TMT-Based Quantitative Proteomics Revealed the Antibacterial Mechanism of Cinnamaldehyde against MRSA.

Natural plant extracts have demonstrated significant potential in alternative antibiotic therapies. Cinnamaldehyde (CA) has garnered considerable attention as a natural antibacterial agent. In this study, Tandem mass tag (TMT) quantitative proteomics combined with Western blot and RT-qPCR methods were employed to explore the antibacterial mechanism of CA against Methicillin-Resistant Staphylococcus aureus (MRSA) at the protein level. The results showed that a total of 254 differentially expressed proteins (DEPs) were identified in the control group and CA treatment group, of which 161 were significantly upregulated and 93 were significantly downregulated. DEPs related to nucleotide synthesis, homeostasis of the internal environment, and protein biosynthesis were significantly upregulated, while DEPs involved in the cell wall, cell membrane, and virulence factors were significantly downregulated. The results of GO and KEGG enrichment analyses demonstrated that CA could exert its antibacterial effects by influencing pyruvate metabolism, the tricarboxylic acid (TCA) cycle, teichoic acid biosynthesis, and the Staphylococcus aureus (S. aureus) infection pathway in MRSA. CA significantly inhibited the expression of recombinant protein MgrA (p < 0.05), significantly reduced the mRNA transcription levels of mgrA, hla, and sdrD genes (p < 0.05), and thermostability migration assays demonstrated that CA can directly interact with MgrA protein, thereby inhibiting its activity. These findings suggest that CA exerts its antibacterial mechanism by regulating the expression of related proteins, providing a theoretical basis for further development of clinical applications of antimicrobial agents derived from natural plant essential oils in the treatment of dairy cow mastitis.

Changes of intestinal barrier in the process of intestinal inflammation induced by Aeromonas hydrophila in snakehead (Channa argus).

Bacterial intestinal inflammation frequently occurs in cultured fish. Nevertheless, research on intestinal barrier dysfunction in the process of intestinal inflammation is deficient. In this study, we explored the changes of intestinal inflammation induced by Aeromonas hydrophila (A. hydrophila) in snakehead and the relationship between intestinal barrier and inflammation. Snakehead [(13.05 ± 2.39) g] were infected via anus with A. hydrophila. Specimens were collected for analysis at 0, 1, 3, 7 and 21 d post-injection. The results showed that with the increase of exposure time, the hindgut underwent stages of normal function, damage, damage deterioration, repair and recovery. Relative to 0 d, the levels of IL-1ß and TNF-α in serum, and the expression of nod1, tlr1, tlr5, nf-κb, tnf-α and il-1ß in intestine were significantly increased, and showed an upward then downward pattern over time. However, the expression of tlr2 and il-10 were markedly decreased, and showed the opposite trend. In addition, with the development of intestinal inflammation, the diversity and richness of species, and the levels of phylum and genus in intestine were obviously altered. The levels of trypsin, LPS, AMS, T-SOD, CAT, GPx, AKP, LZM and C3 in intestine were markedly reduced, and displayed a trend of first decreasing and then rebounding. The ultrastructure observation showed that the microvilli and tight junction structure of intestinal epithelial cells experienced normal function initially, then damage, and finally recovery over time. The expression of claudin-3 and zo-1 in intestine were significantly decreased, and showed a trend of first decreasing and then rebounding. Conversely, the expression of mhc-i, igm, igt and pigr in intestine were markedly increased, and displayed a trend of increasing first and then decreasing. The above results revealed the changes in intestinal barrier during the occurrence and development of intestinal inflammation, which provided a theoretical basis for explaining the relationship between the two.

Transcriptomic Analysis of the Effect of Glabridin on Biofilm Formation in Staphylococcus Aureus.

Staphylococcus aureus (S. aureus) is among the major skin infection-causing pathogens in animals and humans. Its ability to form biofilms has become a foremost cause of bacterial infections and the extensive spread of drug resistance, which poses a great difficulty in clinical treatment. Glabridin (Glb), an extract of licorice with antibacterial and anti-infective properties, has a partially understood biofilm-inhibitory mechanism. This study investigated the inhibitory and antibiofilm activities of subinhibitory concentrations of Glb against S. aureus. The crystal violet assay revealed that Glb significantly suppressed biofilm expression. Scanning electron microscopy observations unveiled that Glb reduced S. aureus adhesion and accumulation by disrupting the spatial structure of the biofilm. In vitro extracellular DNA (eDNA) inhibition assays demonstrated that Glb inhibited biofilm formation by S. aureus by suppressing eDNA secretion. In total, 184 differentially expressed genes were obtained through transcriptomic (RNA-seq) sequencing, of which 81 and 103 genes were upregulated and downregulated, respectively. Glb regulated the transcript levels of biofilm-related genes through the phosphatase transfer system, two-component regulatory system, and nitrogen metabolism. The qPCR analysis was performed to confirm whether Glb interfered with the expression of regulatory genes involved in S. aureus biofilm formation (SarA, ArlR, FnbA, ClfA, icaD, and icaR) as well as the virulence gene Hla. In conclusion, this study demonstrates that Glb has a significant inhibitory effect on biofilm activity and is expected to be a good antibiofilm drug.

