Establishment of a Steatosis Model in LMH Cells, Chicken Embryo Hepatocytes, and Liver Tissues Based on a Mixture of Sodium Oleate and Palmitic Acid.

Research on hepatic steatosis in animal husbandry has been a prominent area of study. Developing an appropriate in vitro cellular steatosis model is crucial for comprehensively investigating the mechanisms involved in liver lipid deposition in poultry and for identifying potential interventions to address abnormalities in lipid metabolism. The research on the methods of in vitro liver steatosis in chickens, particularly the effects of different fat mixtures, is still lacking. In this study, LMH cells were utilized to investigate the effects of OA, SO, PA, SP, and their pairwise combinations on steatosis development, with the aim of identifying the optimal conditions for inducing steatosis. Analysis of triglyceride (TG) content in LMH cells revealed that OA and SP had limited efficacy in increasing TG content, while a combination of SO and PA in a 1:2 ratio exhibited the highest TG content. Moreover, Oil Red O staining results in LMH cells demonstrated that the combination treatment had a more pronounced induction effect compared to 0.375 mM SO. Additionally, RNA-seq analysis showed that 0.375 mM SO significantly influenced the expression of genes associated with fatty acid metabolism compared to the control group, whereas the combination of SO and PA led to an enrichment of key GO terms associated with programmed cell death. These findings suggest that varying conditions of cellular steatosis could lead to distinct disruptions in gene expression. The optimal conditions for inducing steatosis in LMH cells were also tested on chicken embryonic liver cells and embryos. TG detection and Oil Red O staining assays showed that the combination of SO and PA successfully induced steatosis. However, the gene expression pattern differed from that of LMH cells. This study lays the foundations for further investigations into avian hepatic steatosis.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken liver tissues and LMH cells.

The liver plays a vital role in lipid synthesis and metabolism in poultry. To study the functional genes more effectively, it is essential to screen of reliable reference genes in the chicken liver, including females, males, embryos, as well as the Leghorn Male Hepatoma (LMH) cell line. Traditional reference gene screening involves selecting commonly used housekeeping genes (HKGs) for RT-qPCR experiments and using different algorithms to identify the most stable ones. However, this approach is limited in selecting the best reference gene from a small pool of HKGs. High-throughput sequencing technology may offer a solution to this limitation. This study aimed to identify the most consistently expressed genes by utilizing multiple published RNA-seq data of chicken liver and LMH cells. Subsequently, the stability of the newly identified reference genes was assessed in comparison to previously validated stable poultry liver expressed reference genes and the commonly employed HKGs using RT-qPCR. The findings indicated that there is a higher degree of similarity in stable expression genes between female and male liver (such as LSM14A and CDC40). In embryonic liver, the optimal new reference genes were SUDS3, TRIM33, and ERAL1. For LMH cells, the optimal new reference genes were ALDH9A1, UGGT1, and C21H1orf174. However, it is noteworthy that most HKGs did not exhibit stable expression across multiple samples, indicating potential instability under diverse conditions. Furthermore, RT-qPCR experiments proved that the stable expression genes identified from RNA-seq data outperformed commonly used HKGs and certain validated reference genes specific to poultry liver. Over all, this study successfully identified new stable reference genes in chicken liver and LMH cells using RNA-seq data, offering researchers a wider range of reference gene options for RT-qPCR in diverse situations.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken oviduct tract.

Utilizing publicly available RNA-seq data to screen for ideal reference genes is more efficient and accurate than traditional methods. Previous studies have identified optimal reference genes in various chicken tissues, but none have specifically focused on the oviduct (including the infundibulum, magnum, isthmus, uterus, and vagina), which is crucial for egg production. Identifying stable reference genes in the oviduct is essential for improving research on gene expression levels. This study investigated genes with consistent expression patterns in the chicken oviduct, encompassing both individual oviduct tract tissues and the entire oviduct, by utilizing multiple RNA-seq datasets. The screening results revealed the discovery of 100 novel reference genes in each segment of oviduct tissues, primarily associated with cell cycle regulation and RNA binding. Moreover, the majority of housekeeping genes (HKGs) showed inconsistent expression levels across distinct samples, suggesting their lack of stability under varying conditions. The stability of the newly identified reference genes was assessed in comparison to previously validated stable reference genes in chicken oviduct and commonly utilized HKGs, employing traditional reference gene screening methods. HERPUD2, CSDE1, VPS35, PBRM1, LSM14A, and YWHAB were identified to be suitable novel reference gene for different parts of the oviduct. HERPUD2 and YWHAB were reliable for gene expression normalization throughout the oviduct tract. Furthermore, overexpression and interference assays in DF1 cells showed LSM14A and YWHAB play a crucial role in cell proliferation, highlighting the importance of these newly reference genes for further research. Overall, this study has expanded the options for reference genes in RT-qPCR experiments in different segments of the chicken oviduct and the entire oviduct.

