Building Haplotype-Resolved 3D Genome Maps of Chicken Skeletal Muscle.

Haplotype-resolved 3D chromatin architecture related to allelic differences in avian skeletal muscle development has not been addressed so far, although chicken husbandry for meat consumption has been prevalent feature of cultures on every continent for more than thousands of years. Here, high-resolution Hi-C diploid maps (1.2-kb maximum resolution) are generated for skeletal muscle tissues in chicken across three developmental stages (embryonic day 15 to day 30 post-hatching). The sequence features governing spatial arrangement of chromosomes and characterize homolog pairing in the nucleus, are identified. Multi-scale characterization of chromatin reorganization between stages from myogenesis in the fetus to myofiber hypertrophy after hatching show concordant changes in transcriptional regulation by relevant signaling pathways. Further interrogation of parent-of-origin-specific chromatin conformation supported that genomic imprinting is absent in birds. This study also reveals promoter-enhancer interaction (PEI) differences between broiler and layer haplotypes in skeletal muscle development-related genes are related to genetic variation between breeds, however, only a minority of breed-specific variations likely contribute to phenotypic divergence in skeletal muscle potentially via allelic PEI rewiring. Beyond defining the haplotype-specific 3D chromatin architecture in chicken, this study provides a rich resource for investigating allelic regulatory divergence among chicken breeds.

Haplotype-resolved 3D chromatin architecture of the hybrid pig.

In diploid mammals, allele-specific three-dimensional (3D) genome architecture may lead to imbalanced gene expression. Through ultradeep in situ Hi-C sequencing of three representative somatic tissues (liver, skeletal muscle, and brain) from hybrid pigs generated by reciprocal crosses of phenotypically and physiologically divergent Berkshire and Tibetan pigs, we uncover extensive chromatin reorganization between homologous chromosomes across multiple scales. Haplotype-based interrogation of multi-omic data revealed the tissue dependence of 3D chromatin conformation, suggesting that parent-of-origin-specific conformation may drive gene imprinting. We quantify the effects of genetic variations and histone modifications on allelic differences of long-range promoter-enhancer contacts, which likely contribute to the phenotypic differences between the parental pig breeds. We also observe the fine structure of somatically paired homologous chromosomes in the pig genome, which has a functional implication genome-wide. This work illustrates how allele-specific chromatin architecture facilitates concomitant shifts in allele-biased gene expression, as well as the possible consequential phenotypic changes in mammals.

Re-Engineering Therapeutic Anti-Aß Monoclonal Antibody to Target Amyloid Light Chain.

Amyloidosis involves the deposition of misfolded proteins. Even though it is caused by different pathogenic mechanisms, in aggregate, it shares similar features. Here, we tested and confirmed a hypothesis that an amyloid antibody can be engineered by a few mutations to target a different species. Amyloid light chain (AL) and ß-amyloid peptide (Aß) are two therapeutic targets that are implicated in amyloid light chain amyloidosis and Alzheimer's disease, respectively. Though crenezumab, an anti-Aß antibody, is currently unsuccessful, we chose it as a model to computationally design and prepare crenezumab variants, aiming to discover a novel antibody with high affinity to AL fibrils and to establish a technology platform for repurposing amyloid monoclonal antibodies. We successfully re-engineered crenezumab to bind both Aß42 oligomers and AL fibrils with high binding affinities. It is capable of reversing Aß42-oligomers-induced cytotoxicity, decreasing the formation of AL fibrils, and alleviating AL-fibrils-induced cytotoxicity in vitro. Our research demonstrated that an amyloid antibody could be engineered by a few mutations to bind new amyloid sequences, providing an efficient way to reposition a therapeutic antibody to target different amyloid diseases.

Identification of a Novel Long Non-Coding RNA G8110 That Modulates Porcine Adipogenic Differentiation and Inflammatory Responses.

