Ion Mobility-Based Enrichment-Free N-Terminomics Analysis Reveals Novel Legumain Substrates in Murine Spleen.

Aberrant levels of the asparaginyl endopeptidase legumain have been linked to inflammation, neurodegeneration, and cancer, yet our understanding of this protease is incomplete. Systematic attempts to identify legumain substrates have been previously confined to in vitro studies, which fail to mirror physiological conditions and obscure biologically relevant cleavage events. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we developed a streamlined approach for proteome and N-terminome analyses without the need for N-termini enrichment. Compared to unfractionated proteomic analysis, we demonstrate FAIMS fractionation improves N-termini identification by >2.5 fold, resulting in the identification of >2882 unique N-termini from limited sample amounts. In murine spleens, this approach identifies 6366 proteins and 2528 unique N-termini, with 235 cleavage events enriched in WT compared to legumain-deficient spleens. Among these, 119 neo-N-termini arose from asparaginyl endopeptidase activities, representing novel putative physiological legumain substrates. The direct cleavage of selected substrates by legumain was confirmed using in vitro assays, providing support for the existence of physiologically relevant extra-lysosomal legumain activity. Combined, these data shed critical light on the functions of legumain and demonstrate the utility of FAIMS as an accessible method to improve depth and quality of N-terminomics studies.

A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ.

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.

Case Series: Hepatic and Splenic Titanium Dioxide Deposition in Association With Intravenous Drug Use.

ABSTRACT: Titanium dioxide is a versatile compound that is found in a variety of consumer products, medical hardware, and pharmaceuticals. Although oral and topical ingestion of this compound is common, intravenous introduction is much less common. We present three cases where significant titanium dioxide deposits were identified in liver and splenic tissue of three decedents, all of whom died of illicit drug overdose in the same geographic area and had fentanyl and its metabolites in blood on postmortem toxicologic testing. At autopsy, liver sections had a granular texture with fine white stippling grossly, and histologic examination of hepatic and splenic tissues showed scattered patches of black granular material with pink birefringence. Energy-dispersive x-ray spectroscopy performed on these tissues revealed the presences of clusters of titanium dioxide. Immunohistochemical staining of both the liver and spleen with CD68 confirmed the titanium dioxide clusters were within macrophages. Intravenous titanium dioxide nanoparticle elimination studies in rats suggest a time sensitive period for this elimination, with a transient period of pigment deposition between 1-58 days following injection. If a time-dependent link between titanium dioxide pigment deposition within tissues and intravenous drug use can be shown, this could be a valuable tool for Pathologists.

Role of Serial Phlebotomy in the Management of Blunt Solid Organ Injury in Adults.

BACKGROUND: The management of blunt spleen and liver trauma has become increasingly nonoperative. There is no consensus on timing or duration of serial hemoglobin and hematocrit monitoring in this patient population. OBJECTIVE: This study examined the clinical utility of serial hemoglobin and hematocrit monitoring. We hypothesized that most interventions occur early in the hospital course, based on hemodynamic instability or physical examination findings rather than serial monitoring. METHODS: We conducted a retrospective cohort study of adult trauma patients with blunt spleen or liver injury from November 2014 through June 2019 at our Level II trauma center. Interventions were classified as no intervention, surgical intervention, angioembolization, or packed red blood cell transfusion. Demographics, length of stay, total blood draws, laboratory values, and clinical triggers preceding intervention were reviewed. RESULTS: A total of 143 patients were studied, of whom 73 (51%) received no intervention, 47 (33%) received an intervention within 4 hr of presentation, and 23 (16%) had interventions beyond 4 hr. Of these 23 patients, 13 received an intervention based on phlebotomy results alone. Most of these patients (n = 12, 92%) received blood transfusion without further intervention. Only one patient underwent operative intervention based on serial hemoglobin results on hospital day 2. CONCLUSION: The majority of patients with these injury patterns either require no intervention or declare themselves promptly after arrival. Serial phlebotomy after initial triage and intervention may add little value in the management of blunt solid organ injury.