Atmosphere teleconnections from abatement of China aerosol emissions exacerbate Northeast Pacific warm blob events.

During 2010 to 2020, Northeast Pacific (NEP) sea surface temperature (SST) experienced the warmest decade ever recorded, manifested in several extreme marine heatwaves, referred to as "warm blob" events, which severely affect marine ecosystems and extreme weather along the west coast of North America. While year-to-year internal climate variability has been suggested as a cause of individual events, the causes of the continuous dramatic NEP SST warming remain elusive. Here, we show that other than the greenhouse gas (GHG) forcing, rapid aerosol abatement in China over the period likely plays an important role. Anomalous tropospheric warming induced by declining aerosols in China generated atmospheric teleconnections from East Asia to the NEP, featuring an intensified and southward-shifted Aleutian Low. The associated atmospheric circulation anomaly weakens the climatological westerlies in the NEP and warms the SST there by suppressing the evaporative cooling. The aerosol-induced mean warming of the NEP SST, along with internal climate variability and the GHG-induced warming, made the warm blob events more frequent and intense during 2010 to 2020. As anthropogenic aerosol emissions continue to decrease, there is likely to be an increase in NEP warm blob events, disproportionately large beyond the direct radiative effects.

Quail GHRL and LEAP2 gene cloning, polymorphism detection, phylogenetic analysis, tissue expression profiling and its association analysis with feed intake.

The GHRL, LEAP2, and GHSR system have recently been identified as important regulators of feed intake in mammals and chickens. However, the complete cloning of the quail GHRL (qGHRL) and quail LEAP2 (qLEAP2) genes, as well as their association with feed intake, remains unclear. This study cloned the entire qGHRL and qLEAP2 cDNA sequence in Chinese yellow quail (Coturnix japonica), including the 5' and 3' untranslated regions. Sanger sequencing analysis revealed no missense mutations in the coding region of qGHRL and qLEAP2. Subsequently, phylogenetic analysis and protein homology alignment were conducted on the qGHRL and qLEAP2 in major poultry species. The findings of this research indicated that the qGHRL and qLEAP2 sequences exhibit a high degree of similarity with those of chicken and turkey. Specifically, the N-terminal 6 amino acids of GHRL mature peptides and all the mature peptide sequence of LEAP2 exhibited consistent patterns across all species examined. The analysis of tissue gene expression profiles indicated that qGHRL was primarily expressed in the proventriculus and brain tissue, whereas qLEAP2 exhibited higher expression levels in the intestinal tissue, kidney, and liver tissue, differing slightly from previous studies conducted on chicken. It is necessary to investigate the significance of elevated expression of qGHRL in brain and qLEAP2 in kidney in the future. Further research has shown that the expression of qLEAP2 can quickly respond to changes in different energy states, whereas qGHRL does not exhibit the same capability. Overall, this study successfully cloned the complete cDNA sequences of qGHRL and qLEAP2, and conducted a comprehensive examination of their tissue expression profiles and gene expression levels in the main expressing organs across different energy states. Our current findings suggested that qLEAP2 is highly expressed in the liver, intestine, and kidney, and its expression level is regulated by feed intake.

Alum/CpG adjuvant promotes immunogenicity of inactivated SARS-CoV-2 Omicron vaccine through enhanced humoral and cellular immunity.