Poly (I:C)-Induced microRNA-30b-5p Negatively Regulates the JAK/STAT Signaling Pathway to Mediate the Antiviral Immune Response in Silver Carp (Hypophthalmichthys molitrix) via Targeting CRFB5.

In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune system, with interferons (IFNs) playing a crucial role in mediating the immune response. With advancements in molecular biology, the role of non-coding RNA (ncRNA), particularly microRNAs (miRNAs), in gene expression has gained increasing attention. While the function of miRNAs in regulating the host immune response has been extensively studied, research on their immunomodulatory effects in teleost fish, including silver carp (Hyphthalmichthys molitrix), is limited. Therefore, this research aimed to investigate the immunomodulatory role of microRNA-30b-5p (miR-30b-5p) in the antiviral immune response of silver carp (Hypophthalmichthys molitrix) by targeting cytokine receptor family B5 (CRFB5) via the JAK/STAT signaling pathway. In this study, silver carp were stimulated with polyinosinic-polycytidylic acid (poly (I:C)), resulting in the identification of an up-regulated miRNA (miR-30b-5p). Through a dual luciferase assay, it was demonstrated that CRFB5, a receptor shared by fish type I interferon, is a novel target of miR-30b-5p. Furthermore, it was found that miR-30b-5p can suppress post-transcriptional CRFB5 expression. Importantly, this study revealed for the first time that miR-30b-5p negatively regulates the JAK/STAT signaling pathway, thereby mediating the antiviral immune response in silver carp by targeting CRFB5 and maintaining immune system stability. These findings not only contribute to the understanding of how miRNAs act as negative feedback regulators in teleost fish antiviral immunity but also suggest their potential therapeutic measures to prevent an excessive immune response.

Study on the inhibitory effect of quercetin combined with gentamicin on the formation of Pseudomonas aeruginosa and its bioenvelope.

OBJECTIVE: The potential effects of quercetin and gentamicin combination on the bacteriostatic activity and biofilm formation of Pseudomonas aeruginosa (PA) were examined, and the findings provided a theoretical basis for the development of quercetin as a new biofilm inhibitor. METHODS: The minimum inhibitory concentration (MIC) of eight PAs was determined by microdilution method and the partial inhibitory concentration index (FICI) of the combined drug was analyzed by micro-dilution method. Thereafter, the lowest film inhibitory concentration (MBIC) of quercetin and gentamicin alone and in combination was evaluated by crystal violet staining. Finally, scanning electron microscopy (SEM) and laser confocal microscopy (CLSM) were used to decipher the inhibitory effect of the combination on biofilm formation. OUTCOME: The antibacterial activity of quercetin alone was relatively weak, but after combination with gentamicin, the antibacterial activity was significantly enhanced, as evident by FICI of 0.28 and 0.53 and manifested as synergistic or additive effect, which indicated that quercetin can enhance gentamicin antibacterial activity. The results of crystal violet staining revealed that quercetin and gentamicin alone exhibited a similar biofilm formation inhibitory effect, but the inhibitory effect was substantially weaker, and the antibiofilm activity was stronger and exhibited a dose-dependent response after the combination of the two with 1/2FICI. The results of scanning electron microscopy and laser confocal microscopy also showed that the treatment of PA biofilm after combining quercetin and gentamicin with 1/2FICI could completely destroy the spatial structure of the complete biofilm, significantly reduce the thickness of bacteria, and markedly reduce the proportion of viable bacteria in the membrane. CONCLUSION: The combination of quercetin and gentamicin can effectively inhibit the formation of PA as well as its biofilm, and exhibit synergistic and additive effects.

Characterization of Bacillus spp. isolated from the intestines of Rhynchocypris lagowskii as a potential probiotic and their effects on fish pathogens.

