Exploration of the molecular characteristics of the tumor-immune interaction and the development of an individualized immune prognostic signature for neuroblastoma.

Neuroblastoma (NBL) exists in a complex tumor-immune microenvironment. Immune cell infiltration and tumor-immune molecules play a critical role in tumor development and significantly impact the prognosis of patients. However, the molecular characteristics describing the NBL-immune interaction and their prognostic potential have yet to be investigated systematically. We first employed multiple machine learning algorithms, such as Gene Sets Enrichment Analysis and cell type identification by estimating relative subsets of RNA transcripts, to identify immunophenotypes and immunological characteristics in NBL patient data from public databases and then investigated the prognostic potential and regulatory networks of identified immune-related genes involved in the NBL-immune interaction. The immunity signature combining nine immunity genes was confirmed as more effective for individual risk stratification and survival outcome prediction in NBL patients than common clinical characteristics (area under the curve [AUC] = 0.819, C-index = 0.718, p < .001). A mechanistic exploration revealed the regulatory network of molecules involved in the NBL-immune interaction. These immune molecules were also discovered to possess a significant correlation with plasma cell infiltration, MYCN status, and the level of chemokines and macrophage-related molecules (p < .001). A nomogram was constructed based on the immune signature and clinical characteristics, which showed high potential for prognosis prediction (AUC = 0.856, C-index = 0.755, p < .001). We systematically elucidated the complex regulatory mechanisms and characteristics of the molecules involved in the NBL-immune interaction and their prognostic potential, which may have important implications for further understanding the molecular mechanism of the NBL-immune interaction and identifying high-risk NBL patients to guide clinical treatment.

Differential virulence of infectious hematopoietic necrosis virus (IHNV) isolated from salmonid fish in Gangwon Province, Korea.

The present study investigated the virulence and expression of innate immunity genes in isolates of infectious hematopoietic necrosis virus (IHNV) in Gangwon province, South Korea, by challenging rainbow trout, Atlantic salmon, and coho salmon. Eight IHNV isolates were used to infect RTG-2 cells for viral replication using plaque assays. Three isolates with the highest replication rates, the RtPc0314g and RtPc0314c isolates of the JRt-Shizuoka type and the RtPc0816g isolate of the JRt-Nagano type, were experimentally infected into the fish. In rainbow trout, both RtPc0314c and RtPc0314g isolates showed 100% cumulative mortality while the RtPc0816g isolate showed 60% cumulative mortality for 14 days. In contrast, all three isolates showed <60% cumulative mortality in Atlantic salmon and coho salmon. The expression of G genes in the kidney was higher than that in the spleen-infected fish, with the highest expression observed in the kidneys of rainbow trout. The relative expression levels of innate immunity genes were higher in rainbow trout than in Atlantic salmon and coho salmon. The expression level of immunoglobulin M increased until day 7, and the expression of type I interferon was higher in the spleen than in other tissues. The expression of Mx-1 was higher in the kidney and liver than other tissues. These results indicate that IHNV isolates from Gangwon province show host-specific virulence in rainbow trout and that their virulence and replication were higher in JRt-Shizuoka type than in JRt-Nagano type isolates.

Effects of graded levels of starch on the non-specific immune responses, antioxidant capacities and intestinal health in Chinese mitten crab, Eriocheir inensis.

A 9-week feeding trial was conducted to investigate the effects of graded levels of dietary starch (12%, 17%, 22%, 27% and 32%) on growth, non-specific immune responses, antioxidant capacities, immunity genes expression levels and pathogen resistance in Chinese mitten crab, Eriocheir inensis (initial body weight: 10.5 ± 0.5 g). Results showed that the highest weight gain rate of crabs was obtained in group containing 22% dietary starch. The highest activity of acid phosphatase, phenoloxidase and lysozyme in blood was found in crabs fed with 22-27% dietary starch. Additionally, 17%-27% dietary starch significantly increased the activities of superoxide dismutase and glutathione peroxidase, reduced malondinaldehyde content and then increased the total antioxidant capacities in hepatopancreas of crabs. The highest activity of alanine aminotransferase and aspartate aminotransferase was found in crabs fed with 32% dietary starch, indicating that excess starch had a negative effect on the liver function of crabs. With the dietary starch level increased, the transcription factors gene expression of the pro-inflammatory factors were significantly up-regulated, and the highest ILF2, IL-16, Relish and ADAM10 was found in crabs fed with dietary 32% starch, which may potentially promote the inflammatory response in intestines. Moreover, with the dietary starch increased, the activity of phenoloxidase and lysozyme, as well as the gene expression of crustin, were all increased in crabs after challenge against Citrobacter freundii, which demonstrated that additional dietary starch could provide immune-protection and help crabs improve their resistance against pathogens. In conclusion, these results suggest that adequate dietary starch can increase growth, enhance innate immune responses and promote disease resistance, reduce oxidative stress and inflammatory response in E. inensis. Taken together, 22-27% dietary starch (25.9-30.8% dietary carbohydrate) was suggested as a digestible energy source in crabs feed.