Long non-coding RNAs (lncRNAs) have been extensively studied, and their crucial roles in adipogenesis, lipid metabolism, and gene expression have been revealed. However, the exact regulatory or other mechanisms by which lncRNAs influence the functioning of mesenteric adipose tissue (MAT) remain largely unknown. In this paper, we report the identification of a new lncRNA, named G8110, from the MAT of Bama pigs. The coordinated expression levels of lncRNA G8110 and NFE2L1 were significantly decreased in the MAT of obese Bama pigs compared with those in the MAT of lean pigs. Using a bone mesenchymal stem cell adipogenic differentiation model, we found that lncRNA G8110 played a role in adipocyte differentiation by positively regulating NFE2L1. We also found that lncRNA G8110 inhibited the formation of intracellular lipid synthesis, promoted lipid metabolism, and inhibited the expression of inflammatory cytokines. Our findings regarding lipid synthesis may further promote the role of lncRNAs in driving adipose tissue remodeling and maintaining metabolic health.

Profiling of Chromatin Accessibility in Pigs across Multiple Tissues and Developmental Stages.

The study of chromatin accessibility across tissues and developmental stages is essential for elucidating the transcriptional regulation of various phenotypes and biological processes. However, the chromatin accessibility profiles of multiple tissues in newborn pigs and across porcine liver development remain poorly investigated. Here, we used ATAC-seq and rRNA-depleted RNA-seq to profile open chromatin maps and transcriptional features of heart, kidney, liver, lung, skeletal muscle, and spleen in newborn pigs and porcine liver tissue in the suckling and adult stages, respectively. Specifically, by analyzing a union set of protein-coding genes (PCGs) and two types of transcripts (lncRNAs and TUCPs), we obtained a comprehensive annotation of consensus ATAC-seq peaks for each tissue and developmental stage. As expected, the PCGs with tissue-specific accessible promoters had active transcription and were relevant to tissue-specific functions. In addition, other non-coding tissue-specific peaks were involved in both physical activity and the morphogenesis of neonatal tissues. We also characterized stage-specific peaks and observed a close association between dynamic chromatin accessibility and hepatic function transition during liver postnatal development. Overall, this study expands our current understanding of epigenetic regulation in mammalian tissues and organ development, which can benefit both economic trait improvement and improve the biomedical usage of pigs.

Dynamic chromatin architecture of the porcine adipose tissues with weight gain and loss.

Using an adult female miniature pig model with diet-induced weight gain/weight loss, we investigated the regulatory mechanisms of three-dimensional (3D) genome architecture in adipose tissues (ATs) associated with obesity. We generated 249 high-resolution in situ Hi-C chromatin contact maps of subcutaneous AT and three visceral ATs, analyzing transcriptomic and chromatin architectural changes under different nutritional treatments. We find that chromatin architecture remodeling underpins transcriptomic divergence in ATs, potentially linked to metabolic risks in obesity development. Analysis of chromatin architecture among subcutaneous ATs of different mammals suggests the presence of transcriptional regulatory divergence that could explain phenotypic, physiological, and functional differences in ATs. Regulatory element conservation analysis in pigs and humans reveals similarities in the regulatory circuitry of genes responsible for the obesity phenotype and identified non-conserved elements in species-specific gene sets that underpin AT specialization. This work provides a data-rich tool for discovering obesity-related regulatory elements in humans and pigs.

A synergistic therapeutic nano-eyedrop for dry eye disease based on ascorbic acid-coupled exosomes.

Dry eye disease (DED), a complex ocular surface disease with a high prevalence rate, is associated with corneal injury, excess oxidative stress and inflammation. Current therapeutic strategies, including artificial tears and anti-inflammatory agents, are unable to address all the deleterious factors or to achieve a clinical cure due to their temporary or side effects. Here, we prepared a multiple-functional eyedrop based on the deposition of gold nanoparticles (AuNPs) reduced by ascorbic acid (AA) onto the exosomal phospholipid membrane of mesenchymal stem cell (mExo)-derived exosomes in situ (mExo@AA). The therapeutic value of mExo@AA for DED was demonstrated in a mouse DED model. Combining the benefits of mExo and AA, mExo@AA effectively improves corneal epithelium recovery and anti-inflammation capacity, decreases corneal reactive oxygen species, and restores tear secretion without adverse effects. Thus, this study suggests that mExo@AA is effective and safe as a therapeutic agent for the treatment of DED.