Advanced Glycation End-Products (AGEs) of Lysine and Effects of Anti-TCR/Anti-TNF-α Antibody-Based Therapy in the LEW.1AR1-iddm Rat, an Animal Model of Human Type 1 Diabetes.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D). Previously, we have shown that combination with anti-TCR/anti-TNF-α antibody-based therapy re-established normoglycemia and increased proteinic arginine-dimethylation in the spleen, yet not in the pancreas. High blood glucose is often associated with elevated formation of advanced glycation end-products (AGEs) which act via their receptor (RAGE). Both anti-TCR and anti-TNF-α are inhibitors of RAGE. The aim of the present work was to investigate potential biochemical changes of anti-TCR/anti-TNF-α therapy in the LEW.1AR1-iddm rat. We determined by stable-isotope dilution gas chromatography-mass spectrometry (GC-MS) the content of free and proteinic AGEs and the Nε-monomethylation of lysine (Lys) residues in proteins of pancreas, kidney, liver, spleen and lymph nodes of normoglycemic control (ngCo, n = 6), acute diabetic (acT1D, n = 6), chronic diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Analyzed biomarkers included Lys and its metabolites Nε-carboxymethyl lysine (CML), furosine and Nε-monomethyl lysine (MML). Other amino acids were also determined. Statistical methods including ANOVA, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to evaluate the effects. Most statistical differences between the study groups were observed for spleen, pancreas and kidney, with liver and lymph nodes showing no such differences. In the pancreas, the groups differed with respect to proteinic furosine (p = 0.0289) and free CML (p = 0.0023). In the kidneys, the groups differed with respect to proteinic furosine (p = 0.0076) and CML (p = 0.0270). In the spleen, group differences were found for proteinic furosine (p = 0.0114) and free furosine (p = 0.0368), as well as for proteinic CML (p = 0.0502) and proteinic MML (p = 0.0191). The acT1D rats had lower furosine, CML and MML levels in the spleen than the rats in all other groups. This observation corresponds to the lower citrullination levels previously measured in these rats. PCA revealed diametric associations between PC1 and PC2 for spleen (r = -0.8271, p < 0.0001) compared to pancreas (r = 0.5805, p = 0.0073) and kidney (r = 0.8692, p < 0.0001). These findings underscore the importance of the spleen in this animal model of human T1D. OPLS-DA showed that in total sixteen amino acids differed in the experimental groups.

Use of a Ferritin L134P Mutant for the Facile Conjugation of Prussian Blue in the Apoferritin Cavity.

Since the bullfrog H-ferritin L134P mutant in which leucine 134 is replaced with proline was found to exhibit a flexible conformation in the C3 axis channel, homologous ferritins with the corresponding mutation have often been studied in terms of a mechanism of iron release from the mineral core within the protein cavity. Meanwhile, a ferritin mutant with the flexible channel is an attractive material in developing a method to encapsulate functional molecules larger than mononuclear ions into the protein cavity. This study describes the clathrate with a horse spleen L-ferritin L134P mutant containing Prussian blue (PB) without a frequently used technique, disassembly and reassembly of the protein subunits. The spherical shell of ferritin was confirmed in a TEM image of the clathrate. The produced clathrate (PB@L134P) was soluble in water and reproduced the spectroscopic and electrochemical properties of PB prepared using the conventional method. The catalytic activity for an oxidoreductive reaction with H2O2, one of the major applications of conventional PB, was also observed for the clathrate. The instability of PB in alkaline solutions, limiting its wide applications in aqueous media, was significantly improved in PB@L134P, showing the protective effect of the protein shell. The method developed here shows that horse spleen L-ferritin L134P is a useful scaffold to produce clathrates of three-dimensional complexes with ferritin.

Distinct kinetics for nucleotide hydrolysis in lymphocytes isolated from blood, spleen and cervical lymph nodes: Characterization of ectonucleotidase activity.