GDF10 and IDO1 as a thyroid cancer prognostic biomarker associated with immune infiltration.

Objection: The aim of this work is to screen the immune-related genes to predict the prognosis and provide a new direction of treatment for patients with thyroid cancer (THCA). Methods: The mRNA and clinical features of THCA patients were collected from the Cancer Genome Atlas (TCGA) databases. The immune-related genes were obtained from the ImmPort databases. The bio-information methods were performed to screen the differential expression genes (DEGs) and genes related to immunity between the THCA patients and normal individuals. On this basis, the hub prognosis immunity genes were screened by Veen. The related genes were obtained by constructing the protein-protein interaction network. The enrichment analyses were performed based on the protein and protein interaction (PPI) related genes. The hub immune checkpoint was screened by correlation analysis. Finally, the hub gene and the immunity checkpoint-miRNA (or transcription factor, drug) interaction network were constructed. A drug-sensitive analysis also was performed. Results: The GDF10 was screened. The PPI genes were enriched in the TGF-beta signaling pathway, signaling pathways regulating, the pluripotency of stem cells, Cytokine-cytokine receptor interaction, and so on. The hub immunity checkpoint IDO1 was obtained. The joint indicator of two hub genes was positively related to the thyroid differentiation score. Three interaction factors were found to be related to the two hub genes, and 7 kinds of drugs screened act on the two hub genes at the same time. Conclusion: This work indicated that immune-related gene GDF10 and immune checkpoint IDO1 are important for the prognosis prediction of THCA patients, and immunity is involved in the proliferation, and differentiation of tumor cells.

Characterization of Prophages in Leuconostoc Derived from Kimchi and Genomic Analysis of the Induced Prophage in Leuconostoc lactis.

Leuconostoc has been used as a principal starter in natural kimchi fermentation, but limited research has been conducted on its phages. In this study, prophage distribution and characterization in kimchi-derived Leuconostoc strains were investigated, and phage induction was performed. Except for one strain, 16 Leuconostoc strains had at least one prophage region with questionable and incomplete regions, which comprised 0.5-6.0% of the bacterial genome. Based on major capsid protein analysis, ten intact prophages and an induced incomplete prophage of Leu. lactis CBA3626 belonged to the Siphoviridae family and were similar to Lc-Nu-like, sha1-like, phiMH1-like, and TPA_asm groups. Bacterial immunology genes, such as superinfection exclusion proteins and methylase, were found on several prophages. One prophage of Leu. lactis CBA3626 was induced using mitomycin C and was confirmed as belonging to the Siphoviridae family. Homology of the induced prophage with 21 reported prophages was not high (< 4%), and 47% identity was confirmed only with TPA_asm from Siphoviridae sp. isolate ct3pk4. Therefore, it is suggested that Leuconostoc from kimchi had diverse prophages with less than 6% genome proportion and some immunological genes. Interestingly, the induced prophage was very different from the reported prophages of other Leuconostoc species.

Homologous recombination between tandem paralogues drives evolution of a subset of type VII secretion system immunity genes in firmicute bacteria.

The type VII secretion system (T7SS) is found in many Gram-positive firmicutes and secretes protein toxins that mediate bacterial antagonism. Two T7SS toxins have been identified in Staphylococcus aureus, EsaD a nuclease toxin that is counteracted by the EsaG immunity protein, and TspA, which has membrane depolarising activity and is neutralised by TsaI. Both toxins are polymorphic, and strings of non-identical esaG and tsaI immunity genes are encoded in all S. aureus strains. To investigate the evolution of esaG repertoires, we analysed the sequences of the tandem esaG genes and their encoded proteins. We identified three blocks of high sequence similarity shared by all esaG genes and identified evidence of extensive recombination events between esaG paralogues facilitated through these conserved sequence blocks. Recombination between these blocks accounts for loss and expansion of esaG genes in S. aureus genomes and we identified evidence of such events during evolution of strains in clonal complex 8. TipC, an immunity protein for the TelC lipid II phosphatase toxin secreted by the streptococcal T7SS, is also encoded by multiple gene paralogues. Two blocks of high sequence similarity locate to the 5' and 3' end of tipC genes, and we found strong evidence for recombination between tipC paralogues encoded by Streptococcus mitis BCC08. By contrast, we found only a single homology block across tsaI genes, and little evidence for intergenic recombination within this gene family. We conclude that homologous recombination is one of the drivers for the evolution of T7SS immunity gene clusters.

