An untargeted analytical workflow based on Kendrick mass defect filtering reveals dysregulations in acylcarnitines in prostate cancer tissue.

BACKGROUND: Metabolomics is nowadays considered one the most powerful analytical for the discovery of metabolic dysregulations associated with the insurgence of cancer, given the reprogramming of the cell metabolism to meet the bioenergetic and biosynthetic demands of the malignant cell. Notwithstanding, several challenges still exist regarding quality control, method standardization, data processing, and compound identification. Therefore, there is a need for effective and straightforward approaches for the untargeted analysis of structurally related classes of compounds, such as acylcarnitines, that have been widely investigated in prostate cancer research for their role in energy metabolism and transport and ß-oxidation of fatty acids. RESULTS: In the present study, an innovative analytical platform was developed for the straightforward albeit comprehensive characterization of acylcarnitines based on high-resolution mass spectrometry, Kendrick mass defect filtering, and confirmation by prediction of their retention time in reversed-phase chromatography. In particular, a customized data processing workflow was set up on Compound Discoverer software to enable the Kendrick mass defect filtering, which allowed filtering out more than 90 % of the initial features resulting from the processing of 25 tumoral and adjacent non-malignant prostate tissues collected from patients undergoing radical prostatectomy. Later, a partial least square-discriminant analysis model validated by repeated double cross-validation was built on the dataset of 74 annotated acylcarnitines, with classification rates higher than 93 % for both groups, and univariate statistical analysis helped elucidate the individual role of the annotated metabolites. SIGNIFICANCE: Hydroxylation of short- and medium-chain minor acylcarnitines appeared to be a significant variable in describing tissue differences, suggesting the hypothesis that the neoplastic growth is linked to oxidation phenomena on selected metabolites and reinforcing the need for effective methods for the annotation of minor metabolites.

An atlas of cells in the human tonsil.

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.

Impact of plastic-related compounds on the gene expression signature of HepG2 cells transfected with CYP3A4.

The presence of plastic and microplastic within the oceans as well as in marine flora and fauna have caused a multitude of problems that have been the topic of numerous investigations for many years. However, their impact on human health remains largely unknown. Such plastic and microplastic particles have been detected in blood and placenta, underlining their ability to enter the human body. Plastics also contain other compounds, such as plasticizers, antioxidants, or dyes, whose impact on human health is currently being studied. Critical enzymes within the metabolism of endogenous molecules, especially of xenobiotics, are the cytochrome P450 monooxygenases (CYPs). Although their importance in maintaining cellular balance has been confirmed, their interactions with plastics and related products are poorly understood. In this study, the possible relationship between different plastic-related compounds and CYP3A4 as one of the most important CYPs was analyzed using hepatic cells overexpressing this enzyme. Beginning with virtual compound screening and molecular docking of more than 1000 plastic-related compounds, several candidates were identified to interact with CYP3A4. In a second step, RNA-sequencing was used to study in detail the transcriptome-wide gene expression levels affected by the selected compounds. Three candidate molecules ((2,2'-methylenebis(6-tert-butyl-4-methylphenol), 1,1-bis(3,5-di-tert-butyl-2-hydroxyphenyl)ethane, and 2,2'-methylenebis(6-cyclohexyl-4-methylphenol)) had an excellent binding affinity to CYP3A4 in-silico as well as cytotoxic effects and interactions with several metabolic pathways in-vitro. We identified common pathways influenced by all three selected plastic-related compounds. In particular, the suppression of pathways related to mitosis and 'DNA-templated DNA replication' which were confirmed by cell cycle analysis and single-cell gel electrophoresis. Furthermore, several mis-regulated metabolic and inflammation-related pathways were identified, suggesting the induction of hepatotoxicity at different levels. These findings imply that these compounds may cause liver problems subsequently affecting the entire organism.

A20 regulates lymphocyte adhesion in murine neuroinflammation by restricting endothelial ICOSL expression in the CNS.