Ectonucleotidases are a plasma membrane-bound enzyme that hydrolyses extracellular adenosine triphosphate (eATP) and adenosine diphosphate (eADP) to adenosine monophosphate (AMP). It regulates normal function of lymphocytes, acts as an inflammatory marker and represents a molecular target for new therapeutics. Thus, this study sought to isolate lymphocytes from blood (BL), spleen (SL) and cervical lymph node (CLL), and characterize the eATP and eADP enzymatic hydrolysis in Wistar rats. The hydrolysis of the nucleotides occurred primarily at pH 8.0, 37°C in the presence of Ca2+ or Mg2+ . Chevillard-plot showed the hydrolysis of eATP and eADP at the same active site. The inhibitors of some classical ATDPases did not cause any significant change on enzymatic activity. Inhibitors of E-NTPDase (-1, -2, -3 isoforms) and E-NPP-1 decrease the enzyme activity in all resident lymphocytes. Furthermore, kinetic parameters (Vmax and Km) revealed that SL had significantly (P < .001) higher enzymatic activity when compared to BL and CLL. In conclusion, this study standardized kinetic values for eATP and eADP hydrolysis for resident lymphocytes isolated from BL, SL and CLL.

In vivo evaluation of anti-Leishmania activity of alkyltriazoles and alkylphosphocholines by oral route.

The failures in the treatment of leishmaniasis is an increasing problem around the world, especially related to resistance. Thus, we describe the synthesis and in vivo anti-Leishmania activity of alkylphosphocholine and alkyltriazoles; besides, their likely action mechanisms stem from some eventual inhibition of parasite enzymes using computational tools. These compounds were tested in an in vivo hamster model infected with Leishmania Leishmania infantum chagasi. Fifty days after parasite inoculation, the two compounds 12-azidedodecylphosphocholine (3) and 3-(1-(12-fluorododecyl)-1H-1,2,3-triazol-1-yl)propano-1-ol (9), were separately administered once a day as oral suspensions (25 and 12.5 mg/kg/day, respectively) during ten days, and their efficacy was compared to the reference compound pentavalent antimonial Glucantime (GLU). Compound 3 significantly reduced the number of parasites in the spleen (4.93 × 102 amastigotes/g) and liver (4.52 × 103 amastigotes/g). Compound 9 reduced the number of amastigotes in the spleen to 1.30 × 104 and 1.36 × 103 amastigotes/g in the liver. GLU was the most effective overall treatment (7.50 × 101 and 2.28 × 102 amastigotes/g in the spleen and liver, respectively). The high activity levels of these compounds in vivo may stem from their high in vitro leishmanicidal activity and lipophilicity. The in silico absorption, distribution, metabolism, and excretion studies also showed some anti-Leishmania potential. Compound 9 had more lipophilic characteristics than those of compound 3. In silico studies of the nine enzymes of compounds 3 and 9 showed significant evidence of interactions with nicotimidase and tyrosine aminotransferase, demonstrating possible inhibition enzymes present in L. (L.) infantum chagasi. These compounds could be a promising template for developing a new class of leishmanicidal agents, by oral route, and deserve further investigation to explore different therapeutic regimens.

A quantitative LC-MS/MS method for the determination of tissue brincidofovir and cidofovir diphosphate in a MuPyV-infected mouse model.

Brincidofovir (BCV) is an investigational lipid conjugate of the nucleotide analog cidofovir (CDV), which is being developed as a medical countermeasure for the treatment of smallpox. BCV is active against double-stranded DNA viruses including BK and JC viruses. Here, we validated procedures for quantifying BCV and its pharmacologically active moiety cidofovir diphosphate (CDV-PP) in mouse kidney, brain and spleen tissue homogenates. Following homogenization, BCV and CDV-PP were extracted from the tissues by protein precipitation with their stable, isotopically labeled internal standards, BCV-d6 and 13 C3 15 N2 -CDV-PP. Then, samples were analyzed for BCV by reverse-phase chromatography on a Waters Xterra MS C18 (50 × 2.1 mm, 3.5 µm particle size) column while CDV-PP was analyzed on a Thermo BioBasic AX (50 × 2.1 mm, 5 µm particle size) column using anion exchange chromatography. Detection was achieved by electrospray ionization in positive ion mode on an AB Sciex API-5000 triple quadrupole mass spectrometer. The calibration curves were linear over a range of 1.00-1,000 ng/ml homogenate and 0.050-50.0 ng/ml homogenate for BCV and CDV-PP, respectively. These methods were validated according to US Food and Drug Administration guidance for industry and may be used to characterize the tissue pharmacology of both analytes to advance its preclinical development.

