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Objective Osteosarcoma is the most common primary bone sarcoma. About 50% of patients develop metastatic disease and their 5-year survival lingers at around 20%–30%. T cell checkpoint blockade immunotherapies have revolutionised cancer treatment in the last decade, but their impact remains limited in osteosarcoma.Methods and analysis In order to reveal potentially novel immunotherapeutic strategies for advanced osteosarcoma, we conducted an immunogenomic characterisation of a unique sample set comprising 30 osteosarcoma samples from seven patients, collected throughout disease progression. We performed RNA-sequencing and imaging mass cytometry analysis on these samples to reveal the immunological landscape during osteosarcoma progression.Results Transcriptional and phenotypical hallmarks of cytotoxic T cell-driven anticancer immunity were enriched in metastatic lesions as compared with primary tumours. Spatial analysis showed T cells infiltrating central regions of osteosarcoma metastases, indicating the absence of an immune excluded environment. In parallel, we found a pronounced increase in the expression of cancer testis antigens, particularly melanoma antigen family A (MAGEA)-related antigens, in osteosarcoma metastases, which was validated in an independent cohort (N=91). In addition, we demonstrated the presentation of MAGE-derived peptides in three out of four osteosarcoma cell lines.Conclusion These findings indicate a concurrent augmentation of cytotoxic antitumour immune responses and expression of MAGEA antigens from primary to metastatic osteosarcoma. This observation suggests the exploration of MAGEA antigens as potential targets for immunotherapy in the treatment of advanced osteosarcoma.

Objective Gut-residing bacteria, such as Escherichia coli, can acetylate their proteome under conditions of amine starvation. It is postulated that the (gut) microbiome is involved in the breach of immune tolerance to modified self-proteins leading to the anti-modified protein antibodies (AMPAs), hallmarking seropositive rheumatoid arthritis (RA). Our aim was to determine whether acetylated bacterial proteins can induce AMPA responses cross-reactive to modified self-proteins and be recognised by human AMPA (hAMPA).Methods E. coli bacteria were grown under amine starvation to generate endogenously acetylated bacterial proteins. Furthermore, E. coli proteins were acetylated chemically. Recognition of these proteins by hAMPA was analysed by western blotting and ELISA; recognition by B cells carrying a modified protein-reactive B cell receptor (BCR) was analysed by pSyk (Syk phosphorylation) activation assay. C57BL/6 mice were immunised with (modified) bacterial protein fractions, and sera were analysed by ELISA.Results Chemically modified bacterial protein fractions contained high levels of acetylated proteins and were readily recognised by hAMPA and able to activate B cells carrying modified protein-reactive BCRs. Likely due to substantially lower levels of acetylation, endogenously acetylated protein fractions were not recognised by hAMPA or hAMPA-expressing B cells. Immunising mice with chemically modified protein fractions induced a strong cross-reactive AMPA response, targeting various modified antigens including citrullinated proteins.Conclusions Acetylated bacterial proteins are recognisable by hAMPA and are capable of inducing cross-reactive AMPA in mice. These observations provide the first conceptual evidence for a novel mechanism involving the (endogenous) acetylation of the bacterial proteome, allowing a breach of tolerance to modified proteins and the formation of cross-reactive AMPA.

AbstractPatients with Inflammatory Bowel Disease (IBD) are at increased risk of developing malignancies. IBD patients with oral cavity cancer may have reduced survival compared with the general population. This article describes two IBD patients, non-smokers, on long-term use of mesalazine with the development of oral cavity cancer. In IBD the clinician should be aware of possible head and neck malignancies and in case of doubt a biopsy should be performed, even in the absence of standard risk factors.

DNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 1 and 2 (IDH1/2) are genes involved in epigenetic regulation, each mutated in 7–23% of patients with acute myeloid leukemia. Here, we investigated whether hotspot mutations in these genes encode neoantigens that can be targeted by immunotherapy. Five human B-lymphoblastoid cell lines expressing common HLA class I alleles were transduced with a minigene construct containing mutations that often occur in DNMT3A or IDH1/2. From these minigene-transduced cell lines, peptides were eluted from HLA class I alleles and analyzed using tandem mass spectrometry. The resulting data are available via ProteomeXchange under the identifier PXD050560. Mass spectrometry revealed an HLA-A*01:01-binding DNMT3AR882H peptide and an HLA-B*07:02-binding IDH2R140Q peptide as potential neoantigens. For these neopeptides, peptide–HLA tetramers were produced to search for specific T-cells in healthy individuals. Various T-cell clones were isolated showing specific reactivity against cell lines transduced with full-length DNMT3AR882H or IDH2R140Q genes, while cell lines transduced with wildtype genes were not recognized. One T-cell clone for DNMT3AR882H also reacted against patient-derived acute myeloid leukemia cells with the mutation, while patient samples without the mutation were not recognized, thereby validating the surface presentation of a DNMT3AR882H neoantigen that can potentially be targeted in acute myeloid leukemia via immunotherapy.