A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20ΔCNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL to be expressed by A20-deficient CNS-ECs, which we found to function as adhesion molecules. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20ΔCNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in WT mice. In addition, blocking of ICOSL on primary mouse brain microvascular ECs impaired the adhesion of T cells in vitro. Taken together, we propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation.

Tumor-infiltrating CCR2+ inflammatory monocytes counteract specific immunotherapy.

Tumor development and progression is shaped by the tumor microenvironment (TME), a heterogeneous assembly of infiltrating and resident host cells, their secreted mediators and intercellular matrix. In this context, tumors are infiltrated by various immune cells with either pro-tumoral or anti-tumoral functions. Recently, we published our non-invasive immunization platform DIVA suitable as a therapeutic vaccination method, further optimized by repeated application (DIVA2). In our present work, we revealed the therapeutic effect of DIVA2 in an MC38 tumor model and specifically focused on the mechanisms induced in the TME after immunization. DIVA2 resulted in transient tumor control followed by an immune evasion phase within three weeks after the initial tumor inoculation. High-dimensional flow cytometry analysis and single-cell mRNA-sequencing of tumor-infiltrating leukocytes revealed cytotoxic CD8+ T cells as key players in the immune control phase. In the immune evasion phase, inflammatory CCR2+ PDL-1+ monocytes with immunosuppressive properties were recruited into the tumor leading to suppression of DIVA2-induced tumor-reactive T cells. Depletion of CCR2+ cells with specific antibodies resulted in prolonged survival revealing CCR2+ monocytes as important for tumor immune escape in the TME. In summary, the present work provides a platform for generating a strong antigen-specific primary and memory T cell immune response using the optimized transcutaneous immunization method DIVA2. This enables protection against tumors by therapeutic immune control of solid tumors and highlights the immunosuppressive influence of tumor infiltrating CCR2+ monocytes that need to be inactivated in addition for successful cancer immunotherapy.

The Effect of Plastic-Related Compounds on Transcriptome-Wide Gene Expression on CYP2C19-Overexpressing HepG2 Cells.

In recent years, plastic and especially microplastic in the oceans have caused huge problems to marine flora and fauna. Recently, such particles have also been detected in blood, breast milk, and placenta, underlining their ability to enter the human body, presumably via the food chain and other yet-unknown mechanisms. In addition, plastic contains plasticizers, antioxidants, or lubricants, whose impact on human health is also under investigation. At the cellular level, the most important enzymes involved in the metabolism of xenobiotic compounds are the cytochrome P450 monooxygenases (CYPs). Despite their extensive characterization in the maintenance of cellular balance, their interactions with plastic and related products are unexplored. In this study, the possible interactions between several plastic-related compounds and one of the most important cytochromes, CYP2C19, were analyzed. By applying virtual compound screening and molecular docking to more than 1000 commercially available plastic-related compounds, we identified candidates that are likely to interact with this protein. A growth inhibition assay confirmed their cytotoxic activity on a CYP2C19-transfected hepatic cell line. Subsequently, we studied the effect of the selected compounds on the transcriptome-wide gene expression level by conducting RNA sequencing. Three candidate molecules were identified, i.e., 2,2'-methylene bis(6-tert-butyl-4-methylphenol), 1,1-bis(3,5-di-tert-butyl-2-hydroxyphenyl) ethane, and 2,2'-methylene bis(6-cyclohexyl-4-methylphenol)), which bound with a high affinity to CYP2C19 in silico. They exerted a profound cytotoxicity in vitro and interacted with several metabolic pathways, of which the 'cholesterol biosynthesis process' was the most affected. In addition, other affected pathways involved mitosis, DNA replication, and inflammation, suggesting an increase in hepatotoxicity. These results indicate that plastic-related compounds could damage the liver by affecting several molecular pathways.

Commensal bacteria weaken the intestinal barrier by suppressing epithelial neuropilin-1 and Hedgehog signaling.