Systematic Review of Cancer Targeting by Nanoparticles Revealed a Global Association between Accumulation in Tumors and Spleen.

Active targeting of nanoparticles toward tumors is one of the most rapidly developing topics in nanomedicine. Typically, this strategy involves the addition of cancer-targeting biomolecules to nanoparticles, and studies on this topic have mainly focused on the localization of such formulations in tumors. Here, the analysis of the factors determining efficient nanoparticle targeting and therapy, various parameters such as types of targeting molecules, nanoparticle type, size, zeta potential, dose, and the circulation time are given. In addition, the important aspects such as how active targeting of nanoparticles alters biodistribution and how non-specific organ uptake influences tumor accumulation of the targeted nanoformulations are discussed. The analysis reveals that an increase in tumor accumulation of targeted nanoparticles is accompanied by a decrease in their uptake by the spleen. There is no association between targeting-induced changes of nanoparticle concentrations in tumors and other organs. The correlation between uptake in tumors and depletion in the spleen is significant for mice with intact immune systems in contrast to nude mice. Noticeably, modulation of splenic and tumor accumulation depends on the targeting molecules and nanoparticle type. The median survival increases with the targeting-induced nanoparticle accumulation in tumors; moreover, combinatorial targeting of nanoparticle drugs demonstrates higher treatment efficiencies. Results of the comprehensive analysis show optimal strategies to enhance the efficiency of actively targeted nanoparticle-based medicines.

Three-Dimensional Chemical Mapping of a Single Protein in the Hydrated State with Atom Probe Tomography.

Unravelling the three-dimensional structures and compositions of biological macromolecules sheds light on their functions and also contributes to the design of future biochemical compounds and processes. Atom probe tomography (APT) is demonstrated in this research as a new and effective approach to explore the structure and chemical composition of a single protein in the hydrated state. By introducing graphene encapsulation, proteins in solution can be immobilized on a metal specimen tip, with an end radius in the range of 50 nm to allow field ionization and evaporation. Using a ferritin particle as an example, analysis of the mass spectrum and reconstructed 3D chemical maps at near-atomic resolution acquired from APT reveals the core consisting of iron and iron oxides, the peptide shell containing amino acids, and the interior interface between the iron core and the peptide shell. The quantitative distribution and proportion of iron isotopes from a single ferritin core have been determined for the first time, as well as identification of the possible sites of amino acids inside the protein shell. The complete experimental protocol is straightforward and lays a foundation for future exploration of various macromolecules in a controlled environment.

Dual Mass Spectrometric Tissue Imaging of Nanocarrier Distributions and Their Biochemical Effects.

Nanomaterial-based drug delivery vehicles are able to deliver therapeutics in a controlled, targeted manner. Currently, however, there are limited analytical methods that can detect both nanomaterial distributions and their biochemical effects concurrently. In this study, we demonstrate that matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP-MSI) can be used together to obtain nanomaterial distributions and biochemical consequences. These studies employ nanoparticle-stabilized capsules (NPSCs) loaded with siRNA as a testbed. MALDI-MSI experiments on spleen tissues from intravenously injected mice indicate that NPSCs loaded with anti-TNF-α siRNA cause changes to the lipid composition in white pulp regions of the spleen, as anticipated, based on pathways known to be affected by TNF-α, whereas NPSCs loaded with scrambled siRNA do not cause the predicted changes. Interestingly, LA-ICP-MSI experiments reveal that the NPSCs primarily localize in the red pulp, suggesting that the observed changes in lipid composition are due to diffusive rather than localized effects on TNF-α production. Such information is only accessible by combining data from the two modalities, which we accomplish by using the heme signals from MALDI-MSI and iron signals from LA-ICP-MSI to overlay the images. Several unexpected changes in lipid composition also occur in regions where the NPSCs are found, suggesting that the NPSCs themselves can influence tissue biochemistry as well.