Introduction: Under the dual opportunities of low-carbon consumption preference and online consumption platforms, vendors’ low-carbon advertising incursions provide opportunities for decarbonization and market position enhancement, as well as further research on the value of low-carbon advertising. This study aims to explore the contractual choices of green vendors’ online channels participating in low-carbon advertising competition under the low-carbon goodwill effect, and to simulate and evaluate the contractual choices of supply chain members.Methods: Using differential games, through the innovative application of the traditional low-carbon goodwill model and the introduction of the low-carbon advertising competition intensity coefficient, we design one-way and two-way cost-sharing contracts under low-carbon competition, coordinate the vicious competition in the supply chain, and provide contractual choices for supply chain participants.Results and discussion: Under the low-carbon advertising competition decision, the manufacturer has an absolute low-carbon market advantage, but the interests of all supply chain members are weakened, and interestingly, the manufacturer, who dominates the market, is the facilitator of the contractual agreement. Second, well-designed pacts can provide manufacturers and traders with more options for low-carbon strategies. Although both one-way and two-way cost-sharing pacts can generate Pareto gains for the supply chain and its members in advertising competition, two-way cost-sharing pacts are superior to one-way pacts in terms of coordination advantages. In addition, an important finding is that greater profit growth can be achieved through contractual cooperation in low-intensity advertising competition. Thus, moderate competition is desirable, while excessive competition can harm the supply chain system. Manufacturers should actively urge retailers to cooperate in order to optimize profits and establish long-term stable partnerships between upstream and downstream firms in green supply chains.

Recurrent disease emerges in the majority of patients with ovarian cancer (OVCA). Adoptive T-cell therapies with T-cell receptors (TCRs) targeting tumor-associated antigens (TAAs) are considered promising solutions for less-immunogenic ‘cold’ ovarian tumors. In order to treat a broader patient population, more TCRs targeting peptides derived from different TAAs binding in various HLA class I molecules are essential. By performing a differential gene expression analysis using mRNA-seq datasets, PRAME, CTCFL and CLDN6 were selected as strictly tumor-specific TAAs, with high expression in ovarian cancer and at least 20-fold lower expression in all healthy tissues of risk. In primary OVCA patient samples and cell lines we confirmed expression and identified naturally expressed TAA-derived peptides in the HLA class I ligandome. Subsequently, high-avidity T-cell clones recognizing these peptides were isolated from the allo-HLA T-cell repertoire of healthy individuals. Three PRAME TCRs and one CTCFL TCR of the most promising T-cell clones were sequenced, and transferred to CD8+ T cells. The PRAME TCR-T cells demonstrated potent and specific antitumor reactivity in vitro and in vivo. The CTCFL TCR-T cells efficiently recognized primary patient-derived OVCA cells, and OVCA cell lines treated with demethylating agent 5-aza-2′-deoxycytidine (DAC). The identified PRAME and CTCFL TCRs are promising candidates for the treatment of patients with ovarian cancer, and are an essential addition to the currently used HLA-A*02:01 restricted PRAME TCRs. Our selection of differentially expressed genes, naturally expressed TAA peptides and potent TCRs can improve and broaden the use of T-cell therapies for patients with ovarian cancer or other PRAME or CTCFL expressing cancers.

Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381.

Mass spectrometry (MS)-based proteomics profiling has undoubtedly increased the knowledge about cellular processes and functions. However, its applicability for paucicellular sample analyses is currently limited. Although new approaches have been developed for single-cell studies, most of them have not (yet) been standardized and/or require highly specific (often home-built) devices, thereby limiting their broad implementation, particularly in non-specialized settings. To select an optimal MS-oriented proteomics approach applicable in translational research and clinical settings, we assessed 10 different sample preparation procedures in paucicellular samples of closely-related cell types. Particularly, five cell lysis protocols using different chemistries and mechanical forces were combined with two sample clean-up techniques (C18 filter- and SP3-based), followed by tandem mass tag (TMT)-based protein quantification. The evaluation was structured in three phases: first, cell lines from hematopoietic (THP-1) and non-hematopoietic (HT-29) origins were used to test the approaches showing the combination of a urea-based lysis buffer with the SP3 bead-based clean-up system as the best performer. Parameters such as reproducibility, accessibility, spatial distribution, ease of use, processing time and cost were considered. In the second phase, the performance of the method was tested on maturation-related cell populations: three different monocyte subsets from peripheral blood and, for the first time, macrophages/microglia (MAC) from glioblastoma samples, together with T cells from both tissues. The analysis of 50,000 cells down to only 2,500 cells revealed different protein expression profiles associated with the distinct cell populations. Accordingly, a closer relationship was observed between non-classical monocytes and MAC, with the latter showing the co-expression of M1 and M2 macrophage markers, although pro-tumoral and anti-inflammatory proteins were more represented. In the third phase, the results were validated by high-end spectral flow cytometry on paired monocyte/MAC samples to further determine the sensitivity of the MS approach selected. Finally, the feasibility of the method was proven in 194 additional samples corresponding to 38 different cell types, including cells from different tissue origins, cellular lineages, maturation stages and stimuli. In summary, we selected a reproducible, easy-to-implement sample preparation method for MS-based proteomic characterization of paucicellular samples, also applicable in the setting of functionally closely-related cell populations.

