Asymmetric Expression of LincGET Biases Cell Fate in Two-Cell Mouse Embryos.

In early mammalian embryos, it remains unclear how the first cell fate bias is initially triggered and amplified toward cell fate segregation. Here, we report that a long noncoding RNA, LincGET, is transiently and asymmetrically expressed in the nucleus of two- to four-cell mouse embryos. Overexpression of LincGET in one of the two-cell blastomeres biases its progeny predominantly toward the inner cell mass (ICM) fate. Mechanistically, LincGET physically binds to CARM1 and promotes the nuclear localization of CARM1, which can further increase the level of H3 methylation at Arginine 26 (H3R26me), activate ICM-specific gene expression, upregulate transposons, and increase global chromatin accessibility. Simultaneous overexpression of LincGET and depletion of Carm1 no longer biased embryonic fate, indicating that the effect of LincGET in directing ICM lineage depends on CARM1. Thus, our data identify LincGET as one of the earliest known lineage regulators to bias cell fate in mammalian 2-cell embryos.

Generation and Application of Mouse-Rat Allodiploid Embryonic Stem Cells.

Mammalian interspecific hybrids provide unique advantages for mechanistic studies of speciation, gene expression regulation, and X chromosome inactivation (XCI) but are constrained by their limited natural resources. Previous artificially generated mammalian interspecific hybrid cells are usually tetraploids with unstable genomes and limited developmental abilities. Here, we report the generation of mouse-rat allodiploid embryonic stem cells (AdESCs) by fusing haploid ESCs of the two species. The AdESCs have a stable allodiploid genome and are capable of differentiating into all three germ layers and early-stage germ cells. Both the mouse and rat alleles have comparable contributions to the expression of most genes. We have proven AdESCs as a powerful tool to study the mechanisms regulating X chromosome inactivation and to identify X inactivation-escaping genes, as well as to efficiently identify genes regulating phenotypic differences between species. A similar method could be used to create hybrid AdESCs of other distantly related species.

Live-cell imaging reveals redox metabolic reprogramming during zygotic genome activation.

Metabolic programming is deeply intertwined with early embryonic development including zygotic genome activation (ZGA), the polarization of zygotic cells, and cell fate commitment. It is crucial to establish a noninvasive imaging technology that spatiotemporally illuminates the cellular metabolism pathways in embryos to track developmental metabolism in situ. In this study, we used two high-quality genetically encoded fluorescent biosensors, SoNar for NADH/NAD+ and iNap1 for NADPH, to characterize the dynamic regulation of energy metabolism and redox homeostasis during early zygotic cleavage. Our imaging results showed that NADH/NAD+ levels decreased from the early to the late two-cell stage, whereas the levels of the reducing equivalent NADPH increased. Mechanistically, transcriptome profiling suggested that during the two-cell stage, zygotic cells downregulated the expression of genes involved in glucose uptake and glycolysis, and upregulated the expression of genes for pyruvate metabolism in mitochondria and oxidative phosphorylation, with a decline in the expression of two peroxiredoxin genes, Prdx1 and Prdx2. Collectively, with the establishment of in situ metabolic monitoring technology, our study revealed the programming of redox metabolism during ZGA.

Impaired lipid metabolism by age-dependent DNA methylation alterations accelerates aging.

Epigenetic alterations and metabolic dysfunction are two hallmarks of aging. However, the mechanism of how their interaction regulates aging, particularly in mammals, remains largely unknown. Here we show ELOVL fatty acid elongase 2 (Elovl2), a gene whose epigenetic alterations are most highly correlated with age prediction, contributes to aging by regulating lipid metabolism. Impaired Elovl2 function disturbs lipid synthesis with increased endoplasmic reticulum stress and mitochondrial dysfunction, leading to key accelerated aging phenotypes. Restoration of mitochondrial activity can rescue age-related macular degeneration (AMD) phenotypes induced by Elovl2 deficiency in human retinal pigmental epithelial (RPE) cells. We revealed an epigenetic-metabolism axis contributing to aging and potentially to antiaging therapy.

Long noncoding RNA lnc-NAP sponges mmu-miR-139-5p to modulate Nanog functions in mouse ESCs and embryos.

