virus infected insect vectors. In the present study, we found that the extension sequence suppressed viral replication in insect vectors by impairing a special structure formed by the two ends of the viral genomes. An endogenous insect small RNA was able to bind the extension sequence to relieve the inhibitory effect. However, the expression of this small RNA was reduced in the presence of RSV to ultimately maintain the inhibitory effect of the extension sequence. This elaborate coordination between virus and vector enables a limited level of RSV replication that does not produce serious damage to vectors, thus reflecting a distinct strategy of adaptation of insect-transmitted plant viruses.

Atherosclerosis is the leading cause of heart disease and stroke, and one of the leading causes of death and disability worldwide. The phenotypic transformation of vascular smooth muscle cells (VSMCs) plays an important role in the pathological process of atherosclerosis. The present study aimed to identify differently expressed mRNAs in atherosclerosis by analyzing GSE6088 database. Our results revealed there were totally 467 increased and 490 decreased differential expressed genes (DEGs) in atherosclerosis. Bioinformatics analysis demonstrated that the DEGs substantially existed in pathways, including Glyoxylate and dicarboxylate metabolism, Tyrosine metabolism, Tryptophan metabolism, Beta-Alanine metabolism, Fatty acid biosynthesis and Starch and sucrose metabolism. Next, we constructed a protein-protein interaction (PPI) network to identify hub genes in atherosclerosis. Also, we identified CDK6 as a key regulator of atherosclerosis. In this study, we found that CDK6 knockdown suppressed HASMC and HUASMC cell proliferation. Circular RNA (CircRNA) is a non-coding RNA which is reported to have an unusual influence on tumorigenesis process and other aspects in the last few years. Previous studies showed circRNAs could act as miRNAs sponging in multiple biological processes. Bioinformatics prediction and luciferase analysis showed that CDK6 were targeted and regulated by circHIPK3/miR-637. Moreover, silencing circHIPK3 could also significantly induce the arrest and apoptosis of cell cycle. In conclusion, this study discovered the important regulatory role of circHIPK3 in the proliferation and apoptosis of VSMCs by influencing the miR-637/CDK6 axis.

Outbreaks of locust plagues result from the long-term accumulation of high-density egg production. The migratory locust, Locusta migratoria, displays dramatic differences in the egg-laid number with dependence on population density, while solitarious locusts lay more eggs compared to gregarious ones. However, the regulatory mechanism for the egg-laid number difference is unclear. Herein, we confirm that oosorption plays a crucial role in the regulation of egg number through the comparison of physiological and molecular biological profiles in gregarious and solitarious locusts. We find that gregarious oocytes display a 15% higher oosorption ratio than solitarious ones. Activinβ (Actβ) is the most highly upregulated gene in the gregarious terminal oocyte (GTO) compared to solitarious terminal oocyte (STO). Meanwhile, Actβ increases sharply from the normal oocyte (N) to resorption body 1 (RB1) stage during oosorption. The knockdown of Actβ significantly reduces the oosorption ratio by 13% in gregarious locusts, resulting in an increase in the egg-laid number. Based on bioinformatic prediction and experimental verification, microRNA-34 with three isoforms can target Actβ. The microRNAs display higher expression levels in STO than those in GTO and contrasting expression patterns of Actβ from the N to RB1 transition. Overexpression of each miR-34 isoform leads to decreased Actβ levels and significantly reduces the oosorption ratio in gregarious locusts. In contrast, inhibition of the miR-34 isoforms results in increased Actβ levels and eventually elevates the oosorption ratio of solitarious locusts. Our study reports an undescribed mechanism of oosorption through miRNA targeting of a TGFβ ligand and provides new insights into the mechanism of density-dependent reproductive adaption in insects., Author summary The continuous accumulation of high-density eggs laid by flying swarms of adults results in huge populations of flightless juveniles, which contributes to the outbreaks of locust plagues. An interesting phenomenon is that locusts have the phenotypic plasticity of reproduction. The gregarious locusts lay fewer big eggs than do solitarious phase locusts. In contrast, the solitarious phase locusts lay more small eggs compared to the gregarious locusts. We find the egg-laid number is not only regulated by the phase status of parents but also controlled by oosorption, a type of oocyte death. Further studies confirmed the phase-related ratio of oocyte death in the mother is regulated by a microRNA, which posttranscriptionally influences the expression level of a TGFβ ligand. This maternal effect on progeny size is especially critical for gregarious locusts to control the population size and maintain population fitness, and for solitarious locusts to enhance chance for gregarization and further enlargement of population size. This is the first study to reveal the molecular mechanism underlying the regulation of a microRNA-gene circuit for locust oocyte death to determine the offspring number. These findings can provide some important clues to develop potential drugs to prevent vast locust reproduction from a plague upsurge.