The gut microbiota influences intestinal barrier integrity through mechanisms that are incompletely understood. Here we show that the commensal microbiota weakens the intestinal barrier by suppressing epithelial neuropilin-1 (NRP1) and Hedgehog (Hh) signaling. Microbial colonization of germ-free mice dampens signaling of the intestinal Hh pathway through epithelial Toll-like receptor (TLR)-2, resulting in decreased epithelial NRP1 protein levels. Following activation via TLR2/TLR6, epithelial NRP1, a positive-feedback regulator of Hh signaling, is lysosomally degraded. Conversely, elevated epithelial NRP1 levels in germ-free mice are associated with a strengthened gut barrier. Functionally, intestinal epithelial cell-specific Nrp1 deficiency (Nrp1ΔIEC) results in decreased Hh pathway activity and a weakened gut barrier. In addition, Nrp1ΔIEC mice have a reduced density of capillary networks in their small intestinal villus structures. Collectively, our results reveal a role for the commensal microbiota and epithelial NRP1 signaling in the regulation of intestinal barrier function through postnatal control of Hh signaling.

Modulation of cellular transcriptome and proteome composition by azidohomoalanine-implications on click chemistry-based secretome analysis.

The analysis of the secretome provides important information on proteins defining intercellular communication and the recruitment and behavior of cells in specific tissues. Especially in the context of tumors, secretome data can support decisions for diagnosis and therapy. The mass spectrometry-based analysis of cell-conditioned media is widely used for the unbiased characterization of cancer secretomes in vitro. Metabolic labeling using azide-containing amino acid analogs in combination with click chemistry facilitates this type of analysis in the presence of serum, preventing serum starvation-induced effects. The modified amino acid analogs, however, are less efficiently incorporated into newly synthesized proteins and may perturb protein folding. Combining transcriptome and proteome analysis, we elucidate in detail the effects of metabolic labeling with the methionine analog azidohomoalanine (AHA) on gene and protein expression. Our data reveal that 15-39% of the proteins detected in the secretome displayed changes in transcript and protein expression induced by AHA labeling. Gene Ontology (GO) analyses indicate that metabolic labeling using AHA leads to induction of cellular stress and apoptosis-related pathways and provide first insights on how this affects the composition of the secretome on a global scale. KEY MESSAGES: Azide-containing amino acid analogs affect gene expression profiles. Azide-containing amino acid analogs influence cellular proteome. Azidohomoalanine labeling induces cellular stress and apoptotic pathways. Secretome consists of proteins with dysregulated expression profiles.

Identification of lncRNAs involved in response to ionizing radiation in fibroblasts of long-term survivors of childhood cancer and cancer-free controls.

Introduction: Long non-coding ribonucleic acids (lncRNAs) are involved in the cellular damage response following exposure to ionizing radiation as applied in radiotherapy. However, the role of lncRNAs in radiation response concerning intrinsic susceptibility to late effects of radiation exposure has not been examined in general or in long-term survivors of childhood cancer with and without potentially radiotherapy-related second primary cancers, in particular. Methods: Primary skin fibroblasts (n=52 each) of long-term childhood cancer survivors with a first primary cancer only (N1), at least one second primary neoplasm (N2+), as well as tumor-free controls (N0) from the KiKme case-control study were matched by sex, age, and additionally by year of diagnosis and entity of the first primary cancer. Fibroblasts were exposed to 0.05 and 2 Gray (Gy) X-rays. Differentially expressed lncRNAs were identified with and without interaction terms for donor group and dose. Weighted co-expression networks of lncRNA and mRNA were constructed using WGCNA. Resulting gene sets (modules) were correlated to the radiation doses and analyzed for biological function. Results: After irradiation with 0.05Gy, few lncRNAs were differentially expressed (N0: AC004801.4; N1: PCCA-DT, AF129075.3, LINC00691, AL158206.1; N2+: LINC02315). In reaction to 2 Gy, the number of differentially expressed lncRNAs was higher (N0: 152, N1: 169, N2+: 146). After 2 Gy, AL109976.1 and AL158206.1 were prominently upregulated in all donor groups. The co-expression analysis identified two modules containing lncRNAs that were associated with 2 Gy (module1: 102 mRNAs and 4 lncRNAs: AL158206.1, AL109976.1, AC092171.5, TYMSOS, associated with p53-mediated reaction to DNA damage; module2: 390 mRNAs, 7 lncRNAs: AC004943.2, AC012073.1, AC026401.3, AC092718.4, MIR31HG, STXBP5-AS1, TMPO-AS1, associated with cell cycle regulation). Discussion: For the first time, we identified the lncRNAs AL158206.1 and AL109976.1 as involved in the radiation response in primary fibroblasts by differential expression analysis. The co-expression analysis revealed a role of these lncRNAs in the DNA damage response and cell cycle regulation post-IR. These transcripts may be targets in cancer therapy against radiosensitivity, as well as provide grounds for the identification of at-risk patients for immediate adverse reactions in healthy tissues. With this work we deliver a broad basis and new leads for the examination of lncRNAs in the radiation response.