The SARS-CoV-2 Omicron variant, which was classified as a variant of concern (VOC) by the World Health Organization on 26 November 2021, has attracted worldwide attention for its high transmissibility and immune evasion ability. The existing COVID-19 vaccine has been shown to be less effective in preventing Omicron variant infection and symptomatic infection, which brings new challenges to vaccine development and application. Here, we evaluated the immunogenicity and safety of an Omicron variant COVID-19 inactivated vaccine containing aluminum and CpG adjuvants in a variety of animal models. The results showed that the vaccine candidate could induce high levels of neutralizing antibodies against the Omicron variant virus and binding antibodies, and significantly promoted cellular immune response. Meanwhile, the vaccine candidate was safe. Therefore, it provided more foundation for the development of aluminum and CpG as a combination adjuvant in human vaccines.

Historical changes in wind-driven ocean circulation drive pattern of Pacific warming.

The tropical Pacific warming pattern since the 1950s exhibits two warming centers in the western Pacific (WP) and eastern Pacific (EP), encompassing an equatorial central Pacific (CP) cooling and a hemispheric asymmetry in the subtropical EP. The underlying mechanisms of this warming pattern remain debated. Here, we conduct ocean heat decompositions of two coupled model large ensembles to unfold the role of wind-driven ocean circulation. When wind changes are suppressed, historical radiative forcing induces a subtropical northeastern Pacific warming, thus causing a hemispheric asymmetry that extends toward the tropical WP. The tropical EP warming is instead induced by the cross-equatorial winds associated with the hemispheric asymmetry, and its driving mechanism is southward warm Ekman advection due to the off-equatorial westerly wind anomalies around 5°N, not vertical thermocline adjustment. Climate models fail to capture the observed CP cooling, suggesting an urgent need to better simulate equatorial oceanic processes and thermal structures.

Differential expression of ASIP transcripts reveals genetic mechanism underpinning black-tail independence from body plumage in yellow-bodied chickens.

The genetic foundation of chicken body plumage color has been extensively studied. However, little attention has been paid to the inheritance patterns and molecular mechanisms underlying the formation of distal feather colors (tail and wingtip). Differences in these colors are common; for example, the Chinese Huiyang Beard chicken has black tail feathers, but yellow body plumage. Here, the hybrid offspring of Huiyang Beard and White Leghorn chickens were used to study the inheritance patterns of tail-feather color. The expression levels of pigment genes in differently colored feather follicles were analyzed using quantitative real-time PCR. The results showed that genetic regulation of tail-feather color was independent of body-plumage color. The Dominant White locus inhibited eumelanin synthesis in tail feathers without affecting the formation of yellow body plumage, whereas the Silver locus had the opposite effect. The expression of agouti signaling protein (ASIP) gene class 1 transcripts was significantly lower in black tail-feather follicles than in yellow body follicles, whereas tyrosinase-related protein 1 (TYRP1) gene expression was significantly higher in black tail feathers. These differentially expressed genes were confirmed to exert an effect on eumelanin and pheomelanin formation in feathers, thus influencing the regulation of chicken tail-feather color. In conclusion, this study lays the foundation for further research on the genetic mechanisms of regional differences in feather color, contributing to a better understanding of plumage pigmentation in chickens.

Increased Asian aerosols drive a slowdown of Atlantic Meridional Overturning Circulation.

Observational evidence and climate model experiments suggest a slowdown of the Atlantic Meridional Overturning Circulation (AMOC) since the mid-1990s. Increased greenhouse gases and the declined anthropogenic aerosols (AAs) over North America and Europe are believed to contribute to the AMOC slowdown. Asian AAs continue to increase but the associated impact has been unclear. Using ensembles of climate simulations, here we show that the radiative cooling resulting from increased Asian AAs drives an AMOC reduction. The increased AAs over Asia generate circumglobal stationary Rossby waves in the northern midlatitudes, which shift the westerly jet stream southward and weaken the subpolar North Atlantic westerlies. Consequently, reduced transport of cold air from North America hinders water mass transformation in the Labrador Sea and thus contributes to the AMOC slowdown. The link between increased Asian AAs and an AMOC slowdown is supported by different models with different configurations. Thus, reducing emissions of Asian AAs will not only lower local air pollution, but also help stabilize the AMOC.

Enhancing Inactivated Yellow Fever 17D Vaccine-Induced Immune Responses in Balb/C Mice Using Alum/CpG.