Dynamic 3D genome reorganization during development and metabolic stress of the porcine liver.

Liver development is a complex process that is regulated by a series of signaling pathways. Three-dimensional (3D) chromatin architecture plays an important role in transcriptional regulation; nonetheless, its dynamics and role in the rapid transition of core liver functions during development and obesity-induced metabolic stress remain largely unexplored. To investigate the dynamic chromatin architecture during liver development and under metabolic stress, we generated high-resolution maps of chromatin architecture for porcine livers across six major developmental stages (from embryonic day 38 to the adult stage) and under a high-fat diet-induced obesity. The characteristically loose chromatin architecture supports a highly plastic genome organization during early liver development, which fundamentally contributes to the rapid functional transitions in the liver after birth. We reveal the multi-scale reorganization of chromatin architecture and its influence on transcriptional regulation of critical signaling processes during liver development, and show its close association with transition in hepatic functions (i.e., from hematopoiesis in the fetus to metabolism and immunity after birth). The limited changes in chromatin structure help explain the observed metabolic adaptation to excessive energy intake in pigs. These results provide a global overview of chromatin architecture dynamics associated with the transition of physiological liver functions between prenatal development and postnatal maturation, and a foundational resource that allows for future in-depth functional characterization.

Efficacy of intravitreal conbercept injection on short- and long-term macular edema in branch retinal vein occlusion.

AIM: To observe the best-corrected visual acuity (BCVA) and central foveal thickness (CFT) repeatedly after the intravitreal injection of conbercept (IVC) for treating cystoid macular edema (CME) in branch retinal vein occlusion (BRVO) and explore the relationship between the duration of CME and visual outcome. METHODS: Subgroup analysis was performed to compare short-term (within 90d of CME onset) and long-term (over 90d of CME onset) macular edema in BRVO. After an initial IVC, a pro re nata (PRN) strategy was performed according to the recurrence of CFT or decrease of BCVA. Analysis of variance using repeated measurements, statistical analysis following indicators including BCVA and CFT collected at baseline and 1, 3, and 6mo after IVC. RESULTS: Among the 60 cases included in this retrospective study, 36 were short-term CME, and 24 were long-term CME. There were statistical significances between and within groups of the BCVAs at different time points (P<0.001). The interaction was found between group and time (P=0.006), indicating the difference in the speed of BCVA improvement between groups. In particular, the improvement speed of BCVA in the short-term CME group was faster than that in the long-term CME group. There were significant differences between and with groups of the CFT at different time points (P<0.001). However, the interaction between group and time in relation to CFT had no significant differences (P=0.59). CONCLUSION: IVC treatment for CME following BRVO is effective and safe. The duration of CME before treatment is a significant predictor of the visual outcomes of patients with BRVO. The improvement of vision might be faster with early IVC treatment than with delayed treatment.

Corrigendum: Artesunate Suppresses Choroidal Melanoma Vasculogenic Mimicry Formation and Angiogenesis via the Wnt/CaMKII Signaling Axis.

[This corrects the article DOI: 10.3389/fonc.2021.714646.].

Dual VEGF/PDGF knockdown suppresses vasculogenic mimicry formation in choroidal melanoma cells via the Wnt5a/ß-catenin/AKT signaling pathway.