The pluripotency of embryonic stem cells (ESCs) is controlled by a multilayer regulatory network, of which the key factors include core pluripotency genes Oct4, Sox2 and Nanog, and multiple microRNAs (miRNAs). Recently, long noncoding RNAs (lncRNAs) have been discovered as a class of new regulators for ESCs, and some lncRNAs could function as competing endogenous RNAs (ceRNAs) to regulate mRNAs by competitively binding to miRNAs. Here, we identify mmu-miR-139-5p as a new regulator for Nanog by targeting Nanog 3' untranslated region (UTR) to repress Nanog expression in mouse ESCs and embryos. Such regulation could be released by an ESC-specifically expressed ceRNA named lnc-NAP. The expression of lnc-NAP is activated by OCT4, SOX2, as well as NANOG through promoter binding. Downregulation of lnc-NAP reduces Nanog abundance, which leads to decreased pluripotency of mouse ESCs and embryonic lethality. These results reveal lnc-NAP as a new regulator for Nanog in mouse ESCs, and uncover a feed-forward regulatory loop of Nanog through the participation of lnc-NAP.

Regulation of [Ca2+]i oscillations and mitochondrial activity by various calcium transporters in mouse oocytes.

Oocyte activation inefficiency is one of the reasons for female infertility and Ca2+ functions play a critical role in the regulation of oocyte activation. We used various inhibitors of Ca2+ channels located on the membrane, including sarcoplasmic/ endoplasmic reticulum Ca2+ATPases (SERCAs, the main Ca2+ pumps which decrease the intracellular Ca2+ level by refilling Ca2+ into the sarcoplasmic reticulum), transient receptor potential (TRP) ion channel subfamily member 7 (TRPM7, a Ca2+/Mg2+-permeable non-selective cation channel), T-type Ca2+ channels and calcium channel Orai1, to investigate their roles in [Ca2+]i oscillation patterns and mitochondrial membrane potential during oocyte activation by real-time recording. Our results showed that SERCAs, TRPM7 and T-type Ca2+ channels were important for initiation and maintenance of [Ca2+]i oscillations, which was required for mitochondrial membrane potential elevation during oocyte activation, as well as oocyte cytoskeleton stability and subsequent embryo development. Increasing the knowledge of calcium transport may provide a theoretical basis for improving oocyte activation in human assisted reproduction clinics.

Rat embryonic stem cells produce fertile offspring through tetraploid complementation.

Pluripotency of embryonic stem cells (ESCs) can be functionally assessed according to the developmental potency. Tetraploid complementation, through which an entire organism is produced from the pluripotent donor cells, is taken as the most stringent test for pluripotency. It remains unclear whether ESCs of other species besides mice can pass this test. Here we show that the rat ESCs derived under 2i (two small molecule inhibitors) conditions at very early passages are able to produce fertile offspring by tetraploid complementation. However, they lose this capacity rapidly during culture due to a nearly complete loss of genomic imprinting. Our findings support that the naïve ground state pluripotency can be captured in rat ESCs but also point to the species-specific differences in its regulation and maintenance, which have implications for the derivation and application of naïve pluripotent stem cells in other species including human.

Effect of oral spray with Lactobacillus on growth performance, intestinal development and microflora population of ducklings.

OBJECTIVE: The aim of this study is to investigate the effect of oral spray with probiotics on the intestinal development and microflora colonization of hatched ducklings. METHODS: In Exp. 1, an one-way factorial design was used to study the antibacterial activity of the probiotics and metabolites on Escherichia coli (E. coli) without antimicrobial resistance. There were four experimental groups including saline as control and Lactobacillus, Bacillus subtilis, combined Lactobacillus and Bacillus subtilis groups. In Exp. 2, 64-day-old ducklings were allotted to 2 treatments with 4 replicated pens. Birds in the control group were fed a basal diet supplemented with Lactobacillus fermentation in the feed whereas birds in the oral spray group were fed the basal diet and administrated Lactobacillus fermentation by oral spray way during the first week. RESULTS: In Exp. 1, the antibacterial activities of probiotics and metabolites on E. coli were determined by the diameter of inhibition zone in order: Lactobacillus>combined Lactobacillusand Bacillus subtilis>Bacillus subtilis. Additionally, compared to E. coli without resistance, E. coli with resistance showed a smaller diameter of inhibition zones. In Exp. 2, compared to control feeding group, oral spray group increased (p<0.05) the final body weight at d 21 and average daily gain for d 1-21 and the absolute weight of the jejunum, ileum and total intestine tract as well as cecum Lactobacillus amount at d 21. CONCLUSION: Lactobacillus exhibited a lower antibacterial activity on E. coli with resistance than E. coli without resistance. Oral spray with Lactobacillus fermentation during the first week of could improve the intestinal development, morphological structure, and microbial balance to promote growth performance of ducklings from hatch to 21 d of age.

A novel long intergenic noncoding RNA indispensable for the cleavage of mouse two-cell embryos.