Probiotics sourced from fish intestinal microbiota have a merit over other bacterial sources due to colonization ability and effective time. This study aimed to evaluate the bacilli isolated from the Rhynchocypris lagowskii intestines and their validity as a probiotic. Three isolates were selected (LSG 2-5, LSG 3-7, and LSG 3-8) and defined by morphological and 16S rRNA analysis as Bacillus velezensis, Bacillus aryabhattai, and Bacillus mojavensis, respectively. Results showed the strain tolerant abilities to gastrointestinal fluid, bile salt, pH, and temperature expotures. Additionally, all bacterial strains showed anti-pathogenic activity against at least four strains out of six tested pathogen strains (Staphylococcus aureus, Aeromonas hydrophila, Escherichia coli, Aeromonas veronii, Edwardsiella, and Aeromonas sobria). The bacterial strains also showed a high percentage of co-aggregation activity, more than 70%, with Aer. hydrophile, Staph. epidermidis, and Klebsiella aerogenes. At the same time, the results of competition, rejection, and substitution activity with Aer. hydrophila and Aer. veronii indicated the ability of the isolated strains to reduce the adhesion of pathogens to mucin. All strains showed safety properties, non-hemolytic, and sensitivity characteristics for most of tested antibiotics. In vivo test after injecting these strains into fish at various concentrations showed no side effects in the internal or external organs of fish compared to controls, proving that this is safe for these fish. Furthermore, the three strains produced lipase, amylase, and protease enzymes. The strains also showed bile salt hydrolase activity and biofilm formation, allowing them to tolerate stressful conditions. Conclusion: Based on these strains characteristics and features, they could be considered a promising candidate probiotic and can be used as an anti-pathogenic, especially in aquaculture.

Beneficial effects of indigenous Bacillus spp. on growth, antioxidants, immunity and disease resistance of Rhynchocypris lagowskii.

This study aimed to investigate the effect of Bacillus aryabhattai (LSG3-7) and Bacillus mojavensis (LSG3-8) on growth performance, antioxidant capacity, and immune response in Rhynchocypris lagowskii (Dybowski, 1869), at the trial and challenge periods. A 630 healthy fish (10.76 ± 0.05) were randomly divided into six groups: control group (D1) was fed the basal diet, D2 and D3 were supplemented with LSG 3-7 and LSG3-8 (1 × 108 CFU/g) for both of them, whereas D4 was supplemented with a mixture of both bacteria (0.5 × 108 CFU/g each), and D5 was supplemented with LSG3-7 0.75 × 108 CFU/g + LSG3-8 0.25 × 108 CFU/g, and D6 supplemented with LSG3-7 0.25 × 108 CFU/g + LSG3-8 0.75 × 108 CFU/g. After the trial, Aeromonas hydrophila was used in a challenge test for 14 days. Treatments showed significant differences (p < 0.05) in growth performance and antioxidant capacity (CAT, CuZn-SOD, GPX) in the liver and intestine compared to the control. The antioxidant-related genes CAT, CuZn-SOD, GPX, and Nrf2 in the liver and intestine showed upregulation compared with the control group. Serum IgM, LZM, C3, C4, and AKP showed a favorable superiority (p < 0.05) in treatments (D2 - D6) at the trial and challenge test compared to controls. In parallel, immune-related genes (IgM, NF-κB, TLR-1, TLR-2, and MyD88) showed an up-regulated level (p < 0.05) in treatments (D2 - D6) compared to the control. In addition, pro-inflammatory cytokines (IL-1, TNF-α) showed a downregulated level in treatments (D2 - D6). After the challenge test, the immune-related genes in the liver and muscle showed an up-regulated level in treatments compared to the controls. The survival rate showed a significant increase (p < 0.05) in the treatment groups (D2 - D6) compared to the control. Overall, individuals and the bacterial mixture of B. aryabhattai and B. mojavensis could improve the growth performance, antioxidant capacity, immune capacity, and survival rate of R. lagowskii and prevent side effects of A. hydrophila. However, B. mojavensis showed a slight improvement compared to B. aryabhattai without a significant difference between them.

Effect of preoperative pupil offset on corneal higher-order aberrations after femtosecond laser-assisted in situ keratomileusis.

BACKGROUND: This study aimed to investigate the relationship between multiple higher-order aberrations (HOAs) subgroups and pupil offset, as well as to analyze the factors affecting postoperative corneal HOAs in patients with different degrees of refractive errors. METHODS: We enrolled 160 patients (316 eyes) aged ≥ 18 years who had undergone femtosecond laser-assisted in situ keratomileusis (FS-LASIK) treatment. Based on the relationship between the preoperative pupil offset and the postoperative ΔHOAs, all patients were divided into two groups: group I (pupil offset ≤ 0.20 mm) and group II (pupil offset > 0.20 mm). All of the eyes had low to high myopia with or without astigmatism (manifest refraction spherical equivalent (MRSE) < -10.00 D). Uncorrected distance visual acuity, corrected distance visual acuity, MRSE, pupil offset, central corneal thickness, corneal HOAs, vertical coma (Z3-1), horizontal coma (Z31), spherical aberration (Z40), trefoil 0° (Z33), and trefoil 30° (Z3-3) over a 6 mm diameter central corneal zone diameter were evaluated preoperatively and at 1 and 3 months postoperatively. RESULTS: Our result revealed significant differences in postoperative corneal total root mean square (RMS) HOAs, RMS vertical coma, RMS horizontal coma, RMS spherical aberration, and RMS trefoil 30° between group I and group II. ΔMRSE was found to be an effective factor for ΔRMS HOAs (R2 = 0.383), ΔRMS horizontal coma (R2 = 0.205), and ΔRMS spherical aberration (R2 = 0.397). In group II, multiple linear regression analysis revealed a significant correlation between preoperative pupillary offset and Δtotal RMS HOAs (R2 = 0.461), ΔRMS horizontal coma (R2 = 0.040), and ΔRMS trefoil 30°(R2 = 0.089). The ΔRMS vertical coma effect factor is the Y-component, and the factor influencing ΔRMS spherical aberration was ΔMRSE (R2 = 0.256). CONCLUSION: A small pupil offset was associated with a lower induction of postoperative corneal HOAs. Efforts to optimize centration are critical for improving surgical outcomes in patients with FS-LASIK.