Amino Acid Profiles in Older Adults with Frailty: Secondary Analysis from MetaboFrail and BIOSPHERE Studies.

An altered amino acid metabolism has been described in frail older adults which may contribute to muscle loss and functional decline associated with frailty. In the present investigation, we compared circulating amino acid profiles of older adults with physical frailty and sarcopenia (PF&S, n = 94), frail/pre-frail older adults with type 2 diabetes mellitus (F-T2DM, n = 66), and robust non-diabetic controls (n = 40). Partial least squares discriminant analysis (PLS-DA) models were built to define the amino acid signatures associated with the different frailty phenotypes. PLS-DA allowed correct classification of participants with 78.2 ± 1.9% accuracy. Older adults with F-T2DM showed an amino acid profile characterized by higher levels of 3-methylhistidine, alanine, arginine, ethanolamine, and glutamic acid. PF&S and control participants were discriminated based on serum concentrations of aminoadipic acid, aspartate, citrulline, cystine, taurine, and tryptophan. These findings suggest that different types of frailty may be characterized by distinct metabolic perturbations. Amino acid profiling may therefore serve as a valuable tool for frailty biomarker discovery.

Gene expression variability in long-term survivors of childhood cancer and cancer-free controls in response to ionizing irradiation.

BACKGROUND: Differential expression analysis is usually adjusted for variation. However, most studies that examined the expression variability (EV) have used computations affected by low expression levels and did not examine healthy tissue. This study aims to calculate and characterize an unbiased EV in primary fibroblasts of childhood cancer survivors and cancer-free controls (N0) in response to ionizing radiation. METHODS: Human skin fibroblasts of 52 donors with a first primary neoplasm in childhood (N1), 52 donors with at least one second primary neoplasm (N2 +), as well as 52 N0 were obtained from the KiKme case-control study and exposed to a high (2 Gray) and a low dose (0.05 Gray) of X-rays and sham- irradiation (0 Gray). Genes were then classified as hypo-, non-, or hyper-variable per donor group and radiation treatment, and then examined for over-represented functional signatures. RESULTS: We found 22 genes with considerable EV differences between donor groups, of which 11 genes were associated with response to ionizing radiation, stress, and DNA repair. The largest number of genes exclusive to one donor group and variability classification combination were all detected in N0: hypo-variable genes after 0 Gray (n = 49), 0.05 Gray (n = 41), and 2 Gray (n = 38), as well as hyper-variable genes after any dose (n = 43). While after 2 Gray positive regulation of cell cycle was hypo-variable in N0, (regulation of) fibroblast proliferation was over-represented in hyper-variable genes of N1 and N2+. In N2+, 30 genes were uniquely classified as hyper-variable after the low dose and were associated with the ERK1/ERK2 cascade. For N1, no exclusive gene sets with functions related to the radiation response were detected in our data. CONCLUSION: N2+ showed high degrees of variability in pathways for the cell fate decision after genotoxic insults that may lead to the transfer and multiplication of DNA-damage via proliferation, where apoptosis and removal of the damaged genome would have been appropriate. Such a deficiency could potentially lead to a higher vulnerability towards side effects of exposure to high doses of ionizing radiation, but following low-dose applications employed in diagnostics, as well.

Proteomics biomarker discovery for individualized prevention of familial pancreatic cancer using statistical learning.

