Immune protection of grass carp by oral vaccination with recombinant Bacillus methylotrophicus expressing the heterologous tolC gene.

In the field of aquaculture, the enhancement of animal health and disease prevention is progressively being tackled using alternatives to antibiotics, including vaccines and probiotics. This study was designed to evaluate the potential of a recombinant Bacillus methylotrophicus, engineered to express the outer membrane channel protein TolC of Aeromonas hydrophila AH3 and the green fluorescent protein GFP, as an oral vaccine. Initially, the genes encoding tolC and GFP were cloned into a prokaryotic expression system, and anti-TolC mouse antiserum was generated. Subsequently, the tolC gene was subcloned into a modified pMDGFP plasmid, which was transformed into B. methylotrophicus WM-1 for protein expression. The recombinant B. methylotrophicus BmT was then administered to grass carp via co-feeding, and its efficacy as an oral vaccine was assessed. Our findings demonstrated successful expression of the 55 kDa TolC and 28 kDa GFP proteins, and the preparation of polyclonal antibodies with high specificity. The BmT exhibited stable expression of the GFP-TolC fusion protein and excellent genetic stability. Following oral immunization, significant elevations were observed in serum-specific IgM levels and the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) in grass carp. Concurrently, significant upregulation of immune-related genes, including IFN-I, IL-10, IL-1ß, TNF-α, and IgT, was noted in the intestines, head kidney, and spleen of the grass carp. Colonization tests further revealed that the BmT persisted in the gut of immunized fish even after a fasting period of 7 days. Notably, oral administration of BmT enhanced the survival rate of grass carp following A. hydrophila infection. These results suggest that the oral BmT vaccine developed in this study holds promise for future applications in aquaculture.

Pathogenicity and inactivated vaccine treatment of Aeromonas veronii JW-4 on crucian carp.

Aeromonas veronii is a common bacterium found in a variety of aquatic environments, capable of causing a diverse array of diseases in both aquatic animals and humans. Therefore, evaluating the pathogenicity of A. veronii and implementing measures to control its spread are essential. In this study, a strain JW-4, identified as A. veronii, was isolated from diseased Scaphesthes macrolepis, a grade Ⅱ protected animal in China. To investigate the pathogenicity of the strain, fish were fed with serial levels JW-4 supplemented diet or basal diet (control group 1, CG1) for 28 days (d). Results showed that JW-4 stimulated an immune response, evidenced by an increase in immune-related enzyme activities (GOT and GPT) of serum and liver and an upregulation of genes expression levels (TNF-α and IFN-γ) of liver and spleen, and these effects gradually decreased over time. Histopathological examination revealed that JW-4 could alter the tissue structure of immune organs, such as liver and kidney. These changes were accompanied by vacuolar degeneration, nuclear dissolution, and an increased lymphocyte count. To assess protective effects of a vaccine against this strain, fish were injected with an inactivated vaccine (immunization group, IG) or 0.85% sterile saline (control group 2, CG2) for 28-day observation period, then challenged with JW-4 on the 28th day. The inactivated vaccine enhanced total and specific IgM to A. veronii levels of the fish, resulting in a relative percentage survival of 75% in IG. These findings provide a foundation for identifying pathogenic bacteria and developing more effective prophylactic strategies in aquaculture.

Manufacture and evaluation of a HER2-positive breast cancer immunotoxin 4D5Fv-PE25.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) positive breast cancer is an aggressive subtype, accounting for around 20% of all breast cancers. The development of HER2-targeted therapy has substantially improved patient outcomes. Nevertheless, the increasing rate of side effects and resistance to targeted drugs limit their efficacy in clinical practice. In this study, we designed and synthesized a new immunotoxin, 4D5Fv-PE25, which targets HER2-positive breast cancer, and evaluated its effectiveness in vitro and in vivo. RESULTS: The 4D5Fv-PE25 was expressed in high-density Escherichia coli (E. coli.) using the fermentor method and refined via hydrophobicity, ion exchange, and filtration chromatography, achieving a 56.06% recovery rate. Additionally, the semi-manufactured product with 96% purity was prepared into freeze-dried powder by the lyophilized process. Flow cytometry was used to detect the expression of HER2 in SK-BR-3, BT-474, MDA-MB-231, and MDA-MB-468 breast cancer cell lines. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method was used for cytotoxicity assay, and the half-maximal inhibitory concentration (IC50) of 4D5Fv-PE25 lyophilized products to HER2-positive cell line SK-BR-3 was 12.53 ng/mL. The 4D5Fv-PE25 was injected into xenograft tumor mice via the tail vein on the 1st, 4th, and 8th day, it indicated that the growth of tumor volume was effectively inhibited for 24 days, although the 4D5Fv-PE25 was metabolized within 60 min by measuring the release of 3 H-Thymidine radiation. CONCLUSION: we succeeded in producing the 4D5Fv-PE25 freeze-dried powder using the prokaryotic expression method, and it could be employed as a potential drug for treating HER2-positive breast cancer.