To survive in the host, pathogenic bacteria need to be able to react to the unfavorable conditions that they encounter, like low pH, elevated temperatures, antimicrobial peptides and many more. These conditions may lead to unfolding of envelope proteins and this may be lethal. One of the mechanisms through which bacteria are able to survive these conditions is through the protease/foldase activity of the high temperature requirement A (HtrA) protein. The gut pathogen Clostridioides difficile encodes one HtrA homolog that is predicted to contain a membrane anchor and a single PDZ domain. The function of HtrA in C. difficile is hitherto unknown but previous work has shown that an insertional mutant of htrA displayed elevated toxin levels, less sporulation and decreased binding to target cells. Here, we show that HtrA is membrane associated and localized on the surface of C. difficile and characterize the requirements for proteolytic activity of recombinant soluble HtrA. In addition, we show that the level of HtrA in the bacteria heavily depends on its proteolytic activity. Finally, we show that proteolytic activity of HtrA is required for survival under acidic conditions. [ABSTRACT FROM AUTHOR]

A group of bacterial proteases, the Pro‐Pro endopeptidases (PPEPs), possess the unique ability to hydrolyze proline‐proline bonds in proteins. Since a protease's function is largely determined by its substrate specificity, methods that can extensively characterize substrate specificity are valuable tools for protease research. Previously, we achieved an in‐depth characterization of PPEP prime‐side specificity. However, PPEP specificity is also determined by the non‐prime‐side residues in the substrate. To gain a more complete insight into the determinants of PPEP specificity, we characterized the non‐prime‐ and prime‐side specificity of various PPEPs using a combination of synthetic combinatorial peptide libraries and mass spectrometry. With this approach, we deepened our understanding of the P3‐P3′ specificities of PPEP‐1 and PPEP‐2, while identifying the endogenous substrate of PPEP‐2 as the most optimal substrate in our library data. Furthermore, by employing the library approach, we investigated the altered specificity of mutants of PPEP‐1 and PPEP‐2. Additionally, we characterized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP‐4. Based on structural comparisons, we hypothesized that PPEP‐4 displays a PPEP‐1‐like prime‐side specificity, which was substantiated by the experimental data. Intriguingly, another putative PPEP from Clostridioides difficile, CD1597, did not display Pro‐Pro endoproteolytic activity. Collectively, we characterized PPEP specificity in detail using our robust peptide library method and, together with additional structural information, provide more insight into the intricate mechanisms that govern protease specificity. [ABSTRACT FROM AUTHOR]

The Maevatanana gold deposit in Madagascar is a quartz-vein type gold deposit hosted in a greenstone belt, and its genesis is highly controversial. This article conducts elemental geochemical analysis and dating of the Maevatanana gold deposit, and explores the mineralization age and process of the Maevatanana gold deposit in Madagascar. The gold content of gneiss is 1.11x10-9 ~ 80.8 x10-9, with an average of 19.55x10-9; pyrite is the main gold-bearing mineral in gneiss, with a Re-Os isotopic age of 752±27 Ma. The gold grade of the gold-bearing quartz vein is 26.44x10-6 ??? 6???? 132.22x10-6, and pyrite is the main gold-bearing mineral. The Re-Os isotopic age of pyrite in the gold-bearing quartz vein is 535±26 Ma, representing the mineralization age of the Maevatanana gold deposit. The Re-Os isotope isochron ages of two phases of pyrite correspond to the time of two collisions during the Pan African orogeny event. The gold content in gneiss is much higher than the average grade of the crust, and encapsulated gold can be seen in pyrite. The Re-Os isotopic age of pyrite indicates intrusion of granite and gabbro at 752±27 Ma. The Madagascar terrane underwent extensive metamorphism from greenschist to amphibolite facies, and gold in volcanic sedimentary rocks was activated, migrated, and enriched to form a source layer. The mineralization age of the gold-bearing quartz vein is 535 ± 26 Ma, which is consistent with the age of the Cambrian post collisional A-type granite in central northern Madagascar. Field evidence and geochemical data indicate a close genetic relationship between the Maevatanana gold deposit and the Cambrian post collisional A-type granite. During the process of magma ascending, the high-Si melts entered the magma and gradually evolved into ore-forming fluids, which enter the host structures and enrich mineralization. [ABSTRACT FROM AUTHOR]