Planthoppers are the most notorious rice pests, because they transmit various rice viruses in a persistent-propagative manner. Protein-protein interactions (PPIs) between virus and vector are crucial for virus transmission by vector insects. However, the number of known PPIs for pairs of rice viruses and planthoppers is restricted by low throughput research methods. In this study, we applied DeNovo, a virus-host sequence-based PPI predictor, to predict potential PPIs at a genome-wide scale between three planthoppers and five rice viruses. PPIs were identified at two different confidence thresholds, referred to as low and high modes. The number of PPIs for the five planthopper-virus pairs ranged from 506 to 1985 in the low mode and from 1254 to 4286 in the high mode. After eliminating the "one-too-many" redundant interacting information, the PPIs with unique planthopper proteins were reduced to 343-724 in the low mode and 758-1671 in the high mode. Homologous analysis showed that 11 sets and 31 sets of homologous planthopper proteins were shared by all planthopper-virus interactions in the two modes, indicating that they are potential conserved vector factors essential for transmission of rice viruses. Ten PPIs between small brown planthopper and rice stripe virus (RSV) were verified using glutathione-S-transferase (GST)/His-pull down or co-immunoprecipitation assay. Five of the ten PPIs were proven positive, and three of the five SBPH proteins were confirmed to interact with RSV. The predicted PPIs provide new clues for further studies of the complicated relationship between rice viruses and their vector insects.

Long noncoding RNAs (lncRNAs) regulate various biological processes from gene expression to animal behavior. Although protein-coding genes, microRNAs, and neuropeptides play important roles in the regulation of phenotypic plasticity in migratory locust, empirical studies on the function of lncRNAs in the process remain limited. Here, we applied high-throughput RNA-seq to characterize the expression patterns of lncRNAs and mRNAs in the time course of the locust phase change. LncRNAs displayed more rapid response at the early stages of the time-course treatments than mRNA expression. Functional annotations demonstrated that early changed lncRNAs employed different pathways in isolation and crowding processes to cope up with the changes in population density. Finally, two overlapping hub lncRNA loci in the crowding and isolation networks were screened to be functionally verified. Experimental validation indicated that LNC1010057 could act as a potential regulator to modulate the locust phase change. This work offers new insights into the mechanism underlying the locust phase change and expands the scope of lncRNA functions in animal behavior.

Background The CRISPR/Cas9 system has been widely used to generate gene knockout/knockin models by inducing frameshift mutants in cell lines and organisms. Several recent studies have reported that such mutants can lead to in-frame exon skipping in cell lines. However, there was little research about post-transcriptional effect of CRISPR-mediated gene editing in vivo. Results We showed that frameshift indels also induced complete or stochastic exon skipping by deleting different regions to influence pre-mRNA splicing in vivo. In the migratory locust, the missing 55 bp at the boundary of intron 3 and exon 4 of an olfactory receptor gene, LmigOr35, resulted in complete exon 4 skipping, whereas the lacking 22 bp in exon 4 of LmigOr35 only resulted in stochastic exon 4 skipping. A single sgRNA induced small insertions or deletions at the boundary of intron and exon to disrupt the 3′ splicing site causing completely exon skipping, or alternatively induce small insertions or deletions in the exon to stochastic alter splicing causing the stochastic exon skipping. Conclusions These results indicated that complete or stochastic exon skipping could result from the CRISPR-mediated genome editing by deleting different regions of the gene. Although exon skipping caused by CRISPR-mediated editing was an unexpected outcome, this finding could be developed as a technology to investigate pre-mRNA splicing or to cure several human diseases caused by splicing mutations.

Heat-shock proteins (Hsps) and their cognates are primary mitigators of cell stress. With increasingly severe impacts of climate change and other human modifications of the biosphere, the ability of the heat-shock system to affect evolutionary fitness in environments outside the laboratory and to evolve in response is topic of growing importance. Since the last major reviews, several advances have occurred. First, demonstrations of the heat-shock response outside the laboratory now include many additional taxa and environments. Many of these demonstrations are only correlative, however. More importantly, technical advances in "omic" quantification of nucleic acids and proteins, genomewide association analysis, and manipulation of genes and their expression have enabled the field to move beyond correlation. Several consequent advances are already evident: The pathway from heat-shock gene expression to stress tolerance in nature can be extremely complex, mediated through multiple biological processes and systems, and even multiple species. The underlying genes are more numerous, diverse and variable than previously appreciated, especially with respect to their regulatory variation and epigenetic changes. The impacts and limitations (e.g., due to trade-offs) of natural selection on these genes have become more obvious and better established. At last, as evolutionary capacitors, Hsps may have distinctive impacts on the evolution of other genes and ecological consequences.