Experimental infection by Neospora caninum in gerbil reduces activity of enzymes involved in energy metabolism.

Neospora caninum is a protozoan that has tropism for the central nervous system. The aim of this study was to determine whether experimental infection of gerbils would interfere with activity of enzymes associated with energy metabolism. We randomized 20 gerbils into two groups (ten animals per group): the control group (healthy animals; uninfected) and the infected group (experimentally infected with dose 7.8 × 102 tachyzoites of N. caninum per gerbil). On day six and twelve post-infection (PI), brain and spleen tissues were collected for biochemical and histopathological analyses. No histopathological lesions were observed in the brains of infected animals; however, inflammatory infiltrates were found in the spleen. Significantly greater levels of reactive oxygen species (ROS) were observed in the brain and spleen of infected gerbils than in the control group at 12 days PI. Cytosolic creatine kinase (CK-CYT), mitochondrial creatine kinase (CK-MIT), and pyruvate kinase (PK) activities were lower in the brains of infected gerbils than in those of the control group on day 12 PI. There was significantly less CK-CYT activity in the spleens of infected gerbils on day 6 and 12 PI. Finally, there was significantly less sodium-potassium ion pump (Na+/K+ ATPase) activity in the brains and spleens of infected gerbils on day 12 PI. These data suggest that experimental infection with N. caninum interfered with energy metabolism associated with ATP homeostasis in the brain and spleen, directly or indirectly, apparently mediated by ROS overproduction, contributing to inhibition of Na+/K+ ATPase activity.

Target gene identification and functional characterization of miR-1704 in chicken.

MiRNAs are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. SNPs in miRNA genes may lead to phenotypic variation by altering miRNA expression and their targets. In this study, miR-1704 expression profiles in nine tissues at 1 d, 6 weeks and 16 weeks old Gushi chickens were detected. MiR-1704 was widely expressed in the detection of tissues. The expression in 1 d and 6 weeks old was low abundance, while its expression at 16 weeks was very high. An rs14668705 (C > G) SNP was detected within the pre-miR-1704 in an F2 resource population of Gushi chicken crossed with Anka broiler. Bioinformatic analysis indicated that the C > G mutation could introduce a base-pair mismatch and cause the change of free energy. Experiments further revealed that the rs14668705 in precursor miR-1704 could significantly affect mature miR-1704 biogenesis and was significantly associated with body weight at the age of 0, 6, 8, 10, and 12 weeks, shank circumference at 4, 8, and 12 weeks, carcass weight, and semi-evisceration weight (p < 0.05). Insulin receptor 2 (IRS2) gene, one of the potential targets of miR-1704 was identified and further confirmed. These data suggested that miR-1704 targeted IRS2 and have an effect on body weight in chicken.

Gestation and breastfeeding in schistosomotic mice differentially alters the expression of histone deacetylases (HDACs) in adult offspring.