Dysfunction of Prkcaa Links Social Behavior Defects with Disturbed Circadian Rhythm in Zebrafish.

Protein kinase Cα (PKCα/PRKCA) is a crucial regulator of circadian rhythm and is associated with human mental illnesses such as autism spectrum disorders and schizophrenia. However, the roles of PRKCA in modulating animal social behavior and the underlying mechanisms remain to be explored. Here we report the generation and characterization of prkcaa-deficient zebrafish (Danio rerio). The results of behavioral tests indicate that a deficiency in Prkcaa led to anxiety-like behavior and impaired social preference in zebrafish. RNA-sequencing analyses revealed the significant effects of the prkcaa mutation on the expression of the morning-preferring circadian genes. The representatives are the immediate early genes, including egr2a, egr4, fosaa, fosab and npas4a. The downregulation of these genes at night was attenuated by Prkcaa dysfunction. Consistently, the mutants demonstrated reversed day-night locomotor rhythm, which are more active at night than in the morning. Our data show the roles of PRKCA in regulating animal social interactions and link the social behavior defects with a disturbed circadian rhythm.

Research progress in relationships between freshwater bivalves and algae.

Eutrophication in freshwater has become increasingly severe around the world, resulting in phytoplankton overgrowth and benthic algae reduction. Bivalves can change the density, dominant species and community structure of phytoplankton, increase available light levels, and provide physical habitats and growth conditions for benthic algae. The nutritional composition, density, community structure, and toxin of algae affect the growth, feeding, digestion, metabolism, immunity of bivalves in return. Interactions of bivalves and algae and effects of environmental factors on these interactions need a synthesis of studies, when using bivalves as a biomanipulation tool to control eutrophication. Whether bivalves can effectively suppress phytoplankton and promote benthic algae is related to the collective filtration and excretion capacity determined by size, species, population densities of bivalves, the quantity and quality of algae, and environmental factors such as temperature, dissolved oxygen, pH, and hydrodynamic. Small scale bivalve biomanipulation experiments are mostly conducted in lakes, urban ponds, and reservoirs with some success, applying in the whole ecosystem should consider more questions such as natural conditions, selection and death or reproduction of bivalves, and ecological disturbances. This review provides new considerations for technical issues such as the sustainable cultivation of bivalves and the implementation of biomanipulation in eutrophic waters.

Calpain-1 contributes to IgE-mediated mast cell activation.

Mast cells play a central role in allergy through secretion of both preformed and newly synthesized mediators. Mast cell mediator secretion is controlled by a complex network of signaling events. Despite intensive studies, signaling pathways in the regulation of mast cell mediator secretion remain incompletely defined. In this study, we examined the role of calpain in IgE-dependent mast cell activation. IgE-mediated activation of mouse bone marrow-derived mast cells enhanced calpain activity. Inhibition of calpain activity by a number of calpain inhibitors reduced IgE-mediated mast cell degranulation both in vitro and in vivo. Calpain inhibitors blocked IgE-mediated TNF and IL-6 production in vitro and reduced late-phase allergic response in vivo. Importantly, mouse calpain-1 null bone marrow-derived mast cells showed reduced IgE-mediated mast cell degranulation in vitro and in vivo, diminished cytokine and chemokine production in vitro, and impaired late-phase allergic response in vivo. Further studies revealed that calpain-1 deficiency led to specific attenuation of IκB-NF-κB pathway and IKK-SNAP23 pathway, whereas calcium flux, MAPK, Akt, and NFAT pathway proceed normally in IgE-activated calpain-1 null mast cells. Thus, calpain-1 is identified as a novel regulator in IgE-mediated mast cell activation and could serve as a potential therapeutic target for the management of allergic inflammation.

Mast cells protect against Pseudomonas aeruginosa-induced lung injury.

Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortality in immune-compromised individuals. Maintaining the integrity of the respiratory epithelium is critical for an effective host response to P. aeruginosa. Given the close spatial relationship between mast cells and the respiratory epithelium, and the importance of tightly regulated epithelial permeability during lung infections, we examined whether mast cells influence airway epithelial integrity during P. aeruginosa lung infection in a mouse model. We found that mast cell-deficient Kit(W-sh)/Kit(W-sh) mice displayed greatly increased epithelial permeability, bacterial dissemination, and neutrophil accumulation compared with wild-type animals after P. aeruginosa infection; these defects were corrected on reconstitution with mast cells. An in vitro Transwell co-culture model further demonstrated that a secreted mast cell factor decreased epithelial cell apoptosis and tumor necrosis factor production after P. aeruginosa infection. Together, our data demonstrate a previously unrecognized role for mast cells in the maintenance of epithelial integrity during P. aeruginosa infection, through a mechanism that likely involves prevention of epithelial apoptosis and tumor necrosis factor production. Our understanding of mechanisms of the host response to P. aeruginosa will open new avenues for the development of successful preventative and treatment strategies.

Regulator of calcineurin 1 suppresses inflammation during respiratory tract infections.

Respiratory tract infection with Pseudomonas aeruginosa is a common cause of hospitalization in immune-compromised individuals. However, the molecular mechanisms involved in the immune response to P. aeruginosa lung infection remain incompletely defined. In this study, we demonstrate that the regulator of calcineurin 1 (RCAN1) is a central negative regulator of inflammation in a mouse model of acute bacterial pneumonia using the opportunistic bacterial pathogen P. aeruginosa. RCAN1-deficient mice display greatly increased mortality following P. aeruginosa lung infection despite enhanced neutrophil recruitment and bacterial clearance. This mortality is associated with higher systemic levels of proinflammatory cytokines in RCAN1-deficient animals. These aberrant inflammatory responses coincide with increased transcriptional activity of proinflammatory RCAN1-target proteins NFAT and NF-κB. In addition, we reveal a novel regulatory role for RCAN1 in the ERK/STAT3 pathway both in vitro and in vivo, suggesting that aberrant STAT3 activity may significantly contribute to delayed resolution of inflammatory responses in our model. Together, these findings demonstrate that RCAN1 is a potent negative regulator of inflammation during respiratory tract infections.

Mast cell FcεRI-induced early growth response 2 regulates CC chemokine ligand 1-dependent CD4+ T cell migration.

Mast cells are well positioned in host tissue for detecting environmental signals, including allergens, leading to activation of the high-affinity IgE receptor FcεRI, and initiating a signaling cascade that perpetuates the production of biologically potent mediators, including chemokines. We have identified a novel target of mast cell FcεRI activity in the transcription factor early growth response 2 (Egr2) and sought to characterize its function therein. Egr2 was transiently activated following FcεRI-mediated signaling, targeted the promoter of the chemokine CCL1, and was critical for allergen-induced mast cell CCL1 production. Egr2-deficient mast cells were incapable of directing CD4(+) T cell migration via the CCL1-CCR8 axis. In a model of allergic asthma, reconstitution of mast cell-deficient mice with Egr2-deficient mast cells demonstrated that mast cell Egr2 was essential for migration of CD4(+) T cells to the inflamed lung. These findings position Egr2 as a critical regulator of mast cell-directed CD4(+) T cell migration.

Calcineurin-Rcan1 interaction contributes to stem cell factor-mediated mast cell activation.

The receptor for stem cell factor (SCF) is expressed on mast cells and hematopoietic progenitors. SCF-induced signaling pathways remain incompletely defined. In this study, we identified calcineurin and regulator of calcineurin 1 (Rcan1) as novel components in SCF signaling. Calcineurin activity was induced in SCF-stimulated primary mouse and human mast cells. NFAT was activated by SCF in bone marrow-derived mast cells (BMMCs) and mouse bone marrow cells, which contain hematopoietic progenitors. SCF-mediated activation also induced expression of Rcan1 in BMMCs. Rcan1-deficient BMMCs showed increased calcineurin activity and enhanced transcriptional activity of NF-κB and NFAT, resulting in increased IL-6 and TNF production following SCF stimulation. These results suggest that Rcan1 suppresses SCF-induced activation of calcineurin and NF-κB. We further demonstrated that SCF-induced Rcan1 expression is dependent on the transcription factor early growth response 1 (Egr1). Interestingly, SCF-induced Egr1 was also suppressed by Rcan1, suggesting a negative regulatory loop between Egr1 and Rcan1. Together, our findings revealed that calcineurin contributes to SCF-induced signaling, leading to NFAT activation, which, together with NF-κB and Egr1, is suppressed by Rcan1. Considering the wide range of biological functions of SCF, these novel regulatory mechanisms in SCF signaling may have broad implications.

