Mouse Urine Samples Research Articles

Deciphering the chemical language of inbred and wild mouse conspecific scents.

In most mammals, conspecific chemosensory communication relies on semiochemical release within complex bodily secretions and subsequent stimulus detection by the vomeronasal organ (VNO). Urine, a rich source of ethologically relevant chemosignals, conveys detailed information about sex, social hierarchy, health, and reproductive state, which becomes accessible to a conspecific via vomeronasal sampling. So far, however, numerous aspects of social chemosignaling along the vomeronasal pathway remain unclear. Moreover, since virtually all research on vomeronasal physiology is based on secretions derived from inbred laboratory mice, it remains uncertain whether such stimuli provide a true representation of potentially more relevant cues found in the wild. Here, we combine a robust low-noise VNO activity assay with comparative molecular profiling of sex- and strain-specific mouse urine samples from two inbred laboratory strains as well as from wild mice. With comprehensive molecular portraits of these secretions, VNO activity analysis now enables us to (i) assess whether and, if so, how much sex/strain-selective 'raw' chemical information in urine is accessible via vomeronasal sampling; (ii) identify which chemicals exhibit sufficient discriminatory power to signal an animal's sex, strain, or both; (iii) determine the extent to which wild mouse secretions are unique; and (iv) analyze whether vomeronasal response profiles differ between strains. We report both sex- and, in particular, strain-selective VNO representations of chemical information. Within the urinary 'secretome', both volatile compounds and proteins exhibit sufficient discriminative power to provide sex- and strain-specific molecular fingerprints. While total protein amount is substantially enriched in male urine, females secrete a larger variety at overall comparatively low concentrations. Surprisingly, the molecular spectrum of wild mouse urine does not dramatically exceed that of inbred strains. Finally, vomeronasal response profiles differ between C57BL/6 and BALB/c animals, with particularly disparate representations of female semiochemicals.

Deciphering the chemical language of inbred and wild mouse conspecific scents

In most mammals, conspecific chemosensory communication relies on semiochemical release within complex bodily secretions and subsequent stimulus detection by the vomeronasal organ (VNO). Urine, a rich source of ethologically relevant chemosignals, conveys detailed information about sex, social hierarchy, health, and reproductive state, which becomes accessible to a conspecific via vomeronasal sampling. So far, however, numerous aspects of social chemosignaling along the vomeronasal pathway remain unclear. Moreover, since virtually all research on vomeronasal physiology is based on secretions derived from inbred laboratory mice, it remains uncertain whether such stimuli provide a true representation of potentially more relevant cues found in the wild. Here, we combine a robust low-noise VNO activity assay with comparative molecular profiling of sex- and strain-specific mouse urine samples from two inbred laboratory strains as well as from wild mice. With comprehensive molecular portraits of these secretions, VNO activity analysis now enables us to (i) assess whether and, if so, how much sex/strain-selective ‘raw’ chemical information in urine is accessible via vomeronasal sampling; (ii) identify which chemicals exhibit sufficient discriminatory power to signal an animal’s sex, strain, or both; (iii) determine the extent to which wild mouse secretions are unique; and (iv) analyze whether vomeronasal response profiles differ between strains. We report both sex- and, in particular, strain-selective VNO representations of chemical information. Within the urinary ‘secretome’, both volatile compounds and proteins exhibit sufficient discriminative power to provide sex- and strain-specific molecular fingerprints. While total protein amount is substantially enriched in male urine, females secrete a larger variety at overall comparatively low concentrations. Surprisingly, the molecular spectrum of wild mouse urine does not dramatically exceed that of inbred strains. Finally, vomeronasal response profiles differ between C57BL/6 and BALB/c animals, with particularly disparate representations of female semiochemicals.

Development of transcriptional factor-based whole-cell biosensors to monitor and degrade antibiotics using mutant cells obtained via adaptive laboratory evolution

With the widespread use of antibiotics and increasing environmental concerns regarding antibiotic abuse, the detection and degradation of antibiotic residues in various samples has become a pressing issue. Transcriptional factor (TF)-based whole-cell biosensors are low-cost, easy-to-use, and flexible tools for detecting chemicals and controlling bioprocesses. However, because of cytotoxicity caused by antibiotics, the application of such biosensors is limited in the presence of antibiotics. In this study, we used antibiotic-tolerant mutants obtained via adaptive laboratory evolution (ALE) to develop TF-based whole-cell biosensors for antibiotic monitoring and degradation. The biosensors had high performance and stability in detecting relatively high concentrations of tetracycline (Tc) and nisin. The ALE mutant-based Tc biosensor exhibited a 10-fold larger linear detection range than the wild-type strain-based biosensor. Then, the Tc biosensor was employed to detect residual amounts of Tc in mouse stool, serum, and urine samples and facilitate Tc biodegradation in mouse stool, demonstrating its high utility. Considering that ALE has been demonstrated to enhance cell tolerance to various toxic chemicals, our strategy might facilitate the development of whole-cell biosensors for most antibiotics and other toxic ligands.