BACKGROUND: The low five-year survival rate of pancreatic ductal adenocarcinoma (PDAC) and the low diagnostic rate of early-stage PDAC via imaging highlight the need to discover novel biomarkers and improve the current screening procedures for early diagnosis. Familial pancreatic cancer (FPC) describes the cases of PDAC that are present in two or more individuals within a circle of first-degree relatives. Using innovative high-throughput proteomics, we were able to quantify the protein profiles of individuals at risk from FPC families in different potential pre-cancer stages. However, the high-dimensional proteomics data structure challenges the use of traditional statistical analysis tools. Hence, we applied advanced statistical learning methods to enhance the analysis and improve the results' interpretability. METHODS: We applied model-based gradient boosting and adaptive lasso to deal with the small, unbalanced study design via simultaneous variable selection and model fitting. In addition, we used stability selection to identify a stable subset of selected biomarkers and, as a result, obtain even more interpretable results. In each step, we compared the performance of the different analytical pipelines and validated our approaches via simulation scenarios. RESULTS: In the simulation study, model-based gradient boosting showed a more accurate prediction performance in the small, unbalanced, and high-dimensional datasets than adaptive lasso and could identify more relevant variables. Furthermore, using model-based gradient boosting, we discovered a subset of promising serum biomarkers that may potentially improve the current screening procedure of FPC. CONCLUSION: Advanced statistical learning methods helped us overcome the shortcomings of an unbalanced study design in a valuable clinical dataset. The discovered serum biomarkers provide us with a clear direction for further investigations and more precise clinical hypotheses regarding the development of FPC and optimal strategies for its early detection.

Circulating Inflammatory, Mitochondrial Dysfunction, and Senescence-Related Markers in Older Adults with Physical Frailty and Sarcopenia: A BIOSPHERE Exploratory Study.

Multisystem derangements encompassing musculoskeletal, stress, and metabolic response have been described in older adults with physical frailty and sarcopenia (PF&S). Whether PF&S is also associated with markers of cellular senescence has yet to be explored. To address this research question, we quantified the serum levels of selected inflammatory, mitochondrial, and senescence-associated secretory phenotype (SASP)-related factors in 22 older adults with PF&S (mean age 75.5 ± 4.7 years; 81.8% women) and 27 nonPF&S controls (mean age 75.0 ± 4.4 years; 62.9% women) and evaluated their association with PF&S. Markers of inflammation (interleukin (IL)1-ß, IL6, and tumor necrosis factor α (TNF-α)), matrix remodeling (Serpin E1, intercellular adhesion molecule 1 (ICAM-1), and tissue inhibitor of metalloproteinases 1 (TIMP-1)), mitochondrial dysfunction (growth/differentiation factor 15 (GDF15) and fibroblast growth factor 21 (FGF21)), Activin A, and glial fibrillary acidic protein (GFAP) were assayed. Serum levels of TNF-α and those of the SASP-related factors ICAM-1 and TIMP-1 were found to be higher, while IL1-ß and IL6 were lower in PF&S participants compared with controls. Partial least squares discriminant analysis allowed discrimination of PF&S from nonPF&S participants with 74.0 ± 3.4% accuracy. Markers that significantly contributed to the classification were ICAM-1, TIMP-1, TNF-α, GFAP, and IL6. Future studies are warranted to establish whether inflammatory and SASP-related pathways are causally linked to the development and progression of PF&S, and may represent new targets for interventions.

Radiation-response in primary fibroblasts of long-term survivors of childhood cancer with and without second primary neoplasms: the KiKme study.