A large number of plant RNA viruses circulate between plants and insects. For RNA viruses, host alternations may impose a differential selective pressure on viral populations and induce variations in viral genomes. Here, we report the variations in the 3′‐terminal regions of the multiple‐segment RNA virus Rice stripe virus (RSV) that were discovered through de novo assembly of the genome using RNA sequencing data from infected host plants and vector insects. The newly assembled RSV genome contained 16‐ and 15‐nt extensions at the 3′‐termini of two genome segments compared with the published reference RSV genome. Our study demonstrated that these extensional sequences were consistently observed in two RSV isolates belonging to distinct genetic subtypes in RSV‐infected rice, wheat and tobacco. Moreover, the de novo assembled genome of Southern rice black‐streaked dwarf virus also contained 3′‐terminal extensions in five RNA segments compared with the reference genome. Time course experiments confirmed that the 3′‐terminal extensions of RSV were enriched in the vector insects, were gradually eliminated in the host plant and potentially affected viral replication. These findings indicate that variations in the 3′‐termini of viral genomes may be different adaptive strategies for plant RNA viruses in insects and plants.

Background Laodelphax striatellus Fallén (Hemiptera: Delphacidae) is one of the most destructive rice pests. L. striatellus is different from 2 other rice planthoppers with a released genome sequence, Sogatella furcifera and Nilaparvata lugens, in many biological characteristics, such as host range, dispersal capacity, and vectoring plant viruses. Deciphering the genome of L. striatellus will further the understanding of the genetic basis of the biological differences among the 3 rice planthoppers. Findings A total of 190 Gb of Illumina data and 32.4 Gb of Pacbio data were generated and used to assemble a high-quality L. striatellus genome sequence, which is 541 Mb in length and has a contig N50 of 118 Kb and a scaffold N50 of 1.08 Mb. Annotated repetitive elements account for 25.7% of the genome. A total of 17 736 protein-coding genes were annotated, capturing 97.6% and 98% of the BUSCO eukaryote and arthropoda genes, respectively. Compared with N. lugens and S. furcifera, L. striatellus has the smallest genome and the lowest gene number. Gene family expansion and transcriptomic analyses provided hints to the genomic basis of the differences in important traits such as host range, migratory habit, and plant virus transmission between L. striatellus and the other 2 planthoppers. Conclusions We report a high-quality genome assembly of L. striatellus, which is an important genomic resource not only for the study of the biology of L. striatellus and its interactions with plant hosts and plant viruses, but also for comparison with other planthoppers.

Notch homolog 1 (Notch 1) signaling is regarded as a potential therapeutic target for modulating the inflammatory response and exhibiting neuroprotective effects in cerebral injury following stroke. N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t‑butylester (DAPT) efficiently inhibits activation of the Notch 1 signaling pathway in microglia and may protect brain tissue from ischemic damage. However, the temporal proliferation and morphological alterations of microglia/macrophages throughout progression of the disease, as well as the comprehensive alterations of the whole brain following DAPT treatment, remain to be elucidated. The present study evaluated the temporal proliferation and the morphological alterations of microglia/macrophages over the period of the subacute and chronic stages, in addition to dynamic alterations of brain tissue, using the magnetic resonance imaging (MRI) method, following DAPT treatment. Sprague‑Dawley rats (n=40) were subjected to 90 min of middle cerebral artery occlusion and were treated with DAPT (n=20) or acted as controls with no treatment (n=20). The two groups of rats underwent MRI scans prior to the induction of stroke symptoms and at 24 h, 7, 14, 21 and 28 days following the stroke. A total of five rats from each group were sacrificed at 7, 14, 21 and 28 days following induction of stroke. Compared with control rats, the MRI data of the ipsilateral striatum in treated rats revealed ameliorated brain edema at the subacute stage and recovered brain tissue at the chronic stage. In addition to this, treatment attenuated the round‑shape and promoted a ramified‑shape of microglia/macrophages. The present study confirmed the protective effect of DAPT treatment by dynamically monitoring the cerebral alterations and indicated the possibility of DAPT treatment to alter microglial characteristics to induce a protective effect, via inhibition of the Notch signaling pathway.

It remains unresolved how insect embryos acquire sufficient oxygen to sustain high rates of respiratory metabolism during embryogenesis in the absence of a fully developed tracheal system. Our previous work showed that the two distinct subunits (Hc1 and Hc2) of haemocyanin (Hc), a copper-containing protein, display embryo-specific high expression that is essential for embryonic development and survival in the migratory locust Locusta migratoria. Here we investigated the role of haemocyanins in oxygen sensing and supply in the embryo of this locust. Putative binding sites for hypoxia-regulated transcription factors were identified in the promoter region of all of the Hc1 and Hc2 genes. Embryonic expression of haemocyanins was highly upregulated by ambient O2 deprivation, up to 10-fold at 13% O2 content. The degree of upregulation of haemocyanins increased with increasing levels of hypoxia. Compared with low-altitude locusts, embryonic expression of haemocyanins in high-altitude locusts from Tibetan plateau was constitutively higher and more robust to oxygen deprivation. These findings strongly suggest an active involvement of haemocyanins in oxygen exchange in embryos. We thus propose a mechanistic model for embryo respiration in which haemocyanin plays a key role by complementing the tracheal system for oxygen transport during embryogenesis.