Identification of an Immune-Related Risk Signature Correlates With Immunophenotype and Predicts Anti-PD-L1 Efficacy of Urothelial Cancer.

Immune checkpoint inhibitor (ICI) treatment has been used to treat advanced urothelial cancer. Molecular markers might improve risk stratification and prediction of ICI benefit for urothelial cancer patients. We analyzed 406 cases of bladder urothelial cancer from The Cancer Genome Atlas (TCGA) data set and identified 161 messenger RNAs (mRNAs) as differentially expressed immunity genes (DEIGs). Using the LASSO Cox regression model, an eight-mRNA-based risk signature was built. We validated the prognostic and predictive accuracy of this immune-related risk signature in 348 metastatic urothelial cancer (mUC) samples treated with anti-PD-L1 (atezolizumab) from IMvigor210. We built an immune-related risk signature based on the eight mRNAs: ANXA1, IL22, IL9R, KLRK1, LRP1, NRG3, SEMA6D, and STAP2. The eight-mRNA-based risk signature successfully categorizes patients into high-risk and low-risk groups. Overall survival was significantly different between these groups, regardless if the initial TCGA training set, the internal TCGA testing set, all TCGA set, or the ICI treatment set. The hazard ratio (HR) of the high-risk group to the low-risk group was 3.65 (p < 0.0001), 2.56 (p < 0.0001), 3.36 (p < 0.0001), and 2.42 (p = 0.0009). The risk signature was an independent prognostic factor for prediction survival. Moreover, the risk signature was related to immunity characteristics. In different tumor mutational burden (TMB) subgroups, it successfully categorizes patients into high-risk and low-risk groups, with significant differences of clinical outcome. Our eight-mRNA-based risk signature is a stable biomarker for urothelial cancer and might be able to predict which patients benefit from ICI treatment. It might play a role in precision individualized immunotherapy.

Effects of lysine and methionine in a low crude protein diet on the growth performance and gene expression of immunity genes in broilers.

Globally, the poultry industry is 1 of the most advanced livestock industries. Feed contributes to the biggest proportion (65-70%) of the production cost. Most feed ingredients in Malaysia are imported, which contributes to the high food bill annually, and alternative feed formulation may help decrease the cost of poultry feed. Feed formulation are improved to efficiently meet the dietary requirements of the broilers and 1 of the ways is by reducing the level of crude protein in the diet while supplementing essential amino acids. In this study, the effects of methionine and lysine, which are the 2 most limiting amino acids in the chicken diet, were supplemented in a low crude protein diet, and its effects on the growth and expression of immunity genes such as MUC2, SLC, GAL6, and LEAP-2 were studied. A total of 300 Cobb500 broilers were tested with 10 different dietary treatments. Experimental treatment diets consist of high, standard, and low levels of methionine and lysine in the diet with reduced crude protein. The control group consists of diet with standard levels of lysine, methionine, and crude protein as recommended for Cobb500 broilers. Ribonucleic acid was extracted from the jejunum, spleen, and liver for gene expression analysis which was performed with real-time polymerase chain reaction using SYBR Green chemistry. Results of the growth performance at 6 wk showed improved feed conversion ratio when lysine was increased by 0.2% in a low crude protein diet at 1.96 ± 0.11. Gene expression of MUC2 gene in the jejunum showed a significant increase across all experimental diets with the treatment with higher lysine in low crude protein diet with the highest increase of 3.8 times as compared with the control diet. The other genes expressed in the spleen and liver were mostly downregulated. It was concluded that supplementation of high lysine with standard methionine in a low crude protein diet performed better in terms of lowest feed conversion ratio and high upregulation of MUC2 gene.

The potential role of nano-selenium and vitamin C on the performances of Nile tilapia (Oreochromis niloticus).

Functional trace elements and vitamins can boost immunity and antioxidative response in aquatic animals without creating environmental hazards. While nano-selenium (Nano-Se) and vitamin C (VC) have been used as immunomodulators and antioxidants in animal and poultry feed, there is little data on Nano-Se and/or VC supplementation in aquatic animals. Thus, the current study evaluated the impact of adding Nano-Se and VC to the diets of Nile tilapia for 8 weeks. Four diets were formulated and offered to the fish: no supplementation (control), 1 mg Nano-Se/kg, 500 mg VC/kg, and 1 mg Nano-Se + 500 mg VC/kg of food. Growth-related parameters (final body weight, weight gain, and specific growth rate) were significantly increased in tilapia fed Nano-Se and VC, with a reduced feed conversion ratio (P < 0.05). Intestinal villus length and width as well as the number of goblet cells were increased in tilapia fed Nano-Se and/or VC (P < 0.05). Additionally, dietary Nano-Se and/or VC significantly increased nitro-blue tetrazolium (NBT) level, superoxide dismutase, glutathione peroxidase, catalase, the phagocytic index, and lysozyme and phagocytic activities (P < 0.05). However, significantly reduced levels of malonaldehyde were observed in fish fed Nano-Se and/or VC (P < 0.05). TNF-α and IL-1ß gene expressions in the liver and spleen of the fish were significantly upregulated by Nano-Se and/or VC (P < 0.05). The results revealed the potential role of Nano-Se and/or VC in enhancing growth, intestinal morphometry, and immune and antioxidative responses in Nile tilapia.