OBJECTIVE: This study aimed to explore the effects of knocking down both vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) on vasculogenic mimicry (VM) formation in choroidal melanoma (CM) cells. METHODS: Cell counting Kit (CCK)-8, monoclonal formation, wound healing, transwell and flow cytometry assays were used to observe the cell effects in CM cell line, ocular choroidal melanoma-1 cells (OCM-1) with respect to proliferation, migration, invasion and apoptosis. Three-dimensional (3D) cultures were also used to characterize VM tube structural effects in OCM-1 cells and western blotting was used to characterize protein expression changes in VM-related markers. RESULTS: Dual VEGF/PDGF knockdown suppressed cell proliferation, migration and invasion, but promoted cell apoptosis. It also reduced VM tube structures in OCM-1 cells. VM associated markers including, VE-cadherin, EphA2 and MT1-MMP were also down-regulated in OCM-1 cells. Similarly, Wnt5a, ß-catenin and phosphorylated-AKT levels were also down-regulated. Western blotting and 3D cultures further demonstrated that combined Wnt5a silencing with dual VEGF/PDGF knockdown significantly decreased VE-cadherin and EphA2 levels and reduced VM tube structures in OCM-1 cells. CONCLUSIONS: Dual VEGF/PDGF knockdown suppressed cell growth and metastasis in OCM-1 cells, and blocked the Wnt5a/ß-catenin/AKT signaling pathway thereby inhibiting VM formation.

Allele-specific regulatory effects on the pig transcriptome.

BACKGROUND: Allele-specific expression (ASE) refers to the preferential expression of one allele over the other and contributes to adaptive phenotypic plasticity. Here, we used a reciprocal cross-model between phenotypically divergent European Berkshire and Asian Tibetan pigs to characterize 2 ASE classes: imprinting (i.e., the unequal expression between parental alleles) and sequence dependent (i.e., unequal expression between breed-specific alleles). We examined 3 transcript types, including protein-coding genes (PCGs), long noncoding RNAs, and transcripts of unknown coding potential, across 7 representative somatic tissues from hybrid pigs generated by reciprocal crosses. RESULTS: We identified a total of 92 putative imprinted transcripts, 69 (75.00%) of which are described here for the first time. By combining the transcriptome from purebred Berkshire and Tibetan pigs, we found ∼6.59% of PCGs are differentially expressed between breeds that are regulated by trans-elements (e.g., transcriptional factors), while only ∼1.35% are attributable to cis (e.g., promoters). The higher prevalence of trans-PCGs indicates the dominated effects of trans-regulation in driving expression differences and shaping adaptive phenotypic plasticity between breeds, which were supported by functional enrichment analysis. We also found strong evidence that expression changes mediated by cis-effects were associated with accumulated variants in promoters. CONCLUSIONS: Our study provides a comprehensive map of expression regulation that constitutes a valuable resource for the agricultural improvement of pig breeds.

Artesunate Suppresses Choroidal Melanoma Vasculogenic Mimicry Formation and Angiogenesis via the Wnt/CaMKII Signaling Axis.

Angiogenesis and vasculogenic mimicry (VM) are considered to be the main processes to ensure tumor blood supply during the proliferation and metastasis of choroidal melanoma (CM). The traditional antimalarial drug artesunate (ART) has some potential anti-CM effects; however, the underlying mechanisms remain unclarified. Recent studies have shown that the Wnt5a/calmodulin-dependent kinase II (CaMKII) signaling pathway has a close correlation with angiogenesis and VM formation. This study demonstrated that ART eliminated VM formation by inhibiting the aforementioned signaling pathway in CM cells. The microvessel sprouting of the mouse aortic rings and the microvessel density of chicken chorioallantoic membrane (CAM) decreased significantly after ART treatment. VM formation assay and periodic acid schiff (PAS) staining revealed that ART inhibited VM formation in CM. Moreover, ART downregulated the expression levels of the angiogenesis-related proteins vascular endothelial growth factor receptor (VEGFR) 2, platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor (VEGF) A, and VM-related proteins ephrin type-A receptor (EphA) 2 and vascular endothelial (VE)-cadherin. The expression of hypoxia-inducible factor (HIF)-1α, Wnt5a, and phosphorylated CaMKII was also downregulated after ART treatment. In addition, we further demonstrated that ART inhibited the proliferation, migration, and invasion of OCM-1 and C918 cells. Collectively, our results suggested that ART inhibited angiogenesis and VM formation of choroidal melanoma likely by regulating the Wnt5a/CaMKII signaling pathway. These findings further supported the feasibility of ART for cancer therapy.

Rhodium-catalysed selective C-C bond activation and borylation of cyclopropanes.