Effect of Bacillus amyloliquefaciens LSG2-8 on the intestinal barrier function of Amur minnow (Rhynchocypris lagowskii).

Probiotics are widely used in aquaculture. This article aims to study the effect of Bacillus amyloliquefaciens LSG2-8 on the intestinal barrier function of Rhynchocypris lagowskii. B. amyloliquefaciens LSG2-8 were added to R. lagowskii basal diets (CK) as additives at four concentrations: 1.0 × 106 (D-6), 1.0 × 107 (D-7), 1.0 × 108 (D-8) and 1.0 × 109 (D-9) CFU g-1 by dry weight of basal diet. After a 56-day feeding experiment, the activities of intestinal digestive enzymes and immunity-related enzymes of R. lagowskii on group D-6, D-7, D-8 and D-9 diet were significantly higher than the control (P < 0.05). In molecular experiments, the authors found that the levels of TGF-ß mRNA, IL-10 mRNA, ZO-1 mRNA and claudin-3 mRNA in group D-8 R. lagowskii were significantly higher (P < 0.05) than those of the control and other groups. Furthermore, the levels of IL-1ß and IL-8 mRNA of R. lagowskii on group D-6, D-7, D-8 and D-9 diet were significantly lower than those of the control (P < 0.05). In addition, the authors found that B. amyloliquefaciens LSG2-8 can regulate the intestinal flora balance and improve the intestinal structure of R. lagowskii. In conclusion, B. amyloliquefaciens LSG2-8 can improve the intestinal barrier function of R. lagowskii and can be used as a feed additive in aquaculture.

Research progress on the association between mastitis and gastrointestinal microbes in dairy cows and the effect of probiotics.

Mastitis in dairy cows affects milk quality and thereby constrains the development of the dairy industry. A clear understanding of the pathogenesis of mastitis can help its treatment. Mastitis is caused by the invasion of pathogenic bacteria into the mammary gland through the mammary ducts. However, recent studies suggested that an endogenous entero-mammary pathway in dairy cattle might also be playing an important role in regulating mastitis. Also, probiotic intervention regulating host gut microbes has become an interesting tool to control mastitis. This review discusses the association of gastrointestinal microbes with mastitis and the mechanism of action of probiotics in dairy cows to provide new ideas for the management of mastitis in large-scale dairy farms.

Analysis of virulence genes, drug resistance detection, and pathogenicity in Enterococcus from farm animals.

This study aimed to investigate the presence of eight virulence genes (ace, asa1, esp, efaA, gelE, cylA, agg, fsr) in Enterococcus from a variety of animals and to explore the drug resistance and pathogenicity. This could provide a theoretical basis for clinical treatment of Enterococcus infections. Anal swabs from pigs, chickens, cattle, and dogs in farms and pet hospitals were collected for Enterococcus isolation and identification. Eight virulence genes were detected (PCR method), and drug resistance was assessed (drug-sensitive paper method). The strains containing different virulence genes were then divided into EV1, EV2, and EV3 groups. The LD50 and pathogenicity was examined by intra-peritoneal injection to infect mice. Differences were found in the detection rates of virulence genes in Enterococcus from the different animals. The highest overall detection rate was for the esp gene (78.0%), and the lowest for the cylA gene (15.5%). Eight genes were detected most frequently in Enterococcus from dogs and least frequently from cattle. Among the Enterococcus strains from four variety of animals, drug resistance was highest against sulfamethoxazole (100%), cefotaxime (>97%), and cefotaxitin (>93%). Drug resistance was lowest against vancomycin (0%), levofloxacin (<12%) and ciprofloxacin (<13%). The LD50 for each of the three groups was EV1LD50=8.71×109CFU， EV2LD50=2.34×1010CFU，and EV3LD50=9.33×1010CFU. The Enterococcus12LD50 dose group caused significant clinical symptoms in mice, with pathological effects on the heart, liver, lungs, and kidneys, and particularly on the urinary system. The abundance of Enterococcus virulence genes, drug resistance, and pathogenicity vary among different animal origins, and the pathology caused by Enterococcus requires effective treatment protocols based on species and regional characteristics.

Inhibitory effect of probiotic Bacillus spp. isolated from the digestive tract of Rhynchocypris Lagowskii on the adhesion of common pathogenic bacteria in the intestinal model.