Aeromonas veronii tolC modulates its virulence and the immune response of freshwater pearl mussels, Hyriopsis cumingii.

Aeromonas veronii is an opportunistic pathogen that causes diseases in aquatic animals, but its key virulence factors remain unclear. We screened the gene tolC with significantly different expression levels in the two isolates, A. veronii GL2 (higher virulence) and A. veronii FO1 (lower virulence). Therefore, we constructed mutant strain ΔtolC and analyzed its immunological properties. ΔtolC exhibited the reduced ability of biofilms formation, inhibited envelope stress response mediated by several antibiotics except cefuroxime, implying the ability to evade host immunity might be restrained. Challenge tests showed that the LD50 of ΔtolC was 10.89-fold than that of GL2. Enzymatic activities of ΔtolC group were significantly lower and peak time was delayed to 12 h, as demonstrated by qRT-PCR results. Histopathological examination displayed that the degree of tissue damage in ΔtolC group was alleviated. The results show that tolC is an important virulence factor of A. veronii, which provides references for live-attenuated vaccine.

Comparative transcriptome reveals importance of export apparatus subunit (ascR) in type III secretion system and its roles on biological properties, gene expression profiles, virulence and colonization of Aeromonas veronii.

Aeromonas veronii, an opportunistic pathogen, is known to cause serious infections across various species. In our previous study, we discovered that A. veronii GL2 exhibited a virulence up to ten times greater than that of FO1. To ascertain the factors contributing to the disparity in virulence between the two strains, we conducted a comparative transcriptome analysis. This analysis reveals a significant upregulation (P < 0.05) of the ascR gene in GL2 compared with FO1. Additionally, six differentially expressed genes (DEGs) were identified within the "Bacterial secretion system" pathway (map03070), with ascR being an essential component of type III secretion system (T3SS). AscR, considered as SctR family export apparatus subunit within the T3SS, has ambiguous roles in the biological properties, gene expression profiles, virulence and colonization of A. veronii. Therefore, we constructed a mutant strain (ΔascR) by homologous recombination. Comparative analysis with the wide-type GL2 reveals no significant differences in terms of colony morphology, growth curve, hemolytic activity and protease activity. However, significant reductions (P < 0.01) were observed in the abilities of biofilm formation and swimming mobility. No remarkable difference was noted in the lengths of flagella. The LD50 value of ΔascR was to be 5.15 times higher than that of GL2. Interestingly, the mRNA expression of ascC, ascD, ascJ and ascI genes in the T3SS, and mshB, mshE, mshK and mshP genes in the MSHA type pili were significantly upregulated (P < 0.05) in ΔascR, potentially due to transcriptional compensation. Further analysis of enzymatic biomarkers revealed that ΔascR might not destruct the recognition of innate immune response in host remarkably, but the colonization levels of A.veronii were significantly suppressed (P < 0.01) in ΔascR group. In conclusion, the ascR gene may be a key determinant in regulating the virulence of A. veronii, and the destruction of the T3SS caused by ascR deficiency results in these notable changes.

Fucoidan alleviated dextran sulfate sodium-induced ulcerative colitis with improved intestinal barrier, reshaped gut microbiota composition, and promoted autophagy in male C57BL/6 mice.