Transition metal (TM)-catalysed directed hydroboration of aliphatic internal olefins which facilitates the construction of complex alkylboronates is an essential synthetic methodology. Here, an efficient method for the borylation of cyclopropanes involving TM-catalysed directed C-C activation has been developed. Upon exposure to neutral Rh(i)-catalyst systems, N-Piv-substituted cyclopropylamines (CPAs) undergo proximal-selective hydroboration with HBpin to provide valuable γ-amino boronates in one step which are otherwise difficult to synthesize by known methods. The enantioenriched substrates can deliver chiral products without erosion of the enantioselectivities. Versatile synthetic utility of the obtained γ-amino boronates is also demonstrated. Experimental and computational mechanistic studies showed the preferred pathway and the origin of this selectivity. This study will enable the further use of CPAs as valuable building blocks for the tunable generation of C-heteroatom or C-C bonds through selective C-C bond activation.

Correction: Rhodium-catalysed selective C-C bond activation and borylation of cyclopropanes.

[This corrects the article DOI: 10.1039/D0SC06186G.].

Evidence for nutrient-dependent regulation of the COPII coat by O-GlcNAcylation.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is a dynamic form of intracellular glycosylation common in animals, plants and other organisms. O-GlcNAcylation is essential in mammalian cells and is dysregulated in myriad human diseases, such as cancer, neurodegeneration and metabolic syndrome. Despite this pathophysiological significance, key aspects of O-GlcNAc signaling remain incompletely understood, including its impact on fundamental cell biological processes. Here, we investigate the role of O-GlcNAcylation in the coat protein II complex (COPII), a system universally conserved in eukaryotes that mediates anterograde vesicle trafficking from the endoplasmic reticulum. We identify new O-GlcNAcylation sites on Sec24C, Sec24D and Sec31A, core components of the COPII system, and provide evidence for potential nutrient-sensitive pathway regulation through site-specific glycosylation. Our work suggests a new connection between metabolism and trafficking through the conduit of COPII protein O-GlcNAcylation.

Future changes in the intensity and frequency of precipitation extremes over China in a warmer world: Insight from a large ensemble.

Sufficient samples of extreme precipitation events are needed in order to obtain reliable estimates of the probability of their occurrence. Here, we use a large ensemble simulation with 50 members from the Canadian Earth System Model (CanESM2) under the representative concentration pathway 8.5 (RCP8.5) scenario to give future projection of the intensity and frequency of extreme precipitation events under different warming levels relative to the current climate over China. A bias-correction method based on quantile mapping is first used to remove systematic biases in the ensemble. The return value and return period are obtained by fitting enough annual maximum precipitation samples with the generalized extreme value to represent the intensity and frequency of extreme events, respectively. The results show that the average intensity of extreme precipitation in China will increase by nearly 8% per 1°C of global warming, which closely follows the Clausius-Clapeyron relation. Rarer extreme events will experience greater changes in frequency, especially under higher warming. The nationally averaged extreme precipitation events, presently expected to occur every 50 years (100 years) under the current climate conditions, are expected to occur approximately every 41 years (82 years), 32 years (62 years), 22 years (42 years) and 15 years (29 years) under warming levels of 1.5, 2.0, 3.0 and 4.0°C, respectively. Northwestern China (NW), southwestern China (SW) and the Yangtze River valley (YZ) exhibit the greatest increase in probability ratio (PR) under future climate condition. The risk of extreme precipitation events, currently expected to occur once every 50 years, will be nearly 11 (21) times more likely to occur under a climate warming by 3.0°C (4.0°C). Limiting warming to 1.5°C will help avoid approximately 40%-50%, 70%-80% and over 90% of the increase in the risk of extreme events in almost all subregions if the global mean surface temperature (GMST) continues warming to 2.0°C, 3.0°C and 4.0°C, respectively. Our study provides a useful information for the understanding the impact of climate change on the future risk of extreme events over China.

Whole-transcriptome analysis of differentially expressed genes in the mutant and normal capitula of Chrysanthemum morifolium.