There are some concerns about the safety of live attenuated yellow fever vaccines (YF-live), particularly viscerotropic adverse events, which have a high mortality rate. The cellular production of the vaccine will not cause these adverse effects and has the potential to extend applicability to those who have allergic reactions, immunosuppression, and age. In this study, inactivated yellow fever (YF) was prepared and adsorbed with Alum/CpG. The cellular and humoral immunities were investigated in a mouse model. The results showed that Alum/CpG (20 µg/mL) could significantly increase the binding and neutralizing activities of the antibodies against YF. Moreover, the antibody level at day 28 after one dose was similar to that of the attenuated vaccine, but significantly higher after two doses. At the same time, Alum/CpG significantly increased the levels of IFN-γ and IL-4 cytokines.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken gastrointestinal tract.

The application of reverse transcription quantitative real-time PCR technology for the production of gene tissue expression profiles is a widely employed approach in molecular biology research. It is imperative to ascertain internal reference genes that exhibit stable expression across diverse tissues to ensure the precision of tissue gene expression profiles. While there have been studies documenting the most suitable reference genes for various tissues in chickens, there is a dearth of research on the identification of reference genes in the gastrointestinal (GI) tract of chickens. This study utilized 4 different algorithms (Delta CT, BestKeeper, NormFinder, and Genorm) to assess the stability of 19 internal reference genes in various GI tract tissues, including individual GI tract tissues, the anterior and posterior GI tract, and the entire GI tissue. The RefFinder software was employed to comprehensively rank these genes. The research findings successfully identified the most appropriate internal reference genes for each type of GI tissue. Furthermore, TBP, DNAJC24, Polr2b, RPL13, andAp2m exhibited stable expression in the entire and posterior GI tract, whereas HMBS, TBP, Ap2m, GUSB, DNAJC24, and RPL13 demonstrated stable expression in the anterior GI tract. However, the internal reference genes commonly utilized, namely ß-Actin, 18s RNA, and ALB, exhibit poor stability and are not advised for future investigations concerning gene expression in the GI region. Consequently, MUC2 and CDX1, 2 genes that specifically express in the gut, were chosen for examination to ascertain the stability of the aforementioned internal reference genes in this particular study. In summary, this study presents a relatively stable set of internal reference genes that can be employed to enhance the precision of quantifying mRNA expression levels in functional genes within the chicken GI tract.

Effects of Glycine Supplementation in Drinking Water on the Growth Performance, Intestinal Development, and Genes Expression in the Jejunum of Chicks.

Glycine, the most basic amino acid found in nature, is considered an essential amino acid for chicks. However, the precise understanding of high concentrations of glycine's significance in promoting the growth performance of chicks, as well as its impact on intestinal development, re-mains limited. Consequently, the objective of this study was to investigate the effects of glycine supplementation in drinking water on growth performance, intestine morphology, and development in newly hatched chicks. In this study, 200 newly born chicks were selected and pro-vided with a supplementation of 0.5%, 1%, and 2% glycine in their drinking water during their first week of life. The results revealed that glycine supplementation in drinking water could significantly increase the average daily gain of chicks from days 7 to 14. Furthermore, a significant difference was observed between the group supplemented with 1% glycine and the control group. Concurrently, this glycine supplementation increased the villus height and the ratio of the villus height to crypt depth in jejunum on both day 7 and day 14. Glycine supplementation in drinking water significantly affected the mRNA expression level of the ZO-1, GCLM, and rBAT genes in jejunum, which may have certain effects on the mucosal immune defense, cellular antioxidant stress capacity, and amino acid absorption. Overall, the findings of this study indicate that glycine supplementation in drinking water can enhance the growth performance of chicks and promote their intestine development.

HOXB8 overexpression induces morphological changes in chicken mandibular skin: an RNA-seq analysis.