Scavenging Glyoxal and Methylglyoxal by Synephrine and Neohesperidin from Flowers of Citrus aurantium L. var. amara Engl. in Mice and Humans.

There is considerable research evidence that α-dicarbonyl compounds, including glyoxal (GO) and methylglyoxal (MGO), are closely related to many chronic diseases. In this work, after comparison of the capture capacity, reaction pathway, and reaction rate of synephrine (SYN) and neohesperidin (NEO) on GO/MGO in vitro, experimental mice were administrated with SYN and NEO alone and in combination. Quantitative data from UHPLC-QQQ-MS/MS revealed that SYN/NEO/HES (hesperetin, the metabolite of NEO) could form the GO/MGO-adducts in mice (except SYN-MGO), and the levels of GO/MGO-adducts in mouse urine and fecal samples were dose-dependent. Moreover, SYN and NEO had a synergistic scavenging effect on GO in vivo by promoting each other to form more GO adducts, while SYN could promote NEO to form more MGO-adducts, although it could not form MGO-adducts. Additionally, human experiments showed that the GO/MGO-adducts of SYN/NEO/HES found in mice were also detected in human urine and fecal samples after drinking flowers of Citrus aurantium L. var. amara Engl. (FCAVA) tea using UHPLC-QTOF-MS/MS. These findings provide a novel strategy to reduce endogenous GO/MGO via the consumption of dietary FCAVA rich in SYN and NEO.

Abstract 4528: Improving carboplatin therapy: A novel albumin-targeted platinum(IV) prodrug with superior anticancer activity in vivo

Abstract Platinum(II)-based chemotherapeutics are among the most commonly used anticancer drugs and are part of nearly every second treatment scheme. However, they lack in tumor specificity, causing severe sides effects, dose-dependent toxicity as well as drug resistance development. Due to their higher tolerability, platinum(IV) prodrugs are currently in the focus of interest. However, comparable to their platinum(II) counterparts, they show insufficient tumor accumulation and are frequently prematurely activated. A promising strategy to improve tumor targeting of anticancer drugs is to exploit the enhanced consumption and accumulation of albumin in the malignant tissue. Thus, we developed a new albumin-targeted maleimide-containing platinum(IV) prodrug, which releases carboplatin in a highly tumor-specific manner. The maleimide moieties enable the drug to selectively bind the endogenous albumin via its free thiol of cysteine 34. The aim of this study was to in-depth characterize the pharmacological behavior and anticancer activity in vivo of the new prodrug. To this end, several xenograft and allograft experiments were performed. To investigate tissue distribution and pharmacokinetics serum, urine, tumor and organ samples of mice were collected after drug treatment and were evaluated by (size-exclusion chromatography) inductively-coupled plasma mass spectrometry and immunohistochemistry (e.g cleaved caspase-3). These experiments revealed that the new prodrug fast and selectively binds to the serum albumin after intravenous injection, leading to an enhanced plasma half-life and an increased tumor accumulation in comparison to its corresponding platinum(II) drug carboplatin. Additionally, the new complex resulted in superior anticancer activity and prolonged overall survival based on the distinctly improved pharmacokinetic profile and enhanced apoptosis induction. In conclusion, these data support that albumin binding is a potent tool to increase the tumor specificity of platinum(IV) drugs and prevent their premature activation in other body compartments. Consequently, this very promising prodrug will be further developed towards clinical phase I testing. Citation Format: Hemma Schueffl, Regina Weinmuellner, Nadine Sommerfeld, Bernhard K. Keppler, Walter Berger, Christian R. Kowol, Petra Heffeter. Improving carboplatin therapy: A novel albumin-targeted platinum(IV) prodrug with superior anticancer activity in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4528.