Although previous research has unveiled the remedial effects of fucoidan, an extract from marine algae, on ulcerative colitis (UC), the precise mechanisms remain elusive. Animal studies have suggested a connection between autophagy and the beneficial influences of fucoidan intervention. We hypothesized that fucoidan's alleviative effects on dextran sulfate sodium (DSS)-induced UC could be ascribed to autophagy. For our study, we chose 36 male C57BL/6 mice and administered 100 or 400 mg/(kg/body weight/day) of fucoidan via gavage for 5 consecutive weeks. During the last week, the mice were given 3% DSS in drinking water to induce UC. In contrast to the DSS-induced UC model, fucoidan intervention prevented DSS-induced body weight loss, mitigated colon shortening, improved colon mucosa damage, enhanced the intestinal barrier, and reduced serum inflammatory factor concentrations. Furthermore, fucoidan intervention reshaped the gut microbiota compositions, increased the relative abundance of Bacteroidota, Muribaculaceae_unclassified, Clostridiales_unclassified, and Lachnospiraceae_NK4A136_group, and decreased the relative abundance of Firmicutes, Proteobacteria, and Escherichia-Shigella, which led to a lower Firmicutes/Bacteroidota ratio. Additionally, fucoidan treatment enhanced autophagy, as evidenced by upregulated protein expressions of BECLIN1, ATG5, ATG7, and an increased microtubule-associated-proteinlight-chain-3-II/microtubule-associated-proteinlight-chain-3-I ratio. Our findings corroborated the ameliorating effects of fucoidan intervention on DSS-induced UC through autophagy activation, reorganization of gut microbiota, and fortification of the intestinal barrier. This lends support to the therapeutic potential of fucoidan as a natural bioactive ingredient for future UC treatments in humans.

Apple polyphenol extract ameliorates sugary-diet-induced depression-like behaviors in male C57BL/6 mice by inhibiting the inflammation of the gut-brain axis.

To explore whether apple polyphenol extract (APE) ameliorates sugary-diet-induced depression-like behaviors, thirty male C57BL/6 mice (3-4 weeks old) were assigned to three groups randomly to receive different treatments for 8 consecutive weeks: (1) control group (CON), (2) S-HSD group (60% high sucrose diet feeding with 0.1 mg mL-1 sucralose solution as drinking water), and (3) S-APE group (S-HSD feeding with 500 mg per (kg bw day) APE solution gavage). The S-HSD group showed significant depression-like behaviors compared with the CON group, which was manifested by an increased number of buried marbles in the marble burying test, prolonged immobility time in both the tail suspension test and forced swimming test, and cognitive impairment based on the Morris water maze test. However, APE intervention significantly improved the depression-like behaviors by reducing serum levels of corticosterone and adrenocorticotropic hormone, and increasing the serum level of IL-10. Moreover, APE intervention inhibited the activation of the NF-κB inflammatory pathway, elevated colonic MUC-2 protein expression, and elevated the colonic and hippocampal tight junction proteins of occludin and ZO-1. Furthermore, APE intervention increased the richness and diversity of gut microbiota by regulating the composition of microbiota, with increased relative abundance of Firmicutes and Bacteroidota, decreased relative abundance of Verrucomicrobiota at the phylum level, significantly lowered relative abundance of Akkermansia at the genus level, and rebalanced abnormal relative abundance of Muribaculaceae_unclassified, Coriobacteriaceae_UCG-002, and Lachnoclostridium induced by S-HSD feeding. Thus, our study supports the potential application of APE as a dietary intervention for ameliorating depression-like behavioral disorders.

Ameliorated Effects of Fucoidan on Dextran Sulfate Sodium-Induced Ulcerative Colitis and Accompanying Anxiety and Depressive Behaviors in Aged C57BL/6 Mice.

Fucoidan has shown better effects on the improvement of acute ulcerative colitis (UC). However, the specific mechanisms by which fucoidan improves UC-related behavioral disorders in aged mice, especially its effect on the gut-brain axis, remain to be further explored. C57BL/6 male mice aged 8 months were gavaged with 400 or 100 mg/kg bw day fucoidan for five consecutive weeks, with UC being induced by ad libitum to dextran sulfate sodium (DSS) solution in the fifth week. The results showed that fucoidan ameliorated UC and accompanying anxiety- and depressive-like behaviors with downregulated expressions of (NOD)-like receptor family and pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), cysteine aspartate-specific protease-1 (Caspase-1) and interlekin-1ß (IL-1ß), and elevated mRNA levels of brain-derived neurotrophic factor (Bdnf) and postsynaptic-density protein 95 (Psd-95) in cortex and hippocampus. Furthermore, fucoidan improved the permeability of intestinal barrier and blood-brain barrier and restored the abnormal structure of the gut microbiota with a significantly decreased ratio of Firmicutes to Bacteroidota (F/B) and obviously increased abundance of Akkermansia. As a diet-derived bioactive ingredient, fucoidan might be a better alternative for the prevention of UC and accompanying anxiety- and depressive-like behaviors.

Comparative genomics of parasitic silkworm microsporidia reveal an association between genome expansion and host adaptation.