BACKGROUND: Breastfeeding or gestation in schistosomotic mothers can cause long-term alterations in the immune response of offspring. OBJECTIVES: Evaluate the expression of histone deacetylases (HDACs) (all classes), the production of cytokines by T and B lymphocytes and macrophages, and the frequency of CD4+CD25+FoxP3+-cells in adult offspring born and/or suckled by schistosomotic mothers. METHODS: We harvested splenocytes from offspring born to (BIM), suckled by (SIM), or born to/suckled by (BSIM) schistosomotic mothers and animals from noninfected mothers (Control) at seven-weeks old and cultured them with/without Concanavalin A. HDAC expression was evaluated by real-time quantitative polymerase chain reaction (qPCR), and cytokines and membrane markers were evaluated by fluorescence-activated cell sorting (FACS). FINDINGS: Compared to Control, BIM mice showed increased expression of HDAC9 and frequency of CD4+IL-10+-cells. The SIM group had increased expression of HDAC1, HDAC2, HDAC6, HDAC7, HDAC10, Sirt2, Sirt5, Sirt6, and Sirt7. The BSIM group only had increased HDAC10 expression. The SIM and BSIM groups exhibited decreased frequencies of CD4+IL-4+-cells and CD4+CD25+FoxP3+-cells, along with a higher frequency of CD14+IL-10+-cells and an increase in CD45R/B220+IL-10+-cells. The BSIM group also showed a high frequency of CD4+IL10+-cells. MAIN CONCLUSIONS: Breastfeeding induced the expression of HDACs from various classes involved in reducing inflammatory responses. However, gestation enhanced the expression of a single HDAC and breastfeeding or gestation appears to favour multiple IL-10-dependent pathways, but not cells with a regulatory phenotype.

Transcriptome Analysis of Paralichthys olivaceus Erythrocytes Reveals Profound Immune Responses Induced by Edwardsiella tarda Infection.

Unlike mammalian red blood cells (RBCs), fish RBCs are nucleated and thus capable of gene expression. Japanese flounder (Paralichthys olivaceus) is a species of marine fish with important economic values. Flounder are susceptible to Edwardsiella tarda, a severe bacterial pathogen that is able to infect and survive in flounder phagocytes. However, the infectivity of and the immune response induced by E. tarda in flounder RBCs are unclear. In the present research, we found that E. tarda was able to invade and replicate inside flounder RBCs in both in vitro and in vivo infections. To investigate the immune response induced by E. tarda in RBCs, transcriptome analysis of the spleen RBCs of flounder challenged with E. tarda was performed. Six sequencing libraries were constructed, and an average of 43 million clean reads per library were obtained, with 85% of the reads being successfully mapped to the genome of flounder. A total of 1720 differentially expressed genes (DEGs) were identified in E. tarda-infected fish. The DEGs were significantly enriched in diverse Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, especially those associated with immunity, disease, and infection. Ninety-one key DEGs involved in 12 immune-related pathways were found to form extensive interaction networks. Twenty-one genes that constituted the hub of the networks were further identified, which were highly regulated by E. tarda and involved in a number of immune processes, notably pathogen recognition and signal transduction, antigen processing and presentation, inflammation, and splicing. These results provide new insights into the immune role of flounder RBCs during bacterial infection.

Formalin pigment artifact deposition in autopsy tissue: predisposing factors, patterns of distribution and methods for removal.

Formalin pigment deposition is a known artifact of autopsy histology, often anecdotally associated with decomposition of bodies. However, there is minimal data within the forensic literature demonstrating an association between formalin pigment deposition and length of postmortem interval. Furthermore, there is minimal data concerning other predisposing factors and patterns of distribution of formalin pigment deposition. In this study, we compare the amount and patterns of formalin deposition on histology slides from three categories of death: 1) decomposed bodies, 2) critically ill at time of death, and 3) sudden cardiac death. We also compare the effectiveness of two relatively simple histology laboratory methods to remove formalin pigment deposition from histology slides. Amongst the three categories of death, formalin deposition was highest in the decomposed category, second highest in the critically ill category, and lowest in the sudden cardiac death category. The organs most severely affected by formalin deposition were liver/spleen/pancreas and kidneys, and the organs least affected were brain and lung. Formalin pigment deposition correlated with length of postmortem interval. Histologic patterns of formalin deposition included the endothelial lining of vessels, perinuclear compartment of neurons and myocytes, and the basal epithelial compartment of renal tubular epithelial cells. The alcoholic ammonium hydroxide method (AAH) was slightly more effective than the alkylphenol ethoxylate (APE) method for removing formalin pigment, though both methods were effective. Because formalin pigment is strongly refractile under polarized light, a polarization filter can also be useful for distinguishing formalin pigment from other pigments.