Increasing coverage of the urinary polar metabolome using ultra high-performance hydrophobic interaction liquid chromatography combined with linear and cyclic travelling wave ion mobility and mass spectrometry

The use of HILIC-based separations for the analysis of polar metabolites in metabolic phenotyping studies is well established. Here, we demonstrate the increased coverage of the polar metabolome obtained by travelling wave (TW) ion mobility (IM) instruments combined with HILIC and mass spectrometry (MS) for metabotyping rat and mouse urine samples. Profiling was performed using either a linear TW IM-MS based instrument with a path length of 40 cm or an instrument with a cyclic travelling wave analyser (cIM) with a path length of 95 cm. Due to the added resolution afforded by using both the linear and cyclic IM geometries with MS detection (IM-MS) significant increases in feature count (m/z-tR pairs) were generally obtained compared to HILIC-MS alone. In addition, the use of both linear and cyclic IM-MS improved the quality of the mass spectra obtained as a result of the separation of co-eluting analytes. As would be expected from the increased path length of the cyclic IM-MS instrument compared to the linear device, the largest gains in feature detection were obtained for the HILIC-cIM-MS combination. By increasing the resolution of coeluting components, the cyclic IM-MS instrumentation also provided the largest improvement in the quality of the mass spectral data obtained. When applied to mouse urines obtained from both control and gefitinib-dosed mice, time-related changes were detected in those obtained from the treated animals that were not seen in the controls. Polar metabolites affected by drug administration included, but were not limited to, hypoxanthine, 1,3-dimethyluracil and acetylcarnitine. The changes seen in the relative concentrations of these endogenous metabolites appeared to be related to drug concentrations in the plasma and urine suggesting a pharmacometabodynamic link.

Proteolytic Activity against the Distal Polybasic Tract of the Gamma Subunit of the Epithelial Sodium Channel ENaC in Nephrotic Urine.

Experimental nephrotic syndrome in mice leads to proteolytic activation of the epithelial sodium channel ENaC, possibly involving the distal polybasic tract of its γ-subunit (183RKRK). We sought to determine if urine samples from both nephrotic mice and a cohort of patients with acute nephrotic syndrome contain a specific proteolytic activity against this region of γ-ENaC. A peptide substrate consisting of amino acids 180-194 of murine γ-ENaC was N-terminally coupled to a fluorophore, yielding AMCA-FTGRKRKISGKIIHK. The substrate was incubated with nephrotic urine samples from mice as well as patients with or without the serine protease inhibitor, aprotinin. The digested peptides were separated on a reverse phase HPLC and detected with a fluorescence detector (350/450 nm). Peptide masses of the peaks were determined with a MALDI-TOF mass spectrometer. In addition, urinary proteolytic activity was quantitated using AMC-coupled substrates reflecting different cleavage sites within the polybasic tract. No significant proteolytic activity against the substrate was found in the urine of healthy humans or mice. Incubation with urine samples of nephrotic patients (n = 8) or mice subjected to three different models of experimental nephrotic syndrome (n = 4 each) led to cleavage of the substrate within the polybasic tract prevented by the serine protease inhibitor aprotinin. The most dominant cleavage product was FTGRKR in both species, which was confirmed using quantitative measurements with FTGRKR- AMC. Nephrotic urine from both humans and mice contains aprotinin-sensitive proteolytic activity against the distal polybasic tract of γ-ENaC, reflecting excretion of active proteases in the urine or proteasuria.

Feature MS fragments-based method for identification of toxic furanoids in biological samples

Numerous furan-containing compounds have been reported to be toxic. The toxicity may be attributed to the metabolic activation of the furan ring to cis-enediones. Identification of unknown furans that undergo bioactivation is challenging. Here, we present a novel approach that enables non-targeted profiling of bioactivation of unknown furanoids both in vitro and in vivo. Cyclic pyrrole-glutathione conjugate was the predominant product of cis-enediones with glutathione. The shared glutathione substructure of conjugates was capable of generating four constant and signature fragments under collision-induced dissociation (CID) in the mass spectrometer, including neutral loss fragments 103.0269 Da and 146.0691 Da and product ions at m/z 130.0499 and 177.0328. The unique structure and high abundance of conjugates in combination with the consistency and specificity of CID fragmentation brought extraordinarily high selectivity and reliability for the four fragments as a fingerprint of bioactivated furanoids. The bioactivated furanoids can be identified by screening the four fragments in high-resolution MS/MS datasets using the neutral loss filtering and diagnostic fragmentation filtering of data post-acquisition software MZmine. The simultaneous formation of four individual signal points in the filtering channel with the same precursor ion and retention time was assigned to be furanoids. The method has been rigorously validated. In the pooled urine samples from nine model furanoids-treated mice, nine cis-enediones from the parent furanoids and two from furanoid metabolites were accurately detected and identified. The method showed great performance in non-targeted profiling bioactivated furanoids and their metabolites in urine samples of herbal extract-treated mice.