BACKGROUND: Microsporidian Nosema bombycis has received much attention because the pébrine disease of domesticated silkworms results in great economic losses in the silkworm industry. So far, no effective treatment could be found for pébrine. Compared to other known Nosema parasites, N. bombycis can unusually parasitize a broad range of hosts. To gain some insights into the underlying genetic mechanism of pathological ability and host range expansion in this parasite, a comparative genomic approach is conducted. The genome of two Nosema parasites, N. bombycis and N. antheraeae (an obligatory parasite to undomesticated silkworms Antheraea pernyi), were sequenced and compared with their distantly related species, N. ceranae (an obligatory parasite to honey bees). RESULTS: Our comparative genomics analysis show that the N. bombycis genome has greatly expanded due to the following three molecular mechanisms: 1) the proliferation of host-derived transposable elements, 2) the acquisition of many horizontally transferred genes from bacteria, and 3) the production of abundnant gene duplications. To our knowledge, duplicated genes derived not only from small-scale events (e.g., tandem duplications) but also from large-scale events (e.g., segmental duplications) have never been seen so abundant in any reported microsporidia genomes. Our relative dating analysis further indicated that these duplication events have arisen recently over very short evolutionary time. Furthermore, several duplicated genes involving in the cytotoxic metabolic pathway were found to undergo positive selection, suggestive of the role of duplicated genes on the adaptive evolution of pathogenic ability. CONCLUSIONS: Genome expansion is rarely considered as the evolutionary outcome acting on those highly reduced and compact parasitic microsporidian genomes. This study, for the first time, demonstrates that the parasitic genomes can expand, instead of shrink, through several common molecular mechanisms such as gene duplication, horizontal gene transfer, and transposable element expansion. We also showed that the duplicated genes can serve as raw materials for evolutionary innovations possibly contributing to the increase of pathologenic ability. Based on our research, we propose that duplicated genes of N. bombycis should be treated as primary targets for treatment designs against pébrine. The genome data and annotation information of N. bombycis and N.antheraeae were submitted to GenBank (Accession numbers ACJZ01000001 -ACJZ01003558).

Preventive Effects of Apple Polyphenol Extract on High-Fat-Diet-Induced Hepatic Steatosis Are Related to the Regulation of Hepatic Lipid Metabolism, Autophagy, and Gut Microbiota in Aged Mice.

Our previous study confirmed that the ameliorated effects of an intervention with an apple polyphenol extract (APE) on hepatic steatosis induced by a high-fat diet (HFD) are dependent on SIRT1. Since SIRT1 expression decreases with age, it remains unclear whether APE intervention is effective against hepatic steatosis in aged mice. Thus, 12-month-old C57BL/6 male mice were fed with an HFD to establish an aging model of hepatic steatosis and treated with 500 mg/(kg·bw·d) APE for 12 weeks. Young mice (two months old) and baseline mice were used as controls to examine the effects of natural aging on hepatic steatosis. Compared with baseline mice, no obvious difference in hepatic histopathological assessment was observed for both young and aged mice on normal diets. Meanwhile, HFD induced much higher nonalcoholic fatty liver disease (NAFLD) activity scores in aged mice than in young mice. APE intervention ameliorated lipid and glucose metabolic disorders and liver injury in HFD-fed aged mice, improved hepatic steatosis, and reduced NAFLD activity scores. The upregulated expressions of SIRT1, HSL, ATG5, Ulk1, and Becn1 and downregulated expressions of HMGCR and FOXO1 suggested improved lipid metabolism and activated autophagy. APE intervention decreased the ratio of Firmicutes/Bacteroidetes and elevated the Akkermansia probiotics abundance. In summary, HFD showed a more significant effect on hepatic steatosis compared to the natural aging process in aged mice, and APE might be a promising dietary ingredient for alleviating hepatic steatosis.

Apple Polyphenol Extract Ameliorates Atherosclerosis and Associated Cognitive Impairment through Alleviating Neuroinflammation by Weakening TLR4 Signaling and NLRP3 Inflammasome in High-Fat/Cholesterol Diet-Fed LDLR-/- Male Mice.