Cuticular proteins (CPs) are vital components of the insects' cuticle that support movement and protect insect from adverse environmental conditions. The CPs exist in a large number and diversiform structures, thus, the accurate annotation is the first step to interpreting their roles in insect growth. The rapid development of sequencing technology has simplified the access to the information on protein sequences, especially for non-model species. Dendrolimus punctatus is a Lepidopteran defoliator, and its periodic outbreaks cause severe damage to the coniferous forests. The transcriptome of D. punctatus integrating the whole developmental periods are available for the potential investigation of CPs. In this study, we identified 216 CPs from D. punctatus, including 147 from CPR family, 4 from TWDL family, 3 from CPF/CPFL families, 22 from CPAP families, 8 low complexity proteins, 1 CPCPC and 31 from other CP families. The putative CPs were compared with homologs in other species such as Bombyx mori, Manduca sexta and Drosophila melanogaster. We further identified five co-orthologous groups have highly similar sequences of CRPs in nine lepidopteran species, which exclusively presented in RR-2 subfamily rather than RR-1. We inferred that in Lepidoptera the difference in RR-2 numbers was maintained by homologs in co-orthologous groups, coincided with observation in Drosophila and Anopheles that gene cluster was the model and source for the expansion of RR-2 genes. In combination with the variation of members in each CP family among different species, these results indicated the evolution of CPs was highly correlated to the adaptation of insect to environment. Furthermore, we compared the amino acid composition of the different types CPRs, and examined the expression patterns of CP genes in various developmental stages. The comprehensive overview of CPs from our study provides an insight into their evolution and the association between them and insect development.

The SET domain is an evolutionarily conserved motif present in histone lysine methyltransferases, which are important in the regulation of chromatin and gene expression in animals. In this study, we searched for SET domain–containing genes (SET genes) in all of the 147 arthropod genomes sequenced at the time of carrying out this experiment to understand the evolutionary history by which SET domains have evolved in insects. Phylogenetic and ancestral state reconstruction analysis revealed an arthropod-specific SET gene family, named SmydA, that is ancestral to arthropod animals and specifically diversified during insect evolution. Considering that pseudogenization is the most probable fate of the new emerging gene copies, we provided experimental and evolutionary evidence to demonstrate their essential functions. Fluorescence in situ hybridization analysis and in vitro methyltransferase activity assays showed that the SmydA-2 gene was transcriptionally active and retained the original histone methylation activity. Expression knockdown by RNA interference significantly increased mortality, implying that the SmydA genes may be essential for insect survival. We further showed predominantly strong purifying selection on the SmydA gene family and a potential association between the regulation of gene expression and insect phenotypic plasticity by transcriptome analysis. Overall, these data suggest that the SmydA gene family retains essential functions that may possibly define novel regulatory pathways in insects. This work provides insights into the roles of lineage-specific domain duplication in insect evolution.

Sweetpotato [Ipomoea batatas (L.) Lam] is a globally important root crop with high industrial value. However, because sweetpotato tuberous roots undergo chilling injuries that negatively affect their quality at temperatures below 10 °C, postharvest damage during the winter season is a major constraint for industrialization. To understand chilling injury response during postharvest low temperature storage, we used next-generation sequencing technology to comprehensive analyze the transcriptome of tuberous roots stored at optimal (13 °C) or low temperature (4 °C) for 6 weeks. From nine cDNA libraries, we produced 298,765,564 clean reads, which were de novo assembled into 58,392 unigenes with an average length of 1100 bp. A total of 3216 differentially expressed genes (DEGs) were detected and categorized into six clusters, of which clusters 2, 4, and 5 (1464 DEGs) were up-regulated under low temperature. The genes in these three clusters are involved in biosynthesis of unsaturated fatty acids, pathogen defense, and phenylalanine metabolism. By contrast, genes in clusters 1, 3, and 6 (1752 DEGs), which were generally down-regulated at low temperature, encode antioxidant enzymes or are involved in glycerophospholipid, carbohydrate, or energy metabolism. We confirmed the results of the transcriptome analysis by quantitative RT-PCR. Our transcriptome analysis will advance our understanding of the comprehensive mechanisms of chilling injury during low temperature storage and facilitate improvements in postharvest storage of sweetpotato tuberous roots.