Dapagliflozin Ameliorates Renal Tubular Ferroptosis in Diabetes via SLC40A1 Stabilization

Tubular injury has been shown to play a critical role in the morbidity of diabetic kidney disease (DKD); ferroptosis often occurs in tubules during renal disease development. This study was aimed at evaluating the inhibitory effects and potential mechanism of dapagliflozin (DAPA) against diabetic-related ferroptosis in the kidney. C57BL/6 mice were fed a high-fat diet (HFD) for 12 weeks, administered a small dose of streptozocin (STZ) for three consecutive days by intraperitoneal injection, and then orally administered dapagliflozin (10 mg/kg/day) for 8 weeks. Mouse blood and urine samples were collected, and their renal cortices were harvested for subsequent investigations. The effects of DAPA were also evaluated in HK-2 cells subjected to simulated diabetic conditions through excess glucose or palmitic acid (PA) administration. DAPA significantly ameliorated tubular injury independently of glycemic control in diabetic model mice. In vivo and in vitro investigations showed that dapagliflozin ameliorated tubular injury by inhibiting ferroptosis. Docking experiments demonstrated that dapagliflozin and SLC40A1 could bind with each other and may consequently reduce ubiquitination degradation. In conclusion, in this study, the tubular protective effects of DAPA, irrespective of glycemic control, were observed in a diabetic mouse model. DAPA ameliorated ferroptosis during diabetic tubular injury via SLC40A1 stabilization, and this may be the mechanism underlying its action. To the best of our knowledge, this is the first study to investigate the ferroptosis inhibitory effects of DAPA in the treatment of DKD.

Pharmacokinetics and Main Metabolites of Anwulignan in Mice.

Anwulignan is a representative component of Chinese traditional medicine Schisandra sphenanthera, with strong pharmacological activities. However, there are few reports on its pharmacokinetics and metabolites in the body. In this study, a metabolomic method based on UHPLC-Q-Orbitrap-MS was used to study the pharmacokinetics of anwulignan in the blood, organs, urine, and feces samples of mice after the intragastric administration of anwulignan (10 mg/kg). The pharmacokinetic parameters were calculated, and the distribution characteristics and main metabolites of anwulignan in the body of mice were analyzed. The results showed that the retention time of anwulignan in the body of mice was longer (t 1/2 = 7.1 h), and anwulignan was widely distributed in the body (Vz/F = 32.81 L/kg), especially in the liver. The order of anwulignan concentration in the tissues of mice from high to low was the liver > heart > brain > kidney > lung > spleen. Anwulignan was mainly excreted through the digestive tract in the form of its prototype and metabolites, indicating that it might experience an enterohepatic circulation. A total of seven metabolites were identified, and the demethylation, hydroxylation, dehydroxylation, and demethoxylation were considered to be the main metabolic ways of anwulignan in the body of mice.

Novel Urinary Metabolites of Aldehydic Lipid Oxidation Products From Heated Soybean Oil Revealed by Aldehyde Abatement and Metabolomic Fingerprinting

ObjectivesHeated cooking oils are a constitutive component of contemporary human diets and a common ingredient in animal feed. Aldehydes, as the most reactive lipid oxidation products (LOPs) in heated oils, are widely considered as a contributor to the adverse health effect associated with heated oil consumption. Aldehydes-derived metabolites in biofluids are valuable for monitoring the exposure of the aldehydes from heated oils, but the efforts for their identification were hampered by the high reactivity of aldehydes as well as the chemical complexity caused by the coexistence of other LOPs in heated oils. To address this challenge, this study investigated the aldehydes-derived metabolites through the metabolomic fingerprinting of mouse urine samples from feeding heated oils with and without aldehyde abatement. MethodsThe heated soybean oil (HSO) was prepared by heating the control soybean oil (CSO) at 185°C for 6 h with constant air flow (50 ml/min). Both HSO and CSO were then mixed with the silica gel with piperazine side chain to remove their aldehyde content, producing the silica gel-treated HSO (HSO-si) and the silica gel-treated CSO (CSO-si), respectively. The urine, serum, fecal samples were collected from the C57BL/6 mice fed with 4 diets containing CSO, CSO-si, HSO, HSO-si, and then analyzed by the liquid chromatography-mass spectrometry (LC-MS) metabolomic analysis. ResultsThe silica gel treatment dramatically reduced the aldehyde contents in HSO. Novel urinary metabolites formed by the reactions between 2,4-decadienal and lysine were identified through the metabolomic comparison between the HSO samples and the samples from 3 other treatment groups. ConclusionsThe identification of novel urinary metabolites of aldehydic LOPs warrants further biochemical examination on the chemical and metabolic processes responsible for their formation. These metabolites could become the biomarkers for monitoring the exposure of aldehydes in humans and animals. Funding SourcesThe research is partially supported by the NIFA project MIN-18–125.