Endogenous retroviruses (ERVs) are transcriptionally active in cleavage stage embryos, yet their functions are unknown. ERV sequences are present in the majority of long intergenic noncoding RNAs (lincRNAs) in mouse and humans, playing key roles in many cellular processes and diseases. Here, we identify LincGET as a nuclear lincRNA that is GLN-, MERVL-, and ERVK-associated and essential for mouse embryonic development beyond the two-cell stage. LincGET is expressed in late two- to four-cell mouse embryos. Its depletion leads to developmental arrest at the late G2 phase of the two-cell stage and to MAPK signaling pathway inhibition. LincGET forms an RNA-protein complex with hnRNP U, FUBP1, and ILF2, promoting the cis-regulatory activity of long terminal repeats (LTRs) in GLN, MERVL, and ERVK (GLKLTRs), and inhibiting RNA alternative splicing, partially by downregulating hnRNP U, FUBP1, and ILF2 protein levels. Hnrnpu or Ilf2 mRNA injection at the pronuclear stage also decreases the preimplantation developmental rate, and Fubp1 mRNA injection at the pronuclear stage causes a block at the two-cell stage. Thus, as the first functional ERV-associated lincRNA, LincGET provides clues for ERV functions in cleavage stage embryonic development.

Diurnal predators in dim light: the ability of mantids to prey for supper.

Many insects rely heavily on visual cues in foraging and other life activities. Mantids are insect predators that usually ambush prey. The sophisticated visual system of mantids allows them to spot, track, and strike at prey with high accuracy. Mantids are categorized as diurnal animals in most cases, while our field observations suggested that they were active in foraging both day and night. Therefore, we hypothesize that predation in dim light is possible for mantids, while mantids are unable to capture prey in complete darkness. In this study, we experimentally examined whether different light conditions could affect the predation success and efficiency of mantid nymphs and adults, Hierodula chinensis Werner (Mantodea: Mantidae), through behavioral observations. Individual mantids were placed in individual chambers in complete darkness, simulated moonlight (0.1 lux), or simulated dusk (50 lux) conditions and were allowed to forage for prey items for 10 min. Our observations showed no evidence that H. chinensis could capture any prey in complete deprivation of light. The proportion of nymphs with successful predation in simulated moonlight was 50% higher than that in complete darkness and 45.83% lower than that in simulated dusk. The proportion of adults with successful predation in simulated moonlight was 42.11% higher than that in complete darkness and 57.89% lower than that in simulated dusk. Overall, the results provide new insights into the behavioral ecology of diurnal predators at night, with potential association with moonlight, starlight, and light pollution.

The water-exiting behavior and survival of predaceous diving beetles in responses to lambda-cyhalothrin, chlorantraniliprole, and thiamethoxam.

Predaceous diving beetles (dytiscids) are important top insect predators in various natural, agricultural, and artificial water bodies. How they respond to human disturbances such as insecticide contamination to their habitats has been understudied. In this study, we investigated the lethal effects of lambda-cyhalothrin, chlorantraniliprole, and thiamethoxam at nominal field concentrations in 3 cm-deep water in a hypothetical paddy field (Ci,3) on adult Dytiscus sinensis Feng (Coleoptera: Dytiscidae). Lambda-cyhalothrin exhibited significant lethal effects on D. sinensis adults with its Ci,3 exceeding the 95 % confidence interval lower limits for 24, 48, 72, and 96 h- LC99. Chlorantraniliprole and thiamethoxam showed no significant lethal effects on D. sinensis adults at their respective Ci,3 at 24, 48, or 72 h. Additionally, we assessed the temporal propensity of D. sinensis adults to exit water contaminated with chlorantraniliprole and thiamethoxam, respectively. There were significantly more individuals that temporally exited the chlorantraniliprole-contaminated water than clean water 30 min after placing a tile island in the test arena. Meanwhile, thiamethoxam-contaminated water did not repel significantly more individuals than clean water when observed at 10, 30, or 60 min. The study highlights the availability and importance of selecting safer insecticides for dytiscid conservation in agricultural and adjacent habitats, considering the potential of these water bodies becoming ecological traps that keep attracting and killing aquatic beneficials. The water-exiting behavior found indicates the capability of some wildlife to effectively avoid further exposure to toxicants.

Performance of Spodoptera litura (Lepidoptera: Noctuidae) in responses to different amplitudes of alternating temperatures across permissive warm temperature regimes.