For the treatment of diseases affecting bones using bone regenerative medicine, there is an urgent need to develop safe, inexpensive drugs that can strongly induce bone formation. In the present study, we systematically investigated the effects of icaritin, a metabolic product of icariin, on the osteogenic differentiation of human bone marrow‑derived mesenchymal stem cells (hBMSCs) and human adipose tissue‑derived stem cells (hADSCs) in vitro. After treatment with icaritin at concentrations of 10‑8-10‑5 M, hBMSCs and hADSCs were examined for alkaline phosphatase activity, osteocalcin (OC) secretion, matrix mineralization and expression levels of bone‑related mRNA and proteins. Data showed that icaritin at concentrations 10‑7-10‑5 M significantly increased alkaline phosphatase activity, OC secretion at different time points, and calcium deposition at day 21. In addition, icaritin upregulated the mRNA expression of genes for bone morphogenetic proteins (BMP‑2, ‑4 and ‑7), bone transcription factors (Runx2 and Dlx5) and bone matrix proteins (ALP, OC and Col‑1). Moreover, icaritin increased the protein levels of BMPs, Runx2 and OC, as detected by western blot analysis. These findings suggest that icaritin enhances the osteogenic differentiation of hBMSCS and hADSCs. Icaritin exerts its potent osteogenic effect possibly by directly stimulating the production of BMPs. Although the osteogenic activity of icaritin in vitro was inferior to that of rhBMP‑2, icaritin displayed better results than icariin. Moreover, the low cost, simple extraction procedure, and an abundance of icaritin make it appealing as a bone regenerative medicine.

Sweetpotato [Ipomoea batatas (L.) Lam], which contains high levels of antioxidants such as ascorbate and carotenoids in its storage root, is one of the healthiest foods, as well as one of the best starch crops for growth on marginal lands. In plants, carotenoid pigments are involved in light harvesting for photosynthesis and are also essential for photo-protection against excess light. As dietary antioxidants in humans, these compounds benefit health by alleviating aging-related diseases. The storage root of sweetpotato is a good source of both carotenoids and carbohydrates for human consumption. Therefore, metabolic engineering of sweetpotato to increase the content of useful carotenoids represents an important agricultural goal. This effort has been facilitated by cloning of most of the carotenoid biosynthetic genes, as well as the Orange gene involved in carotenoid accumulation. In this review, we describe our current understanding of the regulation of biosynthesis, accumulation and catabolism of carotenoids in sweetpotato. A deeper understanding of these topics should contribute to development of new sweetpotato cultivars with higher levels of nutritional carotenoids and abiotic stress tolerance.

Locusts are important agricultural pests worldwide and regarded as study models for entomology. However, the absence of targeted gene manipulation systems for locusts has restricted their applications for research. Herein, we report the successful use of the CRISPR/Cas9 system to induce a targeted heritable mutagenesis of the migratory locust, Locusta migratoria. The target sequence of gRNA was designed to disrupt the gene encoding the odorant receptor co-receptor (Orco) and examine the roles of the odorant receptor pathway in the locust. Microinjection of the mixture of Cas9-mRNA and Orco-gRNA into the locust eggs resulted in efficient target-gene editing at a rate of 71.7% in G0 animals and achieved a germline efficiency of up to 88.1% in G1 animals. By a crossing strategy, we successfully established stable Orco mutant lines. EAGs and SSRs indicated that the fourth-instar nymphs of the Orco mutants showed severely impaired electrophysiological responses to multiple odors. The Orco mutant locusts lost an attraction response to aggregation pheromones under the crowding conditions. The locomotor activity and body coloration of the Orco mutant locusts did not significantly differ from those of the two other genotypes. This study provides an easy and effective approach by using the CRISPR/Cas9 system for generating loss-of-function mutants for functional genetic studies of locusts and for managing insect pests.