Peculiarities of Collagen Turnover in Aging BALB/c Mice.

Examination of collagen turnover in the liver, lungs, and spleen of BALB/c mice revealed the age-related differences in the levels of hydroxyproline and its fractions. The highest level of total hydroxyproline was observed in the lungs and spleen of young (2-month-old) mice. In the liver, this level attained maximum at the age of 6 months at the expense of elevation of protein-bound hydroxyproline relatively its level in 2-month-old mice. At the age of 12 months, the levels of total hydroxyproline in the liver and spleen were lower than in 6-month-old mice. The decrease in the collagen turnover rate in the liver of 12-month-old mice reflected lower levels of hydroxyproline fractions in comparison with the corresponding values in 6-month-old mice. The rates of collagen turnover in organs differed in mice of different ages: it was maximum in the lungs and spleen of young animals and in the liver of middle-aged mice.

Assessing Automated Sample Preparation Technologies for High-Throughput Proteomics of Frozen Well Characterized Tissues from Swedish Biobanks.

Large cohorts of carefully collected clinical tissue materials play a central role in acquiring sufficient depth and statistical power to discover disease-related mechanisms and biomarkers of clinical significance. Manual preparation of such large sample cohorts requires experienced laboratory personnel. This carries other possible downsides such as low throughput, high risk of errors, and low reproducibility. In this work, three automated technologies for high-throughput proteomics of frozen sectioned tissues were compared. The instruments evaluated included the Bioruptor for tissue disruption and protein extraction; the Barocycler, which is able to disrupt tissues and digest the proteins; and the AssayMAP Bravo, a microchromatography platform for protein digestion, peptide desalting, and fractionation. Wide varieties of tissue samples from rat spleen, malignant melanoma, and pancreatic tumors were used for the assessment. The three instruments displayed reproducible and consistent results, as was proven by high correlations and low coefficients of variation between technical replicates and even more importantly, between replicates that were processed in different batches or at different time points. The results from this study allowed us to integrate these technologies into an automated sample preparation workflow for large-scale proteomic studies that are currently ongoing. Data are available via ProteomeXchange with identifiers PXD010296 and PXD011295.

Natural Spleen Cell Ligandome in Transporter Antigen Processing-Deficient Mice.

Peptides generated by proteases in the cytosol must be translocated to endoplasmic reticulum lumen by the transporter associated with antigen processing (TAP) prior to their assembly with major histocompatibility complex (MHC) class I molecules. Nonfunctional TAP complexes produce a drastic decrease of the MHC class I/peptide complexes presented on the cell surface. Previously, the cellular MHC class I ligandome from TAP-deficient cell lines was determined, but similar analysis from normal tissues remains incomplete. Using high-throughput mass spectrometry to analyze the MHC-bound peptide pools isolated from ex vivo spleen cells of TAP-deficient mice, we identified 210 TAP-independent ligands naturally presented by murine MHC class I molecules. This ligandome showed increased peptide lengths, presence of multiple nested set peptides, and low theoretical MHC binding affinity. The gene ontology enrichment analysis of parental proteins of this TAP-independent subligandome showed almost exclusively enrichment in tissue-specific biological processes related to the immune system as would be expected. Also, cellular components of the extracellular space (namely proteins outside the cell but still within the organism excluding the extracellular matrix) were specifically associated with TAP-independent antigen processing from these ex vivo mice cells. In addition, functional protein association network analysis revealed low protein-protein interactions between parental proteins from the TAP-independent ligandome. Finally, predominant endoproteolytic peptidase specificity for Leu/Phe residues in the P1 position of the scissile bond at both ligand termini was found for the ex vivo TAP-independent ligands. These data indicate that the TAP-independent ligandome from ex vivo cells derives from a more diverse collection of both endoprotease activities and parental proteins and where the cell origin and contribution of the extracellular environment are more relevant than in its equivalent cell lines.