BACKGROUND: The etiology and most risk factors for a sporadic first primary neoplasm in childhood or subsequent second primary neoplasms are still unknown. One established causal factor for therapy-associated second primary neoplasms is the exposure to ionizing radiation during radiation therapy as a mainstay of cancer treatment. Second primary neoplasms occur in 8% of all cancer survivors within 30 years after the first diagnosis in Germany, but the underlying factors for intrinsic susceptibilities have not yet been clarified. Thus, the purpose of this nested case-control study was the investigation and comparison of gene expression and affected pathways in primary fibroblasts of childhood cancer survivors with a first primary neoplasm only or with at least one subsequent second primary neoplasm, and controls without neoplasms after exposure to a low and a high dose of ionizing radiation. METHODS: Primary fibroblasts were obtained from skin biopsies from 52 adult donors with a first primary neoplasm in childhood (N1), 52 with at least one additional primary neoplasm (N2+), as well as 52 without cancer (N0) from the KiKme study. Cultured fibroblasts were exposed to a high [2 Gray (Gy)] and a low dose (0.05 Gy) of X-rays. Messenger ribonucleic acid was extracted 4 h after exposure and Illumina-sequenced. Differentially expressed genes (DEGs) were computed using limma for R, selected at a false discovery rate level of 0.05, and further analyzed for pathway enrichment (right-tailed Fisher's Exact Test) and (in-) activation (z ≥|2|) using Ingenuity Pathway Analysis. RESULTS: After 0.05 Gy, least DEGs were found in N0 (n = 236), compared to N1 (n = 653) and N2+ (n = 694). The top DEGs with regard to the adjusted p-value were upregulated in fibroblasts across all donor groups (SESN1, MDM2, CDKN1A, TIGAR, BTG2, BLOC1S2, PPM1D, PHLDB3, FBXO22, AEN, TRIAP1, and POLH). Here, we observed activation of p53 Signaling in N0 and to a lesser extent in N1, but not in N2+. Only in N0, DNA (excision-) repair (involved genes: CDKN1A, PPM1D, and DDB2) was predicted to be a downstream function, while molecular networks in N2+ were associated with cancer, as well as injury and abnormalities (among others, downregulation of MSH6, CCNE2, and CHUK). After 2 Gy, the number of DEGs was similar in fibroblasts of all donor groups and genes with the highest absolute log2 fold-change were upregulated throughout (CDKN1A, TIGAR, HSPA4L, MDM2, BLOC1SD2, PPM1D, SESN1, BTG2, FBXO22, PCNA, and TRIAP1). Here, the p53 Signaling-Pathway was activated in fibroblasts of all donor groups. The Mitotic Roles of Polo Like Kinase-Pathway was inactivated in N1 and N2+. Molecular Mechanisms of Cancer were affected in fibroblasts of all donor groups. P53 was predicted to be an upstream regulator in fibroblasts of all donor groups and E2F1 in N1 and N2+. Results of the downstream analysis were senescence in N0 and N2+, transformation of cells in N0, and no significant effects in N1. Seven genes were differentially expressed in reaction to 2 Gy dependent on the donor group (LINC00601, COBLL1, SESN2, BIN3, TNFRSF10A, EEF1AKNMT, and BTG2). CONCLUSION: Our results show dose-dependent differences in the radiation response between N1/N2+ and N0. While mechanisms against genotoxic stress were activated to the same extent after a high dose in all groups, the radiation response was impaired after a low dose in N1/N2+, suggesting an increased risk for adverse effects including carcinogenesis, particularly in N2+.

MALDI Mass Spectrometry Imaging Highlights Specific Metabolome and Lipidome Profiles in Salivary Gland Tumor Tissues.

Salivary gland tumors are relatively uncommon neoplasms that represent less than 5% of head and neck tumors, and about 90% are in the parotid gland. The wide variety of histologies and tumor characteristics makes diagnosis and treatment challenging. In the present study, Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to discriminate the pathological regions of patient-derived biopsies of parotid neoplasms by metabolomic and lipidomic profiles. Fresh frozen parotid tissues were analyzed by MALDI time-of-flight (TOF) MSI, both in positive and negative ionization modes, and additional MALDI-Fourier-transform ion cyclotron resonance (FT-ICR) MSI was carried out for metabolite annotation. MALDI-TOF-MSI spatial segmentation maps with different molecular signatures were compared with the histologic annotation. To maximize the information related to specific alterations between the pathological and healthy tissues, unsupervised (principal component analysis, PCA) and supervised (partial least squares-discriminant analysis, PLS-DA) multivariate analyses were performed presenting a 95.00% accuracy in cross-validation. Glycerophospholipids significantly increased in tumor tissues, while sphingomyelins and triacylglycerols, key players in the signaling pathway and energy production, were sensibly reduced. In addition, a significant increase of amino acids and nucleotide intermediates, consistent with the bioenergetics request of tumor cells, was observed. These results underline the potential of MALDI-MSI as a complementary diagnostic tool to improve the specificity of diagnosis and monitoring of pharmacological therapies.