An integrated approach for investigating pharmacodynamic material basis of Lingguizhugan Decoction in the treatment of heart failure

Ethnopharmacological relevanceAs a classical formula of traditional Chinese medicine (TCM), Lingguizhugan Decoction (LGZGD) has been used for treating heart failure (HF) because it has an efficiency of yang-warming and fluid-dispersing. However, the pharmacodynamic material basis of LGZGD responsible for the therapeutic benefits is not well understood. Aim of the studyThe aim of this study was to elucidate the pharmacodynamic material basis of LGZGD by an integrated approach. Materials and methodsFollowing oral administration of LGZGD in mice, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) was used to identify prototype substances. A heart failure (HF) model was established, followed by an untargeted metabolomics study to determine potential targets of LGZGD. The network pharmacology method was performed to screen substances that interacted with potential targets of LGZGD treating HF. Molecular docking technology was applied to further screen substances based on binding energy. Cell viability assays were conducted to verify pharmacodynamic effects of selected substances. ResultsIn all, forty-two prototype substances were identified in the blood, urine, and fecal samples of mice. A total of fifty-five differential metabolites were identified using heart tissue untargeted metabolomics. Twenty-five substances of LGZGD were screened relating to thirty-three targets treating HF. Twenty-two substances were filtered according to their binding energy using molecular docking technology. Cell experiments revealed cinnamaldehyde, glycyrrhetinic acid, kaempferol, daidzein, caffeic acid, and catechin could significantly improve the survival rate of H9c2 cells, which might be the pharmacodynamic material basis of LGZGD. ConclusionsA scientific approach that integrated in vivo substances identification, metabolomics, network pharmacology, molecular docking, and cell pharmacodynamic assay has been developed to study the pharmacodynamic material basis of LGZGD in the treatment of HF.

Ligation-free isothermal nucleic acid amplification

In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.

Identification of in vivo metabolites of a potential anti-rheumatoid arthritis compound, the quinazolinone derivative PD110, using ultra-high performance liquid chromatography coupled with Q-Exactive plus mass spectrometrys

The objective of this study was to identify metabolites of PD110 by UHPLC-Q-Exactive Plus MS and determine its metabolic pathways in vivo. Mouse urine, faeces, and plasma samples were collected after an intraperitoneal administration of PD110 at a single dose of 30 mg·kg−1. The metabolites were detected and identified by UHPLC-Q-Exactive Plus MS and Compound DiscovererTM 2.0 software. In total, 44 metabolites (including 31 phase-I and 13 phase-II metabolites) were preliminarily identified according to the mass accuracy (<5 ppm) and comparison of their mass spectrometry profiles. Oxidation, glucuronide conjugation, and glucoside conjugation were the main metabolic pathways of PD110 in mice.This research first focussed on the biotransformation of PD110 in mice, and its metabolites may provide pivotal information for further pharmacological and clinical studies.

Comparison of Microflow and Analytical Flow Liquid Chromatography Coupled to Mass Spectrometry Global Metabolomics Methods Using a Urea Cycle Disorder Mouse Model.

Microscale-based separations are increasingly being applied in the field of metabolomics for the analysis of small-molecule metabolites. These methods have the potential to provide improved sensitivity, less solvent waste, and reduced sample-size requirements. Ion-pair free microflow-based global metabolomics methods, which we recently reported, were further compared to analytical flow ion-pairing reagent containing methods using a sample set from a urea cycle disorder (UCD) mouse model. Mouse urine and brain homogenate samples representing healthy, diseased, and disease-treated animals were analyzed by both methods. Data processing was performed using univariate and multivariate techniques followed by analyte trend analysis. The microflow methods performed comparably to the analytical flow ion-pairing methods with the ability to separate the three sample groups when analyzed by partial least-squares analysis. The number of detected metabolic features present after each data processing step was similar between the microflow-based methods and the ion-pairing methods in the negative ionization mode. The observed analyte trend and coverage of known UCD biomarkers were the same for both evaluated approaches. The 12.5-fold reduction in sample injection volume required for the microflow-based separations highlights the potential of this method to support studies with sample-size limitations.

Diversity and complexity of mouse allergens in urine, house dust, and allergen extracts assessed with an immuno-allergomic approach.

Mouse allergy is an important cause of indoor asthma and allergic rhinoconjunctivitis. The major mouse allergen, Mus m 1, is a complex of homologous pheromone-binding lipocalins called major urinary proteins (MUPs). We analyzed the proteome of MUPs in mouse urine, commercial mouse epithