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.

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.

Rise in Postprandial GLP-1 Levels After Roux-en-Y Gastric Bypass: Involvement of the Vagus Nerve-Spleen Anti-inflammatory Axis in Type 2 Diabetic Rats.

PURPOSE: The mechanism underlying postprandial glucagon-like peptide-1 (GLP-1) changes after metabolic surgery remains mostly unclarified. This investigation aimed to address whether the vagus nerve-spleen anti-inflammatory axis is involved in the rise in postprandial GLP-1 levels in type 2 diabetes mellitus (T2DM) rats following metabolic surgery. MATERIALS AND METHODS: T2DM rat model was established with a high-fat diet and a low dose of streptozotocin and subjected to Roux-en-Y gastric bypass (RYGB) and splenic denervation. A mixed-meal tolerance test for postprandial GLP-1 response was performed. TNF-α in the plasma, spleen, and ileum was measured by ELISA, and alpha 7 nicotinic acetylcholine receptor (α7nAChR) expression in the spleen was analyzed by Western blot. RESULTS: Postprandial GLP-1 improvement by RYGB was accompanied by the reduction of TNF-α levels in spleen and ileum and up-regulation of splenic α7nAChR in T2DM rats. Splenic denervation abrogates a rise in postprandial GLP-1 levels in response to the mixed-meal challenge, along with higher TNF-α levels in spleen and ileum and down-regulation of splenicα7nAChR, compared with denervated sham rats. CONCLUSION: Our results reveal that the vagus nerve-spleen anti-inflammatory axis mediates the rise of postprandial GLP-1 response after RYGB through lowering TNF-α contents in the intestinal tissue in T2DM rats.

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.

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.

Eosinophils Suppress the Migration of T Cells Into the Brain of Plasmodium berghei-Infected Ifnar1-/- Mice and Protect Them From Experimental Cerebral Malaria.

Cerebral malaria is a potentially lethal disease, which is caused by excessive inflammatory responses to Plasmodium parasites. Here we use a newly developed transgenic Plasmodium berghei ANKA (PbAAma1OVA) parasite that can be used to study parasite-specific T cell responses. Our present study demonstrates that Ifnar1-/- mice, which lack type I interferon receptor-dependent signaling, are protected from experimental cerebral malaria (ECM) when infected with this novel parasite. Although CD8+ T cell responses generated in the spleen are essential for the development of ECM, we measured comparable parasite-specific cytotoxic T cell responses in ECM-protected Ifnar1-/- mice and wild type mice suffering from ECM. Importantly, CD8+ T cells were increased in the spleens of ECM-protected Ifnar1-/- mice and the blood-brain-barrier remained intact. This was associated with elevated splenic levels of CCL5, a T cell and eosinophil chemotactic chemokine, which was mainly produced by eosinophils, and an increase in eosinophil numbers. Depletion of eosinophils enhanced CD8+ T cell infiltration into the brain and increased ECM induction in PbAAma1OVA-infected Ifnar1-/- mice. However, eosinophil-depletion did not reduce the CD8+ T cell population in the spleen or reduce splenic CCL5 concentrations. Our study demonstrates that eosinophils impact CD8+ T cell migration and proliferation during PbAAma1OVA-infection in Ifnar1-/- mice and thereby are contributing to the protection from ECM.

Chemicalome and metabolome profiling of Chai-Gui Decoction using an integrated strategy based on UHPLC-Q-TOF-MS/MS analysis.

Traditional Chinese medicine prescriptions are widely believed to exert therapeutic benefits via a multiple-component and multiple-target mode. The systemic profiling of their in vitro chemicalome and in vivo metabolome is of great importance for further understanding their clinical value. Herein, an integrated strategy using ultra-performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry was proposed to profile the chemicalome and metabolome of Chai-Gui Decoction. Particularly, an approach combined mass defect filter, characteristic product ion filter, and neutral loss filter was adopted to identify metabolites in plasma, urine, bile, and feces by MetabolitePilot. Consequently, a total of 174 constituents were identified or tentatively characterized and 70 metabolites that related to 21 representative structural components were matched in rat biofluids. Among them, 19 prototypes and 7 metabolites that contributed to flavonoids, monoterpenes, and phenylpropanoids were detected distribution in brain, heart, kidney, liver, lung or spleen. This study provided a generally applicable approach to comprehensive investigation on chemicalome and metabolome of traditional Chinese medicine prescriptions, and offered reasonable guidelines for further screening of quality control indicators of Chai-Gui Decoction.

Surimi-like protein ingredient from porcine spleen as lean meat replacer in emulsion-type sausages.

The aim of this work was to assess the influence of a porcine spleen surimi-like protein ingredient as pork meat replacer in emulsified cooked meat products (frankfurter-type sausages). The effects of the addition of porcine spleen protein isolate (SPI) in substitution of lean meat at concentrations of 5%, 10% and 15% on the physicochemical characteristics, microstructure, textural, and sensorial properties of the sausages were investigated. The addition of SPI did not affect the emulsion stability of raw meat batters nor the proximate composition of the cooked sausages, provided that sausages are formulated considering the differences in protein and fat content between pork meat and spleen protein fraction. Results showed that SPI was successfully applied as a meat replacer up to 15% of substitution level without producing significant modification on the physicochemical and techno-functional properties (water holding capacity and instrumental texture) of sausages. Meat replacement with SPI resulted in the formation of a stable and homogeneous protein gel network. Moreover, there were no negative effects on the sensory attributes in the cooked sausages containing 15% SPI as compared to the control ones. Therefore, the results of this study confirm that SPI up to 15% can be successfully used as a lean meat substitute in meat products.

Macroscopic identification of visceral titanium pigment in an intravenous drug user.

Autopsy findings in intravenous drug addicts are quite variable and may involve a number of organ systems. Reports of the macroscopic identification at autopsy of components of tablets that have been crushed and injected are, however, exceedingly rare. The case of 34-year-old man who died of zolpidem toxicity on a background of pulmonary hypertension attributed to intravenous injections of crushed tablets is described. A very unusual finding was very fine white stippling on the cut surfaces of both the liver and spleen which was shown on energy-dispersive x-ray spectroscopy (EDS) to be titanium dioxide most likely from the coating of the zolpidem tablets. This case is significant in demonstrating titanium dioxide accumulation within organs at both macroscopic and microscopic levels, with confirmation of exposure by EDS analysis. The clinical significance of exposure to such high levels of titanium dioxide is unclear.

Molecular characterization, expression and functional analysis of yak IFITM3 gene.

IFITM3 is interferon-induced transmembrane 3, which plays an extremely key role in anti-proliferation, anti-virus and anti-tumor diseases. In this study, the yak (Bos grunniens) IFITM3 (BgIFITM3) gene contained a 5'-untranslated region (UTR) (25 bp), a coding region (441 bp), and a 3'-UTR (115 bp). The expression of BgIFITM3 gene in liver was significantly higher than that in heart, spleen, lung and kidney (P < 0.01). BgIFITM3 protein was localized on the yak hepatocyte plasma membrane, and its expression was significantly different between 1 day and 15 months of age (P < 0.05). Moreover, the prokaryotic expression vector of BgIFITM3 protein was constructed and expressed successfully, with a molecular weight of 19.5 kDa. The activities of yak hepatocyte were significantly inhibited after treating with BgIFITM3 protein (10 and 20 µg/mL) (P < 0.01). The expression levels of ERBB-2, IRS-1, PI3KR-1, AKT-1 and MAPK-3 were significantly lower after treating with 20 µg/mL BgIFITM3 protein (P < 0.05). Besides, the activities of HepG2 cells were significantly inhibited after treating with BgIFITM3 protein (1, 10 and 20 µg/mL) (P < 0.05). While, the cloning ability and migration ability of HepG2 cells were significantly inhibited after treating with 10 µg/mL BgIFITM3 protein (P < 0.05). Finally, the mitochondria of HepG2 cells was concentrated, cristae widened, and the double film density of mitochondria was increased after treating with 10 µg/mL BgIFITM3 protein. After 10 µg/mL BgIFITM3 protein treating, the expression levels of VDAC-2, VDAC-3 and p53 genes were significantly increased, but the expression level of GPX-4 gene was significantly decreased (P < 0.01). Taken together, the BgIFITM3 protein could inhibit the proliferations of yak hepatocyte and HepG2 cells by regulating the PI3K/Akt pathway or ferroptosis-related genes, respectively. These results benefit for further study of the function of BgIFITM3 protein.

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.

Characterization of pepcan-23 as pro-peptide of RVD-hemopressin (pepcan-12) and stability of hemopressins in mice.

Hemopressins ((x)-PVNFKLLSH) or peptide endocannabinoids (pepcans) can bind to cannabinoid receptors. RVD-hemopressin (pepcan-12) was shown to act as endogenous allosteric modulator of cannabinoid receptors, with opposite effects on CB1 and CB2, respectively. Moreover, the N-terminally elongated pepcan-23 was detected in different tissues and was postulated to be the pro-peptide of RVD-hemopressin. Currently, data about the pharmacokinetics, tissue distribution and stability of hemopressin-type peptides are lacking. Here we investigated the secondary structure and physiological role of pepcan-23 as precursor of RVD-hemopressin. We assessed the metabolic stability of these peptides, including hemopressin. Using LC-ESI-MS/MS, pepcan-23 was measured in mouse tissues and human whole blood (~50 pmol/mL) and in plasma was the most stable endogenous peptide containing the hemopressin sequence. Using peptide spiked human whole blood, mouse adrenal gland and liver homogenates demonstrate that pepcan-23 acts as endogenous pro-peptide of RVD-hemopressin. Furthermore, administered pepcan-23 converted to RVD-hemopressin in mice. In circular dichroism spectroscopy, pepcan-23 showed a helix-unordered-helix structure and efficiently formed complexes with divalent metal ions, in particular Cu(II) and Ni(II). Hemopressin and RVD-hemopressin were not bioavailable to the brain and showed poor stability in plasma, in agreement with their overall poor biodistribution. Acute hemopressin administration (100 mg/kg) did not modulate endogenous RVD-hemopressin/pepcan-23 levels or influence the endocannabinoid lipidome but increased 1-stearoyl-2-arachidonoyl-sn-glycerol. Overall, we show that pepcan-23 is a biological pro-peptide of RVD-hemopressin and divalent metal ions may regulate this process. Given the lack of metabolic stability of hemopressins, administration of pepcan-23 as pro-peptide may be suitable in pharmacological experiments as it is converted to RVD-hemopressin in vivo.

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.

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.

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.

Cannabinoid receptor Type 1 densities reflect social organization in Microtus.

Across many species, endocannabinoids play an important role in regulating social play, reward, and anxiety. These processes are mediated through at least two distinct cannabinoid receptors (CB), CB1 and CB2. CB1 expression is found in appreciable densities across regions of the brain that integrate memory with socio-spatial information; many of these regions have been directly linked to the neurobiology of pair bonding in monogamous species. Using receptor autoradiography, we provide the first distributional map of CB1 within the brains of closely related monogamous prairie voles and promiscuous meadow voles, and compare receptor densities across sexes and species in limbic regions. We observe CB1-specific signal using [3H] CP-55,940 and [3H] SR141716A, though the latter exhibited a lower signal to noise ratio. We confirmed the presence of CB2 in prairie vole spleen tissue using [3H] CP-55,940. However, we found no evidence of CB2 in the brain using either [3H] CP-55,940 or [3H] A-836,339. The overall distribution of putative CB1 in the brain was similar across vole species and followed the pattern of CB1 expression observed in other species-high intensity binding within the telencephalon, moderate binding within the diencephalon, and mild binding within the mesencephalon and metencephalon (aside from the cerebellar cortex). However, we found profound differences in CB1 densities across species, with prairie voles having higher CB1 binding in regions implicated in social attachment and spatial memory (e.g., periaqueductal gray, hippocampus). These findings suggest that CB1 densities, but not distribution, correlate with the social systems of vole species.