Metarhizium is a group of insect-pathogenic fungi that can produce insecticidal metabolites, such as destruxins. Interestingly, the acridid-specific fungus Metarhizium acridum (MAC) can kill locusts faster than the generalist fungus Metarhizium robertsii (MAA) even without destruxin. However, the underlying mechanisms of different pathogenesis between host-generalist and host-specialist fungi remain unknown. This study compared transcriptomes and metabolite profiles to analyze the difference in responsiveness of locusts to MAA and MAC infections. Results confirmed that the detoxification and tryptamine catabolic pathways were significantly enriched in locusts after MAC infection compared with MAA infection and that high levels of tryptamine could kill locusts. Furthermore, tryptamine was found to be capable of activating the aryl hydrocarbon receptor of locusts (LmAhR) to produce damaging effects by inducing reactive oxygen species production and immune suppression. Therefore, reducing LmAhR expression by RNAi or inhibitor (SR1) attenuates the lethal effects of tryptamine on locusts. In addition, MAA, not MAC, possessed the monoamine oxidase (Mao) genes in tryptamine catabolism. Hence, deleting MrMao-1 could increase the virulence of generalist MAA on locusts and other insects. Therefore, our study provides a rather feasible way to design novel mycoinsecticides by deleting a gene instead of introducing any exogenous gene or domain., Author summary Mycoinsecticides are widely used in place of chemical pesticides to protect crops from pest damage. Metarhizium spp. fungi specifically live inside the body cavity of insects and can produce insecticidal metabolites, such as beauvericin, destruxins, and taxol. The variable virulence between host-generalist fungus Metarhizium robertsii (MAA) and host-specialist fungus Metarhizium acridum (MAC) to locusts was studied. We found that MAC is more virulent than MAA on locusts, and MAC-infected locusts display higher amounts of tryptamine than do MAA-infected locusts. Furthermore, MAC cannot produce destruxins, but can produce abundant tryptamine to kill locusts when accumulated owing to the absence of a gene for tryptamine catabolism in the MAC genome. Tryptamine activates the aryl hydrocarbon receptor of locusts (LmAhR) that produces damaging effects by regulating reactive oxygen species production and suppressing the immune system of locusts. Therefore, the deletion of MrMao-1 in the generalist fungus MAA significantly improves the virulence of the fungus to locusts and other insect species. The resulting new insights into the core metabolism of high virulence of host-specialist fungus can provide an improved basis for designing mycoinsecticide strains.

Holometabolous insects stop feeding at the final larval instar stage and then undergo metamorphosis; however, the mechanism is unclear. In the present study, using the serious lepidopteran agricultural pest Helicoverpa armigera as a model, we revealed that 20-hydroxyecdysone (20E) binds to the dopamine receptor (DopEcR), a G protein-coupled receptor, to stop larval feeding and promote pupation. DopEcR was expressed in various tissues and its level increased during metamorphic molting under 20E regulation. The 20E titer was low during larval feeding stages and high during wandering stages. By contrast, the dopamine (DA) titer was high during larval feeding stages and low during the wandering stages. Injection of 20E or blocking dopamine receptors using the inhibitor flupentixol decreased larval food consumption and body weight. Knockdown of DopEcR repressed larval feeding, growth, and pupation. 20E, via DopEcR, promoted apoptosis; and DA, via DopEcR, induced cell proliferation. 20E opposed DA function by repressing DA-induced cell proliferation and AKT phosphorylation. 20E, via DopEcR, induced gene expression and a rapid increase in intracellular calcium ions and cAMP. 20E induced the interaction of DopEcR with G proteins αs and αq. 20E, via DopEcR, induced protein phosphorylation and binding of the EcRB1-USP1 transcription complex to the ecdysone response element. DopEcR could bind 20E inside the cell membrane or after being isolated from the cell membrane. Mutation of DopEcR decreased 20E binding levels and related cellular responses. 20E competed with DA to bind to DopEcR. The results of the present study suggested that 20E, via binding to DopEcR, arrests larval feeding and promotes pupation., Author summary The steroid hormone 20-hydroxyecdysone (20E) represses insect larval feeding and promotes metamorphosis; however, the mechanism is unclear. The dopamine receptor plays important roles in animal motor function and reward-motivated behavior. Using the serious lepidopteran agricultural pest Helicoverpa armigera as a model, we revealed that 20E binds to DopEcR to block the dopamine pathway and initiates the 20E pathway. Dopamine (DA) binds to the dopamine receptor (DopEcR), a G protein-coupled receptor (GPCR), to regulate cell proliferation, larval feeding, and growth. However, 20E competes with DA to bind to DopEcR, which represses larval feeding and triggers the 20E-pathway, leading to metamorphosis. The results suggested that 20E, via binding to DopEcR, stops larval feeding and promotes pupation, which presented an example of the steroid hormone regulating dopamine receptor and behavior. Our study showed that GPCRs can bind 20E and function as 20E cell membrane receptors.

Host alternation, an obligatory seasonal shifting between host plants of distant genetic relationship, has had significant consequences for the diversification and success of the superfamily of aphids. However, the underlying molecular mechanism remains unclear. In this study, the molecular mechanism of host alternation was explored through a large-scale gene expression analysis of the mealy aphid Hyalopterus persikonus on winter and summer host plants. More than four times as many unigenes of the mealy aphid were significantly upregulated on summer host Phragmites australis than on winter host Rosaceae plants. In order to identify gene candidates related to host alternation, the differentially expressed unigenes of H. persikonus were compared to salivary gland expressed genes and secretome of Acyrthosiphon pisum. Genes involved in ribosome and oxidative phosphorylation and with molecular functions of heme-copper terminal oxidase activity, hydrolase activity and ribosome binding were potentially upregulated in salivary glands of H. persikonus on the summer host. Putative secretory proteins, such as detoxification enzymes (carboxylesterases and cytochrome P450s), antioxidant enzymes (peroxidase and superoxide dismutase), glutathione peroxidase, glucose dehydrogenase, angiotensin-converting enzyme, cadherin, and calreticulin, were highly expressed in H. persikonus on the summer host, while a SCP GAPR-1-like family protein and a salivary sheath protein were highly expressed in the aphids on winter hosts. These results shed light on phenotypic plasticity in host utilization and seasonal adaptation of aphids.