Diseases of fish caused by pathogenic bacteria are an important constraint on aquaculture production. Antibiotics have been widely used to control infectious diseases, but this has led to the emergence of drug-resistant bacteria and affected human health. In this context, probiotics are used as an alternative to antibiotics for the prevention and control of diseases in aquaculture. The aim of this study was to obtain probiotic candidate strains of Bacillus spp. from the gut of Rhynchocypris Lagowskii. Strains were screened by enzyme-producing ability, antagonism assay and antibiotic susceptibility. The safety of the strains to host fish has also been established. The isolated Bacillus licheniformis (LSG1-1) and Bacillus subtilis (LSG2-1) were characterized and performed well in tolerance experiments. In addition, LSG1-1 and LSG2-1 were detected to have higher self-aggregation ability and surface hydrophobicity. In the in vitro adhesion model, LSG1-1 and LSG2-1 showed good adhesion ability and had obvious adhesion inhibitory effect on three pathogens of Aeromonas. Based on the characteristics observed so far, Bacillus licheniformis LSG1-1 and Bacillus subtilis LSG2-1 could form potential probiotic candidates in the digestive tract of R. lagowskii to help combat diseases in aquaculture.

Dietary α-lipoic acid alleviates deltamethrin-induced immunosuppression and oxidative stress in northern snakehead (Channa argus) via Nrf2/NF-κB signaling pathway.

The goal of the study was to determine the ameliorative effects of dietary alpha-lipoic acid (α-LA) on deltamethrin (DEL)-induced immunosuppression and oxidative stress in northern snakehead (Channa argus). The northern snakeheads (15.38 ± 0.09 g) were exposed to DEL (0.242 µg/L) and fed with diets supplemented α-LA at 300, 600, and 900 mg/kg. After the 28-day exposure test, we obtained the following results: i) α-LA alleviates DEL-induced liver injury by reversing the increase of the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and liver cytochrome P450 enzymes (Cytochrome P450 (cyp)1a and cyp1b) expression levels. ii) α-LA can reverse the DEL-induced reduction of serum complement 4 (C4), C3, immunoglobulin M (IgM), and lysozyme (LYS) levels and the increase of liver and intestine nuclear factor kappa B (nf-κb) p65, tumor necrosis factor (tnf)-α, interleukin (il)-1ß, il-8, and il-6 gene expressions, while il-10 expression levels showed the opposite result. iii) α-LA reversed the reduction of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione-S-transferase (GST) and glutathione peroxidase (GSH-Px) levels in the liver and intestine induced by DEL, while malondialdehyde (MDA) showed the opposite result. iv) α-LA reversed the reduction of Cu/Zn sod, nuclear factor erythroid 2-related factor 2 (nrf2), NAD (P)H: quinone oxidoreductase (nqo)1, and heme oxygenase (ho)-1 antioxidant gene expression levels in the liver and intestine induced by DEL. Therefore, our study indicated that optimal α-LA (600 mg/kg) could attenuate DEL-induced toxicity (including liver damage, immunotoxicity, and oxidative stress) in northern snakehead via Nrf2/NF-κB signaling pathway. This is the first research that explores the alleviated effects of α-LA on DEL-induced toxicity damage in fish. This study provides a positive measure to reduce the toxicity damage caused by DEL to aquatic animals, and provides a theoretical basis for exploring the regulation mechanism of α-LA in toxic substances.

The effects of dietary supplementation of α-lipoic acid on the growth performance, antioxidant capacity, immune response, and disease resistance of northern snakehead, Channa argus.

The study was the first time to explore the positive effects of α-LA on growth performance, antioxidant capability, immunity, and disease resistance of northern snakehead (Channa argus). Five hundred and forty northern snakehead fish (initial body weight: 8.74 ± 0.12 g (mean ± SE)) were randomly allocated into six groups with three replicates each. Six diets supplemented with α-LA at doses of 0 (CON), 300 (LA300), 600 (LA600), 900 (LA900), 1200 (LA1200), and 1500 (LA1500) mg/kg were fed to northern snakehead for 8 weeks. The results demonstrated that, when compared with the control group, optimal dietary α-LA increased the weight gain (WG), protein efficiency ratio (PER), and specific growth rate (SGR) and reduced the feed conversion ratio (FCR) of the fish (P < 0.05). Also, optimal dietary α-LA enhanced the immune-related parameters and antioxidant enzyme parameters levels in the head kidney, spleen, and liver of northern snakehead (P < 0.05). Dietary α-LA upregulated the mRNA expression levels of anti-inflammatory cytokines (il10 and tgfß) and antioxidant related genes (gst, gsh-px, gr and Cu/Zn sod), down-regulated the pro-inflammatory cytokines (il1ß, il8, il12 and tnfα) mRNA levels in the liver, spleen and head kidney of the northern snakehead (P < 0.05). The above results demonstrated that optimal dietary α-LA showed enhancement effects on the growth, antioxidant and anti-inflammatory capability, and immune response of northern snakehead. The survival rates in all α-LA treatments were significantly raised after the challenge with Aeromonas veronii (P < 0.05). Based on the quadratic regression analysis of WG, GSH-Px, LYS, and il1ß, the optimal dietary α-LA levels were estimated to be 737.0, 775.0, 890.0, and 916.7 mg/kg, respectively. Considering the overall responses in growth performance, antioxidant status, immune response, and inflammatory factors, the recommended dose of α-LA in the diet of fish is 737.0-916.7 mg/kg.