The Effect of High-Fat Diet and Exercise Intervention on the TNF-α Level in Rat Spleen.

High-fat diet (HFD) consumption can trigger chronic inflammation in some tissues. However, it remains unclear if HFD induces chronic inflammation in the spleen. This investigation aims to address the effect of HFD consumption and exercise intervention on the level of tumor necrosis factor alpha (TNF-α) in the spleen. Rats were subjected to HFD feeding and/or moderate-intensity treadmill running. The TNF-α levels in plasma and spleen were detected by ELISA. The mass and total cell numbers of the spleen were measured. In addition, the expression of TNF-α and its relevant gene mRNAs in macrophages from the spleen were analyzed by qRT-PCR. We found that HFD consumption did not significantly affect the mass and total cell numbers of the spleen. However, HFD consumption significantly increased splenic TNF-α level, the expression of TNF-α, toll-like receptor 4, and nuclear factor κB p65 mRNAs. In contrast, the expression of nicotinic acetylcholine receptor alpha 7 subunit (α7nAChR) mRNA in macrophages was downregulated. Additionally, exercise abolished the increase in splenic TNF-α level as well as the abnormal expression of TNF-α and related gene mRNAs in macrophages in HFD-fed rats. In conclusion, our results reveal that HFD consumption increases TNF-α level in the spleen, which is along with upregulation of the expression of TLR4 and NF-κB mRNAs as well as downregulation of the expression of α7nAChR mRNA in splenic macrophages in rats. Exercise abolished detrimental effects of HFD on TNF-α level in the spleen and prevented abnormal expression of these genes in the macrophages from rat spleen.

Prolonged residence of an albumin-IL-4 fusion protein in secondary lymphoid organs ameliorates experimental autoimmune encephalomyelitis.

Interleukin-4 (IL-4) suppresses the development of multiple sclerosis in a murine model of experimental autoimmune encephalomyelitis (EAE). Here, we show that, in mice with EAE, the accumulation and persistence in the lymph nodes and spleen of a systemically administered serum albumin (SA)-IL-4 fusion protein leads to higher efficacy in preventing disease development than the administration of wild-type IL-4 or of the clinically approved drug fingolimod. We also show that the SA-IL-4 fusion protein prevents immune-cell infiltration in the spinal cord, decreases integrin expression in antigen-specific CD4+ T cells, increases the number of granulocyte-like myeloid-derived suppressor cells (and their expression of programmed-death-ligand-1) in spinal cord-draining lymph nodes and decreases the number of T helper 17 cells, a pathogenic cell population in EAE. In mice with chronic EAE, SA-IL-4 inhibits immune-cell infiltration into the spinal cord and completely abrogates immune responses to myelin antigen in the spleen. The SA-IL-4 fusion protein may be prophylactically and therapeutically advantageous in the treatment of multiple sclerosis.

Unexpected death in a young man associated with a unilateral swollen leg: Pathological and toxicological findings in a fatal snakebite from Deinagkistrodon acutus (Chinese moccasin).

Deinagkistrodon acutus (D. acutus), also known as the Chinese moccasin, is a viper species found throughout the southeastern parts of China, northern Vietnam and Laos. D. acutus envenomation can result in coagulopathy and lead to death if not treated correctly. A 20-year-old man was discovered with a severely swollen left thigh with overlying dark purple, discolored skin. He was immediately transported to hospital. Laboratory examinations revealed dysfunctional coagulation and fluid-electrolyte imbalances. He died 2 h later despite resuscitation efforts. Surveillance footage revealed that he had walked through a grass field while returning home that night. Autopsy and pathological examination findings revealed a large area of muscle necrosis of the left thigh, renal tubular necrosis, and hepatocyte necrosis. Potential fang marks were found on the decedent's jeans. Due to our suspicions, we performed specific enzyme-linked immunosorbent assays (ELISA) and detected D. acutus venom in the kidneys, left thigh muscle, liver, lung, spleen, and heart tissues of the decedent. In conclusion, the clinical manifestations, autopsy, histopathological examination, ELISA, and investigation results confirmed D. acutus envenomation.