Climate change involves changes in the pattern of temperature fluctuations and the frequency and magnitude of thermal extremes. It is essential to investigate how insects respond to increased temperature variations, especially for species that usually experience diel temperature variations extending to thermal performance limits. To explicitly elucidate the effects of sizes of amplitudes of temperature variations, we used daily alternating temperatures with an equivalent mean of 28 °C to investigate the impact of different fluctuation amplitudes (±â€…0 °C,  ±â€…2 °C,  ±â€…4 °C, and  ±â€…6 °C) across permissive temperature regimes on the performance of Spodoptera lituraFabricius, a highly destructive polyphagous pest of crops and vegetables in tropical and temperate climates. Amplitudes of temperature fluctuations significantly affected developmental durations, adult life spans, pupal weights, fecundity, and fertility of S. litura. The survival rates from the egg stage to the adult eclosion did not differ significantly across different temperature treatments. The developmental durations of individual life cycles significantly increased with larger amplitudes in 3 fluctuating temperature treatments. The pupal weight was significantly lower and the adult life span was significantly shorter in the ±â€…6 °C treatment in all treatments. Fecundity and fertility were significantly reduced in the ±â€…6 °C treatment, making the continuation of generations nearly impossible. The results should provide critical information for understanding the ecology of this widespread pest under diel, seasonal, and global climate changes.

Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12iMax.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12iMax, a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12iMax in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163, and MSTN via Cas12iMax in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12iMax for gene editing in livestock animals and demonstrated the potential application of Cas12iMax in the field of animal trait improvement for agricultural production.

Functional mouse hepatocytes derived from interspecies chimeric livers effectively mitigate chronic liver fibrosis.

Liver disease is a major global health challenge. There is a shortage of liver donors worldwide, and hepatocyte transplantation (HT) may be an effective treatment to overcome this problem. However, the present approaches for generation of hepatocytes are associated with challenges, and interspecies chimera-derived hepatocytes produced by interspecies blastocyst complementation (IBC) may be promising donor hepatocytes because of their more comprehensive hepatic functions. In this study, we isolated mouse hepatocytes from mouse-rat chimeric livers using IBC and found that interspecies chimera-derived hepatocytes exhibited mature hepatic functions in terms of lipid accumulation, glycogen storage, and urea synthesis. Meanwhile, they were more similar to endogenous hepatocytes than hepatocytes derived in vitro. Interspecies chimera-derived hepatocytes could relieve chronic liver fibrosis and reside in the injured liver after transplantation. Our results suggest that interspecies chimera-derived hepatocytes are a potentially reliable source of hepatocytes and can be applied as a therapeutic approach for HT.

Alternative splicing of CARM1 regulated by LincGET-guided paraspeckles biases the first cell fate in mammalian early embryos.

The heterogeneity of CARM1 controls first cell fate bias during early mouse development. However, how this heterogeneity is established is unknown. Here, we show that Carm1 mRNA is of a variety of specific exon-skipping splicing (ESS) isoforms in mouse two-cell to four-cell embryos that contribute to CARM1 heterogeneity. Disruption of paraspeckles promotes the ESS of Carm1 precursor mRNAs (pre-mRNAs). LincGET, but not Neat1, is required for paraspeckle assembly and inhibits the ESS of Carm1 pre-mRNAs in mouse two-cell to four-cell embryos. We further find that LincGET recruits paraspeckles to the Carm1 gene locus through HNRNPU. Interestingly, PCBP1 binds the Carm1 pre-mRNAs and promotes its ESS in the absence of LincGET. Finally, we find that the ESS seen in mouse two-cell to four-cell embryos decreases CARM1 protein levels and leads to trophectoderm fate bias. Our findings demonstrate that alternative splicing of CARM1 has an important role in first cell fate determination.

Sex dependent transcriptome responses of the diamondback moth, Plutella xylostella L. to cold stress.

Temperature has fundamental influences on the performance and distribution of insects. While considerable attention has been devoted to extreme conditions, particularly extreme cold conditions, few studies have investigated effects of mild cold conditions on insects. We examined the transcriptomic changes in mid-fourth instar larvae of both sexes reared at 10 °C and 25 °C to investigate sex-dependent responses of Plutella xylostella to mild cold stress. There were 624 differentially expressed genes (DEGs) in females, the majority of which (n = 386) were down-regulated. In males 3239 genes were differentially expressed and the majority (n = 2341) were up-regulated. Only 280 DEGs were common to both sexes. In females, there were no DEGs encoding heat shock or cold shock proteins, but six of these DEGs were found in males. These differences suggest that females and males might adopt some different strategies to cope with cold stress and/or that they were affected by rearing under cold conditions to different degrees and in different ways. In addition, DEGs encoding antimicrobial peptides, cytochrome P450 monooxygenases, fatty acid-related enzymes, cuticle proteins, myofilament, and hormone-related proteins were found in both sexes under cold stress. The transcriptome study reveals unexpected sex-dependent thermal responses and provides new information of how an insect that does not diapause copes with low temperatures.