The effect of adding Bacillus amyloliquefaciens LSG2-8 in diets on the growth, immune function, antioxidant capacity, and disease resistance of Rhynchocypris lagowskii.

The purpose of this study was to investigate the effect of Bacillus amyloliquefaciens LSG2-8 on the growth performance, immune function, antioxidant capacity, and disease resistance of Rhynchocypris lagowskii. Fish were fed with the feed containing five levels such as 0, 1.0 × 106, 1.0 × 107, 1.0 × 108, and 1.0 × 109 CFU/g of the B. amyloliquefaciens LSG2-8 for 56 days. After 56 days of feeding, twenty four fish were randomly selected to test various growth, immune and antioxidant parameters. Ten fish were challenged with Aeromonas hydrophila for 14 days; the mortality rate was recorded 14 days after infection. The results showed that B. amyloliquefaciens LSG2-8 could significantly increase the growth parameters of R. lagowskii's, such as final body weight, weight gain rate, specific growth rate, and feed efficiency (p < 0.05). Further examination revealed the activity of antioxidant enzymes, Nrf-2 mRNA, and Keap-1 mRNA gene expression in the intestine and liver, and the serum immune index of R. lagowskii in the 1.0 × 108 CFU/g were all significantly higher compared to the other groups. Furthermore, fish fed a diet supplemented with B. amyloliquefaciens LSG2-8 had a significantly lower (p < 0.05) post-challenge mortality rate than the control fish. In summary, the research results showed that B. amyloliquefaciens LSG2-8 could improve the growth performance, immune function, antioxidant capacity, and disease resistance of R. lagowskii and be used in aquaculture.

The immuneoreaction and antioxidant status of Chinese mitten crab (Eriocheir sinensis) involve protein metabolism and the response of mTOR signaling pathway to dietary methionine levels.

To study the effects of dietary methionine on growth performance, immunity, antioxidant capacity, protein metabolism, inflammatory response and apoptosis factors in Chinese mitten crabs (Eriocheir sinensis). Five diets with different methionine levels (0.63%, 0.85%, 1.06%, 1.25% and 1.47%) were fed to E. sinensis for 8 weeks. Results showed that in the 1.25% Met group, both growth performance and feed utilization were significantly increased. The crude protein content of crab muscle in the 1.06% and 1.25% Met groups was significantly higher than that in the control group. The immune and antioxidant enzyme activities, as well as gene expression levels of anti-lipopolysaccharide factor 1 (ALF1), Crustin-1, prophenoloxidase (proPO), cap 'n' collar isoform C (CncC) in 1.25% Met group were significantly higher than other groups. The activities of adenosine deaminase (ADA) and glutamate transaminase (GPT) in serum decreased first and then increased with the increase of methionine content, while the changes of ADA and GPT in hepatopancreas increased first and then decreased. 1.25% Met group exhibited significantly increased levels of GOT, GPT, and ADA compared to the control group. 1.25% Met diet group significantly up-regulated protein synthesis and anti-apoptotic factors, and significantly down-regulated inflammatory and pro-apoptotic factors in hepatopancreas. At 1.25% in the diet, methionine was found to boost E. sinensis growth, muscle protein deposition and immunity, as well as its antioxidant capacity. Combined with the above results, based on the expression of factors involved in the mammalian target of rapamycin (mTOR) signaling pathway and the mitogen-activated protein kinase (MAPK) signaling pathway, it is proved that methionine can not only promote protein metabolism, improve feed utilization, but also alleviate the inflammatory response and apoptosis caused by oxidative stress in the body.

Dietary supplementation of two indigenous Bacillus spp on the intestinal morphology, intestinal immune barrier and intestinal microbial diversity of Rhynchocypris lagowskii.