IRF4 deficiency vulnerates B-cell progeny for leukemogenesis via somatically acquired Jak3 mutations conferring IL-7 hypersensitivity.

The processes leading from disturbed B-cell development to adult B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) remain poorly understood. Here, we describe Irf4-/- mice as prone to developing BCP-ALL with age. Irf4-/- preB-I cells exhibited impaired differentiation but enhanced proliferation in response to IL-7, along with reduced retention in the IL-7 providing bone marrow niche due to decreased CXCL12 responsiveness. Thus selected, preB-I cells acquired Jak3 mutations, probably following irregular AID activity, resulting in malignant transformation. We demonstrate heightened IL-7 sensitivity due to Jak3 mutants, devise a model to explain it, and describe structural and functional similarities to Jak2 mutations often occurring in human Ph-like ALL. Finally, targeting JAK signaling with Ruxolitinib in vivo prolonged survival of mice bearing established Irf4-/- leukemia. Intriguingly, organ infiltration including leukemic meningeosis was selectively reduced without affecting blood blast counts. In this work, we present spontaneous leukemogenesis following IRF4 deficiency with potential implications for high-risk BCP-ALL in adult humans.

Utility of Plasma Protein Biomarkers and Mid-infrared Spectroscopy for Diagnosing Fracture-related Infections: A Pilot Study.

OBJECTIVES: To compare a large panel of plasma protein inflammatory biomarkers and mid-infrared (MIR) spectral patterns in patients with confirmed fracture-related infections (FRIs) with those in controls without infection. DESIGN: Prospective case-control study. SETTING: Academic, Level 1 trauma center. PATIENTS: Thirteen patients meeting confirmatory FRI criteria were matched to 13 controls based on age, time after surgery, and fracture region. INTERVENTION: Plasma levels of 49 proteins were measured using enzyme-linked immunosorbent assay techniques. Fourier transform infrared spectroscopy of dried films was used to obtain MIR spectra of plasma samples. MAIN OUTCOME MEASUREMENTS: The main outcome measurements included plasma protein levels and MIR spectra of samples. RESULTS: Multivariate analysis-based predictive model developed using enzyme-linked immunosorbent assay-based biomarkers had sensitivity, specificity, and accuracy of 69.2% ± 0.0%, 99.9% ± 1.0%, and 84.5% ± 0.6%, respectively, with platelet-derived growth factor-AB/BB, C-reactive protein, and MIG selected as the minimum number of variables explaining group differences ( P < 0.05). Sensitivity, specificity, and accuracy of the predictive model based on MIR spectra were 69.9% ± 6.2%, 71.9% ± 5.9%, and 70.9% ± 4.8%, respectively, with 6 wavenumbers as explanatory variables ( P < 0.05). CONCLUSIONS: This pilot study demonstrates the feasibility of using a select panel of plasma proteins and Fourier transform infrared spectroscopy to diagnose FRIs. Preliminary data suggest that the measurement of these select proteins and MIR spectra may be potential clinical tools to detect FRIs. Further investigation of these biomarkers in a larger cohort of patients is warranted. LEVEL OF EVIDENCE: Diagnostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Dose response to methylating agents in the γH2AX, SCE and colony formation assays: Effect of MGMT and MPG overexpression.