Some long noncoding RNAs (lncRNAs) are specifically expressed in brain cells, implying their neural and behavioural functions. However, how lncRNAs contribute to neural regulatory networks governing the precise behaviour of animals is less explored. Here, we report the regulatory mechanism of the nuclear-enriched lncRNA PAHAL for dopamine biosynthesis and behavioural adjustment in migratory locusts (Locusta migratoria), a species with extreme behavioral plasticity. PAHAL is transcribed from the sense (coding) strand of the gene encoding phenylalanine hydroxylase (PAH), which is responsible for the synthesis of dopamine from phenylalanine. PAHAL positively regulates PAH expression resulting in dopamine production in the brain. In addition, PAHAL modulates locust behavioral aggregation in a population density-dependent manner. Mechanistically, PAHAL mediates PAH transcriptional activation by recruiting serine/arginine-rich splicing factor 2 (SRSF2), a transcription/splicing factor, to the PAH proximal promoter. The co-activation effect of PAHAL requires the interaction of the PAHAL/SRSF2 complex with the promoter-associated nascent RNA of PAH. Thus, the data support a model of feedback modulation of animal behavioural plasticity by an lncRNA. In this model, the lncRNA mediates neurotransmitter metabolism through orchestrating a local transcriptional loop., Author summary The neurotransmitter dopamine is crucial for the neuronal and behavioral response in animals. Phenylalanine hydroxylase (PAH) is involved in dopamine biosynthesis and behavioral regulation in the migratory locust. However, the molecular mechanism for the fine tuning of PAH expression in behavioral response remains ambiguous. Here we discovered a nuclear-enriched lncRNA PAHAL that is transcribed from the coding strand of the PAH gene in the locust (i.e., sense lncRNA). PAHAL positively regulated PAH expression and dopamine production in the brain. In addition, PAHAL modulated behavioral aggregation of the locust. Mechanistically, PAHAL mediated the transcriptional activation of PAH by recruiting SRSF2, a transcription/splicing factor, to the promoter-associated nascent RNA of PAH. These data support a model of feedback modulation of dopamine biosynthesis and behavioral plasticity via a sense lncRNA in the catecholamine metabolic pathway.

Carotenoids are required for many biological processes in plants and humans. Lycopene e-cyclase (LCY-e) catalyzes the conversion of lycopene into lutein via the α-branch carotenoid biosynthesis pathway. Down-regulation of IbLCY-e by RNAi increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato calli. As the role of IbLCY-e in carotenoid biosynthesis and environmental stress responses in whole plants is poorly understood, transgenic sweetpotato (RLE plants) with reduced expression of IbLCY-e were developed. RLE plants contained higher levels of total carotenoid and β-carotene, due to an elevated β-carotene/lutein ratio rather than increased de novo biosynthesis. RLE plants showed high reactive oxygen species/radical-scavenging activity. They also exhibited an enhanced tolerance of both salt and drought stress, which was associated with lower membrane permeability and a higher photosynthetic rate, respectively. Elevated carotenoid accumulation in RLE plants mitigated the reductions in leaf photosystem II efficiency and chlorophyll induced by abiotic stress. Expression of the carotenoid cleavage genes 9-cis-epoxycarotenoid dioxygenase, carotenoid cleavage dioxygenase 1 (CCD1) and CCD4 was higher in RLE plants, as was abscisic acid accumulation. IbLCY-e silencing thus offers an effective approach for developing sweetpotato plants with increased tolerance to abiotic stress that will grow on global marginal lands with no reduction in nutritional value.

Epigenetic gene control is maintained by chromatin-associated Polycomb group (PcG) and Trithorax group (TrxG) genes, which act antagonistically via the interplay between PcG and TrxG regulation to generate silenced or permissive transcriptional states. In this study, we searched for PcG/TrxG genes in 180 arthropod genomes, covering all the sequenced arthropod genomes at the time of conducting this study, to perform a global investigation of PcG/TrxG genes in a phylogenetic frame. Results of ancestral state reconstruction analysis revealed that the ancestor of arthropod species has an almost complete repertoire of PcG/TrxG genes, and most of these genes were seldom lost above order level. The domain diversity analysis indicated that the PcG/TrxG genes show variable extent of domain structure changes; some of these changes could be associated with lineage-specific events. The likelihood ratio tests for selection pressure detected a number of PcG/TrxG genes which underwent episodic positive selection on the branch leading to the insects with holometabolous development. These results suggest that, despite their high conservation across arthropod species, different members of PcG/TrxG genes showed considerable differences in domain structure and sequence divergence in arthropod evolution. Our cross species comparisons using large-scale genomic data provide insights into divergent evolutionary pattern on highly conserved genes in arthropods.