This study evaluated the effects of dietary administration of two indigenous Bacillus (A: basal control diet; B: 0.15 g/kg of Bacillus subtilis; C: 0.1 g/kg of Bacillus subtilis and 0.05 g/kg of Bacillus licheniformis; D: 0.05 g/kg of Bacillus subtilis and 0.1 g/kg of Bacillus licheniformis; E: 0.15 g/kg of Bacillus licheniformis) on the digestive enzyme activities, intestinal morphology, intestinal immune and barrier-related genes relative expression levels, and intestinal flora of Rhynchocypris lagowskii. The results showed that the fold height, lamina propria width, and muscle layer thickness of midgut and hindgut in group C were significantly higher than that of group A (P < 0.05). The activities of protease, amylase, and lipase in group C were significantly higher than those of group A (P < 0.05). The relative expression levels of IL-1ß and IL-8 in the intestine of group C were significantly downregulated, and the relative expression levels of IL-10 and TGF-ß were significantly upregulated (P < 0.05). The relative expression levels of Claudin-2 in group A significantly increased and the relative expression levels of Claudin-4 in group A significantly reduced compared with other groups (P < 0.05). The relative expression levels of ZO-1 in groups C and D were significantly higher than those of other groups (P < 0.05). The Bacillus in the intestine of group C has the highest relative abundance among all groups. Overall, it can generally be concluded that dietary supplementation of indigenous Bacillus subtilis and Bacillus licheniformis (group C) can improve the intestinal morphology, digestion, and absorption enzyme activities, enhance intestinal mucosal immunity and barrier function, and maintain the intestinal microbial balance of R. lagowskii.

Changes in asphericity of anterior and posterior corneal surfaces for mild-moderate and high myopia after topography-guided FS-LASIK.

PURPOSE: To compare changes in asphericity of anterior and posterior corneal surfaces for different myopia patients after corneal topography-guided femtosecond-assisted laser in situ keratomileuses (FS-LASIK), and to analyze correlations between asphericity of corneal surfaces and preoperative spherical equivalence (SEQ). METHODS: In this prospective study, 59 patients who underwent corneal topography-guided FS-LASIK surgery were enrolled and divided into the mild-moderate myopia group (67 eyes) and the high myopia group (44 eyes). Postoperative follow-ups were performed at 1, 3, and 6 months. Postoperative changes in aspherical coefficient (Q values), corneal higher-order aberrations (HOAs), and spherical aberrations (Z40) were compared between the two groups. Relevance between Q value changes and SEQ, HOAs, and Z40 as well as between SEQ and changes of HOAs and Z40 was analyzed. RESULTS: There was a significant increase in Q values of the anterior (each diameter) and posterior (6-8 mm) corneal surface in both groups than before surgery (P < 0.001). Q values of corneal anterior (each diameter) and posterior (7-9 mm) surface in the high group were considerably larger than the mild-moderate group (P < 0.05). Corneal anterior surface HOAs and Z40 values in the high group largely exceeded those of the mild-moderate group (P < 0.001). The preoperative SEQ was linearly correlated with postoperative anterior Q change (ΔQ), HOAs change (ΔHOAs), and spherical aberration change (ΔZ40). CONCLUSION: The changes of corneal asphericity in patients with high myopia were greater than mild-moderate myopia, with more corneal HOAs and Z40 introduced when corneal topography-guided FS-LASIK was conducted.

Disruption of SIRT7 Increases the Efficacy of Checkpoint Inhibitor via MEF2D Regulation of Programmed Cell Death 1 Ligand 1 in Hepatocellular Carcinoma Cells.

