Predictive model using four ferroptosis-related genes accurately predicts gastric cancer prognosis

BACKGROUND Gastric cancer(GC)is a common malignancy of the digestive system.According to global 2018 cancer data,GC has the fifth-highest incidence and the thirdhighest fatality rate among malignant tumors.More than 60%of GC are linked to infection with Helicobacter pylori(H.pylori),a gram-negative,active,microaerophilic,and helical bacterium.This parasite induces GC by producing toxic factors,such as cytotoxin-related gene A,vacuolar cytotoxin A,and outer membrane proteins.Ferroptosis,or iron-dependent programmed cell death,has been linked to GC,although there has been little research on the link between H.pylori infection-related GC and ferroptosis.AIM To identify coregulated differentially expressed genes among ferroptosis-related genes(FRGs)in GC patients and develop a ferroptosis-related prognostic model with discrimination ability.METHODS Gene expression profiles of GC patients and those with H.pylori-associated GC were obtained from The Cancer Genome Atlas and Gene Expression Omnibus(GEO)databases.The FRGs were acquired from the FerrDb database.A ferroptosis-related gene prognostic index(FRGPI)was created using least absolute shrinkage and selection operator–Cox regression.The predictive ability of the FRGPI was validated in the GEO cohort.Finally,we verified the expression of the hub genes and the activity of the ferroptosis inducer FIN56 in GC cell lines and tissues.RESULTS Four hub genes were identified(NOX4,MTCH1,GABARAPL2,and SLC2A3)and shown to accurately predict GC and H.pylori-associated GC.The FRGPI based on the hub genes could independently predict GC patient survival;GC patients in the high-risk group had considerably worse overall survival than did those in the low-risk group.The FRGPI was a significant predictor of GC prognosis and was strongly correlated with disease progression.Moreover,the gene expression levels of common immune checkpoint proteins dramatically increased in the highrisk subgroup of the FRGPI cohort.The hub genes were also confirmed to be highly overexpressed in GC cell lines and tissues and were found to be primarily localized at the cell membrane.The ferroptosis inducer FIN56 inhibited GC cell proliferation in a dose-dependent manner.CONCLUSION In this study,we developed a predictive model based on four FRGs that can accurately predict the prognosis of GC patients and the efficacy of immunotherapy in this population.

Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.

STUDY ON THE ANTI-TUMOR EFFICACY INDUCED BY HEAT SHOCK PROTEIN 70-PEPTIDE COMPLEXES DERIVED FROM TUMOR CELLS

OBJECTIVE: To study the efficacy and explore the mechanism of the anti-tumor immunity elicited by heat shock protein 70-peptide complexes (HSP70-PC) derived from tumor cells. METHODS: Cells culture, flow cytometric analysis, affinity chromatography for protein purification, SDS-PAGE, Western-blotting and animal experiment were used. RESULTS: HSP70-PC immunization rendered protective effect to both naive tumorl-bearing mice. All of the naive mice obtained complete resistance to Hcaf cell attack; 40% of the tumor-bearing mice survived for over 90 days, whereas the mice of control group died within 2 weeks (P

Insight on BRAF^(V600E)mutated colorectal cancer immune microenvironment

Colorectal cancer(CRC)is the second deadliest malignancy for both sexes.The BRAF^(V600E)mutation,one of the most common driver mutations in CRC,is known for its poor prognosis due to the increased risk of metastasis.The effect of the BRAF^(V600E)mutation on the tumor microenvironment was the topic of the study reported in World Journal of Gastrointestinal Oncology,with special focus on immune status.The authors presented insightful findings that were exclusively based on macrophage polarity and cytokine levels,without investigating other relevant immune elements.A more comprehensive look into the dynamic immune activity of cancer environments will warrant more meaningful practical findings.In this letter,we discuss other significant immune factors and their possible implications on the tumor microenvironment of BRAF-mutated CRC.

Neospora caninum immune mapped protein 1(NcIMP1) is a novel vaccine candidate against neosporosis

The Neospora caninum immune mapped protein 1(Nc IMP1) was identified as a membrane protein,and a previous study indicated that Nc IMP1 could be a promising vaccine candidate against neosporosis. In this study, the immune response and protection efficacy of Nc IMP1 were evaluated. The coding sequence of Nc IMP1 was inserted into the eukaryotic expression vector pc DNA3.1(+), resulting in the recombination plasmid pc DNAIMP1, which was used for the intramuscular immunization of BALB/c mice. After immunization, the immune response was evaluated using a lymphoproliferative assay and cytokine and antibody measurements. Quantification of the cerebral parasite burden of mice challenged with 2106 N. caninum was performed 14 days after the last immunization. The results showed that the mice immunized with pc DNA-IMP1 developed a high level of specific antibody responses against recombinant Nc IMP1,with a mixed Ig G1/Ig G2 a response and a predominance of Ig G2 a production. The cellular immune response was associated with the production of IFN-γ, IL-2, IL-4 and IL-10 cytokines. The experiment was terminated 30 days p.i.,and the cerebral parasite burden in each mouse was assessed by quantitative PCR. The parasite burden was significantly reduced in the pc DNA-IMP1-vaccinated mice. These data suggest that IMP1 is a promising vaccine candidate against neosporosis.