The Huiyang beard chicken is a well-known Chinese local breed known for its elongated feathers gathered from both sides of the face (muffs) and below the beak (beard), as well as short wattles (SW). The muff and beard (Mb) mutation is caused by ectopic upregulation of the homeobox B8 (HOXB8) gene in the mandibular skin; and the chi-square test showed a significant correlation between SW and Mb genotypes. However, the underlying molecular mechanisms that regulate Mb and SW variations remain unclear. In this study, we investigated the transcriptomes of the mandibular skin and wattles of chickens with and without the Mb genotype to elucidate the molecular basis of these traits. Our results show that HOXB8 is expressed at significantly higher levels in both the mandibular skin and wattles of Mb chickens than in those of wild-type chickens, indicating that HOXB8 regulates both the Mb and SW phenotypes. Key genes for keratin synthesis were highly expressed in the mandibular skin of Mb chickens, suggesting that HOXB8 may play a role in feather development. In wattles, changes in the expression of extracellular matrix synthesis genes may contribute to SW traits. DNA-binding motif analyses revealed that differentially expressed genes were likely to be directly regulated by HOXB8 binding, indicating that HOXB8 may directly or indirectly regulate feather follicle development and wattle growth. Our study identified both known and novel targets, including several genes not previously implicated in feather development and mesenchymal formation. These findings provide insights into the molecular mechanisms of skin appendage variation in birds and offer potential applications in breeding poultry breeds with unique phenotypes.

Selective inhibition of glycolysis in hepatic stellate cells and suppression of liver fibrogenesis with vitamin A-derivative decorated camptothecin micelles.

The persistent transformation of quiescent hepatic stellate cells (HSCs) into myofibroblasts (MFs) and the excessive proliferation of MF-HSCs in the liver contribute to the pathogenesis of liver fibrosis, cirrhosis, and liver cancer. Glycolysis inhibition of MF-HSCs can reverse their MF phenotype and suppress their abnormal expansion. Here, we have developed vitamin A-derivative (VA) decorated PEG-PCL polymeric micelles to encapsulate the labile and hydrophobic camptothecin (CPT) and direct its active attack on HSCs, selectively inhibiting of HIF-1α and cellular glycolysis, ultimately repressing hepatic fibrogenesis. The obtained micelles exhibited a good stability, biocompatibility, pH sensitivity, and exceptional HSC-targetability, allowing an efficient accumulation of their carried CPT in acutely and chronically injured livers. On their intracellular release of CPT specifically in MF-HSCs, these CPT micelles nicely inhibited the HIF-1α and a series of glycolytic players in MF-HSCs and prominently suppressed their proliferation and MF phenotypic characteristics. Accordingly, on in vitro administration to the mice challenged by CCl4 or subjected to bile duct ligation, these VA-decorated CPT micelles ameliorated the pathological symptoms of the livers, as evidenced by the significant reduction in serum levels of ALT and AST, infiltration of inflammatory cells, and collagen accumulation, the drastic down-regulation of multiple fibrotic genes, and the good recovery of attenuated hepatocyte CYP2E1 and lipogenesis regulator PPARγ. Overall, the CPT carried by VA-decorated PEG-PCL polymeric micelles can selectively inhibit the glycolysis and expansion of HSCs and thus suppress fibrogenesis, providing an original and effective approach for anti-fibrotic therapy. STATEMENT OF SIGNIFICANCE: Our work introduces an innovative antifibrotic drug system that is developed upon the active targeting of CPT and aims for the fate reversal of HSCs. Through HSC-targeted delivery achieved by PEG-PCL polymeric micelles decorated with vitamin A-derivatives, CPT significantly suppressed the expressions of HIF-1α and glycolytic enzymes in MF-HSCs, as well as their pathologic expansion in mouse livers. It effectively ameliorated chronic liver fibrosis in mice induced by CCl4 injection or BDL and restored the damaged liver structure and function. These compelling findings demonstrate the therapeutic potential of glycolytic HSC-targeting in combating fibrosis and related disorders and thus provide new promise for future clinical management of such prevalent and life-threatening conditions.

Hepatoprotective effects of chlorogenic acid on mice exposed to aflatoxin B1: Modulation of oxidative stress and inflammation.

Aflatoxin B1 (AFB1) is widely distributed in crops and feeds, and ingestion of AFB1-contaminated crops is harmful to human/animal health. This study was designed to investigate hepatoprotective effects of chlorogenic acid (CGA), due to its excellent antioxidant and anti-inflammatory activities, on mice exposed to AFB1. Male Kunming mice were orally fed with CGA prior to daily AFB1 exposure for 18 consecutive days. The results showed that CGA treatment reduced the serum activity of aspartate aminotransferase, hepatic malondialdehyde content and pro-inflammatory cytokines synthesis, prevented histopathological changes of the liver, increased hepatic glutathione level, catalase activity and IL10 mRNA expression in mice subjected to AFB1. Taken together, CGA exerted the protective effect on AFB1-induced hepatic damage by modulating redox status and inflammation, suggesting that CGA may be a candidate compound for the treatment of aflatoxicosis.