Cells have developed diverse protective mechanisms that enable them to tolerate low doses of genotoxic compounds. DNA repair processes attenuate the mutagenic and carcinogenic effects of alkylating agents, and multiple studies indicate a key role of specific DNA repair factors and pathways in establishing non-linear dose response relationships. Using an overexpression approach, we investigated the impact of O6-methylguanine-DNA-methyltransferase (MGMT), which repairs O6-methylguanine (O6MeG) in a damage reversal reaction, and N-methylpurine-DNA glycosylase (MPG), which acts as an apical enzyme in the BER pathway, on the DNA damage response to the alkylating agents MNNG and MMS. Our data indicate a clear protective effect of MGMT against MNNG-induced nuclear γH2AX foci formation, sister chromatid exchanges (SCE) and cytotoxicity, as determined in the colony formation assay. MGMT protected with similar efficiency against MMS-induced cytotoxicity and γH2AX foci formation, but suppressed SCE induction only weakly, which indicates that recombination events induced by MMS result from other lesions than O6MeG. In contrast, overexpression of MPG had only a very mild protective effect on the cellular defense against MMS and MNNG. Collectively, our data indicate that overexpression of MGMT results in non-linear DNA damage responses to O6MeG inducers. In contrast, MPG overexpression has only minor impact on the DNA damage response to alkylating drugs, indicating that other downstream enzymes in the BER pathway are limiting.

Comprehensive biomarker profiles and chemometric filtering of urinary metabolomics for effective discrimination of prostate carcinoma from benign hyperplasia.

Prostate cancer (PCa) is the most commonly diagnosed cancer in male individuals, principally affecting men over 50 years old, and is the leading cause of cancer-related deaths. Actually, the measurement of prostate-specific antigen level in blood is affected by limited sensitivity and specificity and cannot discriminate PCa from benign prostatic hyperplasia patients (BPH). In the present paper, 20 urine samples from BPH patients and 20 from PCa patients were investigated to develop a metabolomics strategy useful to distinguish malignancy from benign hyperplasia. A UHPLC-HRMS untargeted approach was carried out to generate two large sets of candidate biomarkers. After mass spectrometric analysis, an innovative chemometric data treatment was employed involving PLS-DA classification with repeated double cross-validation and permutation test to provide a rigorously validated PLS-DA model. Simultaneously, this chemometric approach filtered out the most effective biomarkers and optimized their relative weights to yield the highest classification efficiency. An unprecedented portfolio of prostate carcinoma biomarkers was tentatively identified including 22 and 47 alleged candidates from positive and negative ion electrospray (ESI+ and ESI-) datasets. The PLS-DA model based on the 22 ESI+ biomarkers provided a sensitivity of 95 ± 1% and a specificity of 83 ± 3%, while that from the 47 ESI- biomarkers yielded an 88 ± 3% sensitivity and a 91 ± 2% specificity. Many alleged biomarkers were annotated, belonging to the classes of carnitine and glutamine metabolites, C21 steroids, amino acids, acetylcholine, carboxyethyl-hydroxychroman, and dihydro(iso)ferulic acid.

pH-degradable, bisphosphonate-loaded nanogels attenuate liver fibrosis by repolarization of M2-type macrophages.

Immune-suppressive (M2-type) macrophages can contribute to the progression of cancer and fibrosis. In chronic liver diseases, M2-type macrophages promote the replacement of functional parenchyma by collagen-rich scar tissue. Here, we aim to prevent liver fibrosis progression by repolarizing liver M2-type macrophages toward a nonfibrotic phenotype by applying a pH-degradable, squaric ester­based nanogel carrier system. This nanotechnology platform enables a selective conjugation of the highly water-soluble bisphosphonate alendronate, a macrophage-repolarizing agent that intrinsically targets bone tissue. The covalent delivery system, however, promotes the drug's safe and efficient delivery to nonparenchymal cells of fibrotic livers after intravenous administration. The bisphosphonate payload does not eliminate but instead reprograms profibrotic M2- toward antifibrotic M1-type macrophages in vitro and potently prevents liver fibrosis progression in vivo, mainly via induction of a fibrolytic phenotype, as demonstrated by transcriptomic and proteomic analyses. Therefore, the alendronate-loaded squaric ester­based nanogels represent an attractive approach for nanotherapeutic interventions in fibrosis and other diseases driven by M2-type macrophages, including cancer.