Identification of atypical T4SS effector proteins mediating bacterial defense

To remain competitive,proteobacteria use various contact-dependent weapon systems to defend against microbial com-petitors.The bacterial-killing type IV secretion system(T4SS)is one such powerful weapon.It commonly controls the killing/competition between species by secreting the lethal T4SS effector(T4E)proteins carrying conserved XVIPCD domains into competing cells.In this study,we sought knowledge to understand whether the bacterial-killing T4SS-producing bacteria encode T4E-like proteins and further explore their biological functions.To achieve this,we designed a T4E-guided approach to discover T4E-like proteins that are designated as atypical T4Es.Initially,this approach required scientists to perform simple BlastP search to identify T4E homologs that lack the XVIPCD domain in the genomes of T4SS-producing bacteria.These homologous genes were then screened in Escherichia coli to identify antibacterial candidates(atypical T4Es)and their neighboring detoxification proteins,followed by testing their gene cotranscription and validating their physical interactions.Using this approach,we did discover two atypical T4E proteins from the plant-beneficial Lysobacter enzymogenes and the phytopathogen Xanthomonas citri.We also provided substantial evidence to show that the atypical T4E protein Le1637-mediated bacterial defense in interspecies interactions between L.enzymogenes and its competitors.Therefore,the newly designed T4E-guided approach holds promise for detecting functional atypical T4E proteins in bacterial cells.

Construction of the recombinant expression vector for CD80-IgG fusion gene and its expression in Chinese hamster ovary cells

To construct the recombinant expression functionally in Chinese hamster ovary cells in order vector for CD80-IgG fusion gene and to express it to be used as an effective method to eliminate the immune escape of leukemic cells, the cDNA encoding the signal and extracellular domains of murine CD80 was generated by PCR amplification from plasmid pcDNMB7 containing the full length cDNA of murine CD80 and those of murine IgG1, in which the Fc fragment was obtained through RT-PCR amplification from murine spleen cells. These two cDNAs were then cloned in tandem into eukaryotic expression vector pcDNA3.0 and the resultant recombinant plasmid pcDNA/CD80-IgG was then transfected to Chinese hamster ovary cells with liposome transfection reagent. The cell clones constitutively expressing CD80-IgG fusion protein were obtained by G418 screening. Western blotting and dot ELISA assay were used to detect the expression of the fusion protein in the supernatants of these cells. Meanwhile, the fusion protein expressed was then purified with affinity chromatography, and its biological activity was demonstrated by flow cytometry, MTr colorimetry and ELISA assay. The experimental resuits showed that these two inserts were successfully cloned into plasmid pcDNA3.0, and the highly purified fusion protein was obtained. This fusion protein was proved to be able to upregulate the density of CD80 on leukemic cells, deliberately promote the proliferative reactions of mouse allogenic lymphocytes and increase the killing activity against WEHI-3 cells from 49.7 % up to 84.6 %. In addition, this fusion protein could also enhance the IL-2 secretion from allogenic lymphocytes activated by tumorspecific antigens. It is concluded that the recombinant vector constructed can be functionally expressed in the mammalian cells, thus providing a solid foundation for the further investigation on the mechanism to eliminate the immune escape of leukemic cells in vivo.

Interaction specific binding hotspots in Endonuclease colicin-immunity protein complex from MD simulations

The binding of Endonuclease colicin 9 (E9) by Immunity protein 9 (Im9) was found to involve some hotspots from helix III of Im9 on protein-protein interface that contribute the dominant binding energy to the complex.In the current work,MD simulations of the WT and three hotspot mutants (D51A,Y54A and Y55A of Im9) of the E9-Im9 complexes were carried out to investigate specific interaction mechanisms of these three hotspot residues.The changes of binding energy between the WT and mutants of the complex were computed by the MM/PBSA method using a polarized force field and were in excellent agreement with experiment values,verifying that these three residues were indeed hotspots of the binding complex.Energy decomposition analysis revealed that binding by D51 to E9 was dominated by electrostatic interaction due to the presence of the carboxyl group of Asp51 which hydrogen bonds to K89.For binding by hotspots Y54 and Y55,van der Waals interaction from the aromatic side chain of tyrosine provided the dominant interaction.For comparison,calculation by using the standard (nonpolarizable) AMBER99SB force field produced binding energy changes from these mutations in opposite direction to the experimental observation.Dynamic hydrogen bond analysis showed that conformations sampled from MD simulation in the standard AMBER force field were distorted from the native state and they disrupted the inter-protein hydrogen bond network of the protein-protein complex.The current work further demonstrated that electrostatic polarization plays a critical role in modulating protein-protein binding.

BEACH-Domain Proteins Act Together in a Cascade to Mediate Vacuolar Protein Trafficking and Disease Resistance in Arabidopsis

Membrane trafficking to the protein storage vacuole （PSV） is a specialized process in seed plants. However, this trafficking mechanism to PSV is poorly understood. Here, we show that three types of Beige and Chediak-Higashi （BEACH）-domain proteins contribute to both vacuolar protein transport and effector- triggered immunity （ETI）. We screened a green fluorescent seed （GFS） library of Arabidopsis mutants with defects in vesicle trafficking and isolated two allelic mutants gfs3 and gfs12 with a defect in seed pro- tein transport to PSV. The gene responsible for the mutant phenotype was found to encode a putative pro- tein belonging to group D of BEACH-domain proteins, which possess kinase domains. Disruption of other BEACH-encoding loci in the gfs12 mutant showed that BEACH homologs acted in a cascading manner for PSV trafficking. The epistatic genetic interactions observed among BEACH homologs were also found in the ETI responses of the gfs12 and gfs12 bchb-1 mutants, which showed elevated avirulent bacterial growth. The GFS12 kinase domain interacted specifically with the pleckstrin homology domain of BchCl. These results suggest that a cascade of multiple BEACH-domain proteins contributes to vacuolar protein transport and plant defense.