BACKGROUND & AIMS: Immune checkpoint inhibitors have some efficacy in the treatment of hepatocellular carcinoma (HCC). Programmed cell death 1 ligand 1 (PD-L1), expressed on some cancer cells, binds to the receptor programmed cell death 1 (PDCD1, also called PD1) on T cells to prevent their proliferation and reduce the antigen-tumor immune response. Immune cells that infiltrate some types of HCCs secrete interferon gamma (IFNG). Some HCC cells express myocyte enhancer factor 2D (MEF2D), which has been associated with shorter survival times of patients. We studied whether HCC cell expression of MEF2D regulates expression of PD-L1 in response to IFNG. METHODS: We analyzed immune cells from 20 fresh HCC tissues by flow cytometry. We analyzed 225 fixed HCC tissues (from 2 cohorts) from patients in China by immunohistochemistry and obtained survival data. We created mice with liver-specific knockout of MEF2D (MEF2DLPC-KO mice). We knocked out or knocked down MEF2D, E1A binding protein p300 (p300), or sirtuin 7 (SIRT7) in SMMC-7721, Huh7, H22, and Hepa1-6 HCC cell lines, some incubated with IFNG. We analyzed liver tissues from mice and cell lines by RNA sequencing, immunoblot, dual luciferase reporter, and chromatin precipitation assays. MEF2D protein acetylation and proteins that interact with MEF2D were identified by coimmunoprecipitation and pull-down assays. H22 cells, with MEF2D knockout or without (controls), were transplanted into BALB/c mice, and some mice were given antibodies to deplete T cells. Mice bearing orthotopic tumors grown from HCC cells, with or without knockout of SIRT7, were given injections of an antibody against PD1. Growth of tumors was measured, and tumors were analyzed by immunohistochemistry and flow cytometry. RESULTS: In human HCC specimens, we found an inverse correlation between level of MEF2D and numbers of CD4+ and CD8+ T cells; level of MEF2D correlated with percentages of PD1-positive or TIM3-positive CD8+ T cells. Knockout of MEF2D from H22 cells reduced their growth as allograft tumors in immune-competent mice but not in immune-deficient mice or mice with depletion of CD8+ T cells. When MEF2D-knockout cells were injected into immune-competent mice, they formed smaller tumors that had increased infiltration and activation of T cells compared with control HCC cells. In human and mouse HCC cells, MEF2D knockdown or knockout reduced expression of PD-L1. MEF2D bound the promoter region of the CD274 gene (encodes PD-L1) and activated its transcription. Overexpression of p300 in HCC cells, or knockout of SIRT7, promoted acetylation of MEF2D and increased its binding, along with acetylated histones, to the promoter region of CD274. Exposure of HCC cells to IFNG induced expression of p300 and its binding MEF2D, which reduced the interaction between MEF2D and SIRT7. MEF2D-induced expression of PD-L1 upon IFNG exposure was independent of interferon-regulatory factors 1 or 9. In HCC cells not exposed to IFNG, SIRT7 formed a complex with MEF2D that attenuated expression of PD-L1. Knockout of SIRT7 reduced proliferation of HCC cells and growth of tumors in immune-deficient mice. Compared with allograft tumors grown from control HCC cells, in immune-competent mice, tumors grown from SIRT7-knockout HCC cells expressed higher levels of PD-L1 and had reduced infiltration and activation of T cells. In immune-competent mice given antibodies to PD1, allograft tumors grew more slowly from SIRT7-knockout HCC cells than from control HCC cells. CONCLUSIONS: Expression of MEF2D by HCC cells increases their expression of PD-L1, which prevents CD8+ T-cell-mediated antitumor immunity. When HCC cells are exposed to IFNG, p300 acetylates MEF2D, causing it to bind the CD274 gene promoter and up-regulate PD-L1 expression. In addition to promoting HCC cell proliferation, SIRT7 reduced acetylation of MEF2D and expression of PD-L1 in HCC cells not exposed to IFNG. Strategies to manipulate this pathway might increase the efficacy of immune therapies for HCC.

Dietary α-lipoic acid can alleviate the bioaccumulation, oxidative stress, cell apoptosis, and inflammation induced by lead (Pb) in Channa argus.

This study aims to evaluate the effects of dietary α-lipoic acid (α-LA) on bioaccumulation, oxidative stress, apoptosis, and inflammation in Channa argus after 28 d of lead (Pb) exposure. A total of 300 fish were divided into five groups: the first group was the control group and the other four groups were exposed to waterborne Pb (800 ppb) and fed α-LA diets supplemented with 0, 300, 600, and 900 mg/kg. The results demonstrated that dietary α-LA effectively reduced the Pb accumulation in the liver, kidney, gill, intestine, and muscle of C. argus after exposure to Pb. Meanwhile, dietary α-LA reversed alterations in the biochemical parameters (Alanine aminotransferase (ALT), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), blood urea nitrogen (BUN), cortisol (COR), and creatinine (CRE)) and immunity parameters (myeloperoxidase (MPO), complement 3 (C3), lysozyme (LYS), complement 4 (C4), C-reactive protein (CRP), and immunoglobulin M (IgM)) in the serum of fish caused by Pb. Pb-induced reduction of antioxidant enzyme activities (Catalase (CAT), glutathione reductase (GR), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px)) was inhibited by dietary α-LA. And malondialdehyde (MDA) and protein carbonyl (PC) content exhibited an opposite trend. Meanwhile, dietary supplemented with α-LA was found to relieve Pb-induced oxidative stress by downregulating Keap1 mRNA expression levels and upregulating the expression levels of CAT, nuclear factor erythroid 2-related factor 2 (Nrf2), GSH-Px, and Cu/Zn SOD. Furthermore, α-LA supplementation reversed Pb-induced upregulation of pro-inflammatory genes (interleukin (IL)-6, IL-1ß, tumor necrosis factor α (TNF-α), and nuclear factor kappa B (NF-κB)), Pro-apoptotic genes (Bcl-2-associated X (Bax), caspase (Cas)-3, and tumor protein p53 (p53)) and Hsp70, and downregulation of anti-inflammatory genes (IL-10, inhibitor of κBα (IκBα), and transforming growth factor ß (TGF-ß)) and anti-apoptosis gene (B-cell lymphoma-2 (Bcl-2)). Overall, dietary α-LA supplementation could enhance the innate immunity and antioxidant capacity of fish, attenuating the Pb accumulation, and cell apoptosis after being exposed to Pb. Furthermore, dietary α-LA could relieve Pb-induced inflammatory response and oxidative stress of fish via regulating NF-κB and Nrf2 signaling, respectively.