Comparative analysis of the immune system of an invasive bark beetle, Dendroctonus valens, infected by an entomopathogenic fungus.

Dendroctonus valens LeConte is one of the most economically important forest pest in China. Leptographium procerum, a mutualistic fungus can assist the host beetle in overcoming the pine's chemical defenses, and Beauveria bassiana, an entomopathogenic fungus has shown high beetle killing efficiency. Considering that the D. valens immune system remains unknown at the genomic level, a mutualistic and antagonistic fungus associated with the beetle provides an ideal model for studying immune interactions between the insect and associated fungi. Here, B. bassiana killed most tested larvae more effectively than L. procerum and Tween. The entomopathogenic fungus provoked stronger responses than the symbiotic fungus at the transcriptome level. We identified 185 immunity-related genes, including pattern recognition receptors, signal modulators, members of immune pathways (Toll, IMD, and JAK/STAT), and immune effectors. Quantitative real-time PCR analysis confirmed that several recognition receptors and effector genes were activated at 1 or 2 days post infection, while the effector genes were suppressed at 4 days post infection by B. bassiana, respectively. In contrast, effector genes were upregulated in response to L. procerum. Together, this study provides a comprehensive sequence resource and insight into the D. valens immune system and lays a basis for understanding the molecular aspects of the interaction between the host and associated fungi.

Characterization of the Maf family of polymorphic toxins in pathogenic Neisseria species.

In addition to harmless commensal species, Neisseria genus encompasses two pathogenic species, N. meningitidis (the meningococcus) and N. gonorrhoeae (the gonococcus), which are responsible for meningitis and genital tract infections, respectively. Since the publication of the first Neisseria genome in 2000, the presence of several genomic islands (GI) comprising maf genes has been intriguing. These GIs account for approximately 2% of the genome of the pathogenic Neisseria species and the function of the proteins encoded by maf genes remained unknown. We showed that maf genes encode a functional toxin-immunity system where MafB is a toxin neutralized by an immunity protein named MafI. A strain can harbor several MafB/MafI modules with distinct toxic activities. MafB toxins are polymorphic toxins with a conserved N-terminal region and a variable C-terminal region. MafB N-terminal regions consist of a signal peptide and a domain named DUF1020 that is only found in the genus Neisseria. MafB C-terminal regions are highly polymorphic and encode toxic activities. We evidenced the presence of MafB in the culture supernatant of meningococcal cells and we observed a competitive advantage for a strain overexpressing a MafB toxin. Therefore, we characterized a highly variable family of toxin-immunity modules found in multiple loci in pathogenic Neisseria species.

Cloning and Characterization of the Gene Cluster Involved in the Production of the Circular Bacteriocin Carnocyclin A.

Carnocyclin A is a circular bacteriocin of 60 amino acids produced by Carnobacterium maltaromaticum UAL307. A region of 12 kb that contained the structural gene for carnocyclin A, cclA, was sequenced using a fosmid library, and 10 genes were identified that could be responsible for carnocyclin A production and immunity. Five of those genes, cclBITCD, were found upstream of cclA: one encodes a protein containing a conserved ATP-binding domain and four encode proteins with putative membrane-spanning domains. CclC shows homology with a family of membrane proteins that contain the domain of unknown function 95 (DUF95). Downstream of cclA four additional genes, cclEFGH, were identified that show similarity to the last four genes, as-48EFGH, of the enterocin AS-48 bacteriocin gene cluster. CclFGH shows sequence homology with As-48FGH. Transformation of C. maltaromaticum UAL26 with cclBITCDA resulted in production of carnocyclin A, indicating that these genes form the minimal requirement for the secretion of fully matured bacteriocin. cclI encodes for a small hydrophobic protein with a high pI, which are characteristic features of known immunity proteins for other circular bacteriocins. Indeed, cloning of cclI behind a constitutive promoter in UAL26 resulted in immunity although the level of resistance was lower than that of UAL26 containing cclBITCDA, indicating that CclI alone is not enough to confer full immunity to carnocyclin A.