BACKGROUND: Chrysanthemum morifolium is one of the most economically important and popular floricultural crops in the family Asteraceae. Chrysanthemum flowers vary considerably in terms of colors and shapes. However, the molecular mechanism controlling the development of chrysanthemum floral colors and shapes remains an enigma. We analyzed a cut-flower chrysanthemum variety that produces normal capitula composed of ray florets with normally developed pistils and purple corollas and mutant capitula comprising ray florets with green corollas and vegetative buds instead of pistils. RESULTS: We conducted a whole-transcriptome analysis of the differentially expressed genes (DEGs) in the mutant and normal capitula using third-generation and second-generation sequencing techniques. We identified the DEGs between the mutant and normal capitula to reveal important regulators underlying the differential development. Many transcription factors and genes related to the photoperiod and GA pathways, floral organ identity, and the anthocyanin biosynthesis pathway were differentially expressed between the normal and mutant capitula. A qualitative analysis of the pigments in the florets of normal and mutant capitula indicated anthocyanins were synthesized and accumulated in the florets of normal capitula, but not in the florets of mutant capitula. These results provide clues regarding the molecular basis of the replacement of Chrysanthemum morifolium ray florets with normally developed pistils and purple corollas with mutant ray florets with green corollas and vegetative buds. Additionally, the study findings will help to elucidate the molecular mechanisms underlying floral organ development and contribute to the development of techniques for studying the regulation of flower shape and color, which may enhance chrysanthemum breeding. CONCLUSIONS: The whole-transcriptome analysis of DEGs in mutant and normal C. morifolium capitula described herein indicates the anthocyanin deficiency of the mutant capitula may be related to the mutation that replaces ray floret pistils with vegetative buds. Moreover, pistils may be required for the anthocyanin biosynthesis in the corollas of chrysanthemum ray florets.

Upconversion luminescence enhancement by Fe3+ doping in CeO2:Yb/Er nanomaterials and their application in dye-sensitized solar cells.

To make use of broad spectrum solar energy remains a main target in the photoelectrochemical area. Novel promising photoelectrode CeO2:Fe/Yb/Er nanomaterials supported on upconversion nanomaterials doped with transition-metal ions are reported to improve broad spectrum absorption and scattering properties in dye-sensitized solar cells (DSSCs) for the first time. The results demonstrate that the materials have stronger upconversion luminescence than CeO2:Yb/Er samples when the Fe3+ ion doping concentration is 2 mol% and 33.5% higher photoelectric conversion efficiency than a pure P25 electrode, which are attributed to the special light scattering properties and excellent dye adsorption capacity of the CeO2:Fe/Yb/Er nanomaterials. Accordingly, doping Fe3+ transition metal ions in the upconversion material CeO2:Yb/Er provides a new research idea for improving the photoelectric conversion efficiency of DSSCs.

Bioprocess development of a stable FUT8-/--CHO cell line to produce defucosylated anti-HER2 antibody.

In recent years, an increasing number of defucosylated therapeutic antibodies have been applied in clinical practices due to their better efficacy compared to fucosylated counterparts. The establishment of stable and clonal manufacturing cell lines is the basis of therapeutic antibodies production. Bioprocess development of a new cell line is necessary for its future applications in the biopharmaceutical industry. We engineered a stable cell line expressing defucosylated anti-HER2 antibody based on an established α-1,6-fucosyltransferase (FUT8) gene knockout CHO-S cell line. The optimization of medium and feed was evaluated in a small-scale culture system. Then the optimal medium and feed were scaled up in a bioreactor system. After fed-batch culture over 13 days, we evaluated the cell growth, antibody yield, glycan compositions and bioactivities. The production of anti-HER2 antibody from the FUT8 gene knockout CHO-S cells in the bioreactor increased by 37% compared to the shake flask system. The N-glycan profile of the produced antibody was consistent between the bioreactor and shake flask system. The antibody-dependent cellular cytotoxicity activity of the defucosylated antibody increased 14-fold compared to the wild-type antibody, which was the same as our previous results. The results of our bioprocess development demonstrated that the engineered cell line could be developed to a biopharmaceutical industrial cell line.