Although studies have supported the beneficial effects of the ingredients of apple polyphenol extract (APE), a polyphenol mixture being extracted from whole fresh apples, on neurodegenerative diseases, the role of APE in atherosclerosis-related cognitive impairment remains unclear. To clarify the role of APE in regulating cognitive dysfunction in mice with atherosclerosis and the underlying mechanisms, high-fat/cholesterol diet-fed male LDLR-/- mice were gavaged with 125 or 500 mg/(kg·bw·d) APE solution or sterile double-distilled water for consecutive 8 weeks, and age-matched C57BL/6 male mice were employed as normal control. APE intervention increased the serum concentration of high-density apolipoprotein cholesterol, improved atherosclerosis, and ameliorated cognitive function of mice by inhibiting the phosphorylation of tau protein, supporting with significantly reduced platform latency and obviously increased swimming distance in the target quadrant according to the Morris water maze test. APE intervention alleviated neuroinflammation by attenuating the activation of microglia and astrocytes and inhibiting TLR4 signaling with reduced protein expression of NF-κB, MyD88, TRIF, and IKKß. Meanwhile, APE intervention inactivated NLRP3 inflammasome with downregulated protein expression of caspase-1, IL-18, and IL-1ß. Additionally, APE intervention improved the damaged brain barrier structure by upregulating the protein expression of ZO-1 and occludin. Therefore, our research supplemented new data, supporting the potential of APE as an effective dietary bioactive ingredient to improve atherosclerosis and associated cognitive impairment.

Molecular Cloning and Expression Analysis of Thyrotropin-Releasing Hormone, and Its Possible Role in Gonadal Differentiation in Rice Field eel Monopterus albus.

Rice field eel (Monopterus albus), a protogynous hermaphrodite fish, is a good model for the research of sex determination and gonadal differentiation in teleosts. In this study, we cloned the full-length cDNA sequence of trh, which encoded a predicted protein with 270 amino acids. Trh mainly expressed in the brain, followed by the ovary, testis, muscle and pituitary, and had low levels in other peripheral tissues. During natural sex reversal, trh mRNA expression levels exhibited a significant increase at the late intersexual stage in the hypothalamus. In the gonad, trh mRNA expression levels showed a trend of increase followed by decrease, and only increased significantly at the middle intersexual stage. No matter static incubation or intraperitoneal (IP) injection, TRH had no significant effect on trh and thyroid-stimulating hormone ßsubunit (tshß) mRNA expression levels, and serum T3, T4 and TRH release. After static incubation of ovarian fragments by TRH, the expression of gonadal soma derived factor (gsdf) was up-regulated significantly at both the doses of 10 and 100 nM. IP injection of TRH stimulated the expression of gsdf, and inhibited the expression of ovarian aromatase gene (cyp19a1a), accompanied by the increase of serum 11-KT levels. The results indicated that TRH may play a novel role in gonadal differentiation by the regulation of gonadal differentiation-related gene expression and sex steroid hormone secretion in rice field eel.

Identification and expression analysis of thyroid-stimulating hormone ß subunit, and effects of T3 on gonadal differentiation-related gene expression in rice field eel, Monopterus albus.

Thyroid-stimulating hormone (TSH) is an important glycoprotein in hypothalamic-pituitary-thyroid axis, which plays a crucial role in the synthesis and release of thyroid hormones in vertebrates. Rice field eel, Monopterus albus, a protogynous hermaphroditic fish, which undergoes sex reversal from a functional female to a male, is an ideal model to investigate the regulation of sex differentiation. In this study, we obtained the cDNA sequence of thyroid-stimulating hormone ß subunit (tshß) from rice field eel, which contained a complete open reading frame and encoded a putative protein of 151 amino acids. Multiple alignment of protein sequences showed that tshß was highly conserved in teleost. The tissue distribution indicated that tshß showed high expression in the pituitary, moderate expression in the brain region, gonad, intestine and liver, and low expression in other peripheral tissues. During natural sex reversal, the expression of tshß had no significant difference in the pituitary. Compared to that in the ovary, the expression of tshß increased significantly in the gonad at late intersexual and male stages. After treatment by different doses of triiodothyronine (T3) (1 µg/g, 10 µg/g and 100 µg/g body weight), serum T3 and free triiodothyronine (FT3) increased sharply, while the expression of tshß were inhibited significantly in the pituitary. Although T3 had no significant effect on the levels of serum E2, it stimulated the release of serum 11-KT at high-dose group. We also detected the effects of T3 on the expression of gonadal differentiation-related genes in rice field eel. T3 treatment inhibited the expression of foxl2, cyp19a1a and dax1, while stimulated the expression of sox9a1. These results indicate that TSH may be involved in sex differentiation, and THs may play roles in the regulation of male development and sex reversal in rice field eel.