Salivary proteins play key roles in the co-evolution of aphids and their host plants. This study characterizes the aphid-specific and small secretory protein ACYPI006346 in the pea aphid Acyrthosiphon pisum . The ACYPI006346 transcript is enriched in the salivary glands, particularly in cell types 5 and 7 of the principal salivary glands. Transcript knockdown by dsRNA injection, which lasts for 24 h does not influence the survival of aphids on plants. The transcript levels of ACYPI006346 were different in three pea aphid colonies adapting to Vicia faba , Vicia villosa , or Medicago truncatula , respectively.

Haemocyanins are commonly known as copper-containing oxygen carriers within the haemolymph of arthropods, and have been found in many orders of insects. However, it remains unresolved why haemocyanins persist in insects that possess elaborate tracheal systems for oxygen diffusion to cells. Here we identified haemocyanins in the migratory locust Locusta migratoria that consists of two distinct subunits, Hc1 and Hc2. Genomic sequence analysis indicated that Hc1 and Hc2 have four and three gene copies, respectively, which may have evolved via gene duplication followed by divergent evolution of introns. The two subunits exhibit abundant and embryonic-specific expression at the mRNA and protein level; their expression peaks in the mid-term embryo and is not detectable in the late nymphal and adult stages. A larger proportion of the haemocyanins is present in the yolk compared with that in the embryo. Immunostaining shows that haemocyanins in the embryo are mainly expressed in the epidermis. Knockdown of Hc1 and Hc2 results in significant embryonic developmental delay and abnormality as well as reduced egg hatchability, ie the proportion of hatched eggs. These results reveal a previously unappreciated and fundamental role for haemocyanins in embryonic development and survival in insects, probably involving the exchange of molecules (eg O2 ) between the embryo and its environment.

Many species exhibit transgenerational plasticity by which environmental cues experienced by either parent can be transmitted to their offspring, resulting in phenotypic variants in offspring to match ancestral environments. However, the manner by which paternal experiences affect offspring plasticity through epigenetic inheritance in animals generally remains unclear. In this study, we examined the transgenerational effects of population density on phase-related traits in the migratory locust Locusta migratoria. Using an experimental design that explicitly controls genetic background, we found that the effects of crowd or isolation rearing on phase plasticity could be inherited to the offspring. The isolation of gregarious locusts resulted in reduced weight in offspring eggs and altered morphometric traits in hatchlings, whereas crowding of solitarious locusts exhibited opposite effects. The consequences of density changes were transmitted by both maternal and paternal inheritance, although the expression of paternal effects was not as pronounced as that of maternal effects. Prominent expression of heat-shock proteins (Hsps), such as Hsp90, Hsp70 and Hsp20.6, could be triggered by density changes. Hsps were significantly upregulated upon crowding but downregulated upon isolation. The variation in parental Hsp expression was also transmitted to the offspring, in which the pattern of inheritance was consistent with that of phase characteristics. These results revealed a paternal effect on phase polyphenism and Hsp expression induced by population density, and defined a model system that could be used to study the paternal epigenetic inheritance of environmental changes.

Understanding the roles of transposable elements (TEs) in the evolution of genome and adaptation is a long-sought goal. Here, we present a new model of TE co-option, in which a TE is harnessed by an essential gene and confers local adaptation through heterozygote advantage. We characterized a human Alu-like TE family, the Lm1 elements, in the genome of the migratory locust Locusta migratoria that harbors 0.7 million copies of the elements. Scanning Lm1 insertions in the natural locust populations revealed the widespread high polymorphism of Lm1. An Lm1 was recruited into the coding region of Heat-shock protein 90 (Hsp90), an important molecular chaperone for diverse signal transduction and developmental pathways. Only heterozygotes of the allele are present in natural populations. Allele frequency increases with decreased latitudes in east coastal China, even increasing up to 76% in southern populations. Regions flanking the Lm1 insertion display clear signatures of a selective sweep linked to Lm1. The Lm1-mediated Hsp90 mutation is consequential for the embryonic development of locust. Heterozygous embryos develop faster than the wild type, particularly when cued by long-day parental photoperiod. The heterozygotes also present a reduced within-population variation in embryonic development, i.e., high developmental synchrony of embryos. The naturally occurring Hsp90 mutation could facilitate multivoltinism and developmental synchronization of the locust in southern tropical region. These results revealed a genetic mechanism behind microevolutionary changes in which balancing selection may have acted to maintain the heterozygote advantage through TE co-option in essential genes.