Glycoproteomic data are often very complex, reflecting the high structural diversity of peptide and glycan portions. The use of glycopeptide-centered glycoproteomics by mass spectrometry is rapidly evolving in many research areas, leading to a demand in reliable data analysis tools. In recent years, several bioinformatic tools were developed to facilitate and improve both the identification and quantification of glycopeptides. Here, a selection of these tools was combined and evaluated with the aim of establishing a robust glycopeptide detection and quantification workflow targeting enriched glycoproteins. For this purpose, a tryptic digest from affinity-purified immunoglobulins G and A was analyzed on a nano-reversed-phase liquid chromatography–tandem mass spectrometry platform with a high-resolution mass analyzer and higher-energy collisional dissociation fragmentation. Initial glycopeptide identification based on MS/MS data was aided by the Byonic software. Additional MS1-based glycopeptide identification relying on accurate mass and retention time differences using GlycopeptideGraphMS considerably expanded the set of confidently annotated glycopeptides. For glycopeptide quantification, the performance of LaCyTools was compared to Skyline, and GlycopeptideGraphMS. All quantification packages resulted in comparable glycosylation profiles but featured differences in terms of robustness and data quality control. Partial cysteine oxidation was identified as an unexpectedly abundant peptide modification and impaired the automated processing of several IgA glycopeptides. Finally, this study presents a semiautomated workflow for reliable glycoproteomic data analysis by the combination of software packages for MS/MS- and MS1-based glycopeptide identification as well as the integration of analyte quality control and quantification.

In the context of HLA-DP-mismatched allogeneic stem cell transplantation, mismatched HLA-DP alleles can provoke profound allo-HLA-DP-specific immune responses from the donor T-cell repertoire leading to graft-versus-leukemia effect and/or graft-versus-host disease in the patient. The magnitude of allo-HLA-DP-specific immune responses has been shown to depend on the specific HLA-DP disparity between donor and patient and the immunogenicity of the mismatched HLA-DP allele(s). HLA-DP peptidome clustering (DPC) was developed to classify the HLA-DP molecules based on similarities and differences in their peptide-binding motifs. To investigate a possible categorization of HLA-DP molecules based on overlap of presented peptides, we identified and compared the peptidomes of the thirteen most frequently expressed HLA-DP molecules. Our categorization based on shared peptides was in line with the DPC classification. We found that the HLA-DP molecules within the previously defined groups DPC-1 or DPC-3 shared the largest numbers of presented peptides. However, the HLA-DP molecules in DPC-2 segregated into two subgroups based on the overlap in presented peptides. Besides overlap in presented peptides within the DPC groups, a substantial number of peptides was also found to be shared between HLA-DP molecules from different DPC groups, especially for groups DPC-1 and -2. The functional relevance of these findings was illustrated by demonstration of cross-reactivity of allo-HLA-DP-reactive T-cell clones not only against HLA-DP molecules within one DPC group, but also across different DPC groups. The promiscuity of peptides presented in various HLA-DP molecules and the cross-reactivity against different HLA-DP molecules demonstrate that these molecules cannot be strictly categorized in immunogenicity groups.

ABSTRACT Phosphorylation is a posttranslational modification that can affect both housekeeping functions and virulence characteristics in bacterial pathogens. In the Gram-positive enteropathogen Clostridioides difficile, the extent and nature of phosphorylation events are poorly characterized, though a protein kinase mutant strain demonstrates pleiotropic phenotypes. Here, we used an immobilized metal affinity chromatography strategy to characterize serine, threonine, and tyrosine phosphorylation in C. difficile. We find limited protein phosphorylation in the exponential growth phase but a sharp increase in the number of phosphopeptides after the onset of the stationary growth phase. Our approach identifies expected targets and phosphorylation sites among the more than 1,500 phosphosites, including the protein kinase PrkC, the anti-sigma-F factor antagonist (SpoIIAA), the anti-sigma-B factor antagonist (RsbV), and HPr kinase/phosphorylase (HprK). Analysis of high-confidence phosphosites shows that phosphorylation on serine residues is most common, followed by threonine and tyrosine phosphorylation. This work forms the basis for a further investigation into the contributions of individual kinases to the overall phosphoproteome of C. difficile and the role of phosphorylation in C. difficile physiology and pathogenesis. IMPORTANCE In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.