Southern Ocean warming and its climatic impacts.

The Southern Ocean has warmed substantially, and up to early 21st century, Antarctic stratospheric ozone depletion and increasing atmospheric CO2 have conspired to intensify Southern Ocean warming. Despite a projected ozone recovery, fluxes to the Southern Ocean of radiative heat and freshwater from enhanced precipitation and melting sea ice, ice shelves, and ice sheets are expected to increase, as is a Southern Ocean westerly poleward intensification. The warming has far-reaching climatic implications for melt of Antarctic ice shelf and ice sheet, sea level rise, and remote circulations such as the intertropical convergence zone and tropical ocean-atmosphere circulations, which affect extreme weathers, agriculture, and ecosystems. The surface warm and freshwater anomalies are advected northward by the mean circulation and deposited into the ocean interior with a zonal-mean maximum at ∼45°S. The increased momentum and buoyancy fluxes enhance the Southern Ocean circulation and water mass transformation, further increasing the heat uptake. Complex processes that operate but poorly understood include interactive ice shelves and ice sheets, oceanic eddies, tropical-polar interactions, and impact of the Southern Ocean response on the climate change forcing itself; in particular, limited observations and low resolution of climate models hinder rapid progress. Thus, projection of Southern Ocean warming will likely remain uncertain, but recent community effort has laid a solid foundation for substantial progress.

Aflatoxin-B1-Exposure-Induced Hepatic Injury Could Be Alleviated by Polydatin through Reducing Oxidative Stress, Inhibiting Inflammation and Improving Mitophagy.

Aflatoxin B1 (AFB1) is a toxic food/feed pollutant, exerting extensive deleterious impacts on the liver. Oxidative stress and inflammation are considered to be vital contributors to AFB1 hepatotoxicity. Polydatin (PD), a naturally occurring polyphenol, has been demonstrated to protect and/or treat liver disorders caused by various factors through its antioxidant and anti-inflammatory properties. However, the role of PD in AFB1-induced liver injury is still elusive. Therefore, this study was designed to investigate the protective effect of PD on hepatic injury in mice subjected to AFB1. Male mice were randomly divided into three groups: control, AFB1 and AFB1-PD groups. The results showed that PD protected against AFB1-induced hepatic injury demonstrated by the reduced serum transaminase activity, the restored hepatic histology and ultrastructure, which could be attributed to the enhanced glutathione level, the reduced interleukin 1 beta and tumor necrosis factor alpha concentrations, the increased interleukin 10 expression at transcriptional level and the up-regulated mRNA expression related to mitophagy. In conclusion, PD could alleviate AFB1-induced hepatic injury by reducing oxidative stress, inhibiting inflammation and improving mitophagy.

Pressure-Gradient Counterwork of Dual-Fuel Driven Nanocarriers in Abnormal Interstitial Fluids for Enhancing Drug Delivery Efficiency.

The abnormal pressure in tumor tissue is a significant limitation on the drug delivery efficiency of tumor therapy. This work reports a gradient-driven nanomotor as drug nanocarrier with the pressure-counterworking function. The dual-fuel nanomotors are formed by co-electrospinning of the photosensitive polymers with calcium peroxide (CaO2 ) and catalase (CAT), followed by ultraviolet (UV) irradiation and bovine serum albumin (BSA) incubation. The UV-responsive cleavage nanomotors can effectively release O2 molecules at the fractures as a driving force to increase the delivery speed and escape the phagocytosis of macrophage system in normal tissues. Furthermore, CAT catalyzes H2 O2 produced by CaO2 and the tumor interstitial fluids to provide stronger power for the nanomotors. Additionally, according to the analysis of directional motions of the nanomotors, the functional relationship between the rotational diffusion coefficient (DR ) and the physiological viscosity is constructed. The dual-fuel nanocarriers enable up to 13.25% of the injected dose (ID)/per gram tissue and significantly improve the penetration in deep tumor. It is of vital importance to design and obtain the adaptive pressure-gradient counterworking nanomotors, which can effectively improve the drug delivery efficiency in vitro and in vivo.