Deformation Behavior of ß Phase in a WE54 Magnesium Alloy.

Second phases play a significant role in the development of high-performance magnesium alloys with rare earth elements. Here, in situ tensile tests combined with synchrotron radiation were carried out to investigate the deformation behavior of ß phases in a WE (Mg-Y-Gd-Nd) alloy. By lattice strain analysis, it was found that micro load continuously transferred from the soft α-Mg matrix to the hard ß phases during the whole plastic deformation, while this behavior was much more obvious at the beginning of deformation. Based on diffraction peak broadening, Williamson-Hall (W-H) plotting was used to study the microstrain of ß phases. The results showed that the microstrain of ß phases increased rapidly within 4% plastic strain and reached the maximum at plastic strain of ~6.5%. Since the ß phases acted as hard phases, the microstrain was considered as a sign of the stress concentration near phase interfaces. It was also suggested that the effective release of local stress concentration at the ß/α-Mg interface benefited the ductility of the WE alloy by the plastic deformation of ß phases and phase interface sliding.

A fast chemical reprogramming system promotes cell identity transition through a diapause-like state.

Cellular reprogramming by only small molecules holds enormous potentials for regenerative medicine. However, chemical reprogramming remains a slow process and labour intensive, hindering its broad applications and the investigation of underlying molecular mechanisms. Here, through screening of over 21,000 conditions, we develop a fast chemical reprogramming (FCR) system, which significantly improves the kinetics of cell identity rewiring. We find that FCR rapidly goes through an interesting route for pluripotent reprogramming, uniquely transitioning through a developmentally diapause-like state. Furthermore, FCR critically enables comprehensive characterizations using multi-omics technologies, and has revealed unexpected important features including key regulatory factors and epigenetic dynamics. Particularly, activation of pluripotency-related endogenous retroviruses via inhibition of heterochromatin significantly enhances reprogramming. Our studies provide critical insights into how only environmental cues are sufficient to rapidly reinstate pluripotency in somatic cells, and make notable technical and conceptual advances for solving the puzzle of regeneration.

Differential temperature responses between Plutella xylostella and its specialist endo-larval parasitoid Diadegma semiclausum-Implications for biological control.

Understanding the thermal dynamics of host-parasitoid interactions is crucial to predicting how biological control of pest insects by parasitoids might be affected by geographic location and climate change. We compared performance traits of Plutella xylostella (Lepidoptera: Plutellidae) and its solitary endo-larval parasitoid Diadegma semiclausum (Hymenoptera: Ichneumonidae), over a wide range of constant rearing temperatures (10-30°C). Parasitoids reared at 30°C experienced reductions in pupation rate, pupal mass, egg load, and adult life span when compared with those reared at lower temperatures. Our analyses of the fate of parasitoids and their hosts and intergenerational population growth at different rearing temperatures show that D. semiclausum and P. xylostella respond differently to temperature, leading to divergent outcomes under different temperature conditions. Some parasitoid larvae could not complete development at 30°C, the temperature at which the host biomass was least and the metabolic demands of the parasitoid could be high, suggesting that parasitoid development might be constrained by lack of host resources at higher temperatures. We discuss the potential mechanisms of parasitoid susceptibility to elevated temperatures, which likely explain the pronounced seasonal dynamics of D. semiclausum in subtropical regions and its failure to establish in lowland tropical regions, where P. xylostella is a serious pest. Similar interactions in other host-parasitoid associations would constrain the efficacy of parasitoids as biological control agents as global temperatures increase.

Lipid metabolism dysfunction induced by age-dependent DNA methylation accelerates aging.

Epigenetic alterations and metabolic dysfunction are two hallmarks of aging. However, the mechanism of how their interaction regulates aging, particularly in mammals, remains largely unknown. Here we show ELOVL fatty acid elongase 2 (Elovl2), a gene whose epigenetic alterations are most highly correlated with age prediction, contributes to aging by regulating lipid metabolism. We applied artificial intelligence to predict the protein structure of ELOVL2 and the interaction with its substrate. Impaired Elovl2 function disturbs lipid synthesis with increased endoplasmic reticulum stress and mitochondrial dysfunction, leading to key aging phenotypes at both cellular and physiological level. Furthermore, restoration of mitochondrial activity can rescue age-related macular degeneration (AMD) phenotypes induced by Elovl2 deficiency in human retinal pigmental epithelial (RPE) cells; this indicates a conservative mechanism in both human and mouse. Taken together, we revealed an epigenetic-metabolism axis contributing to aging and illustrate the power of an AI-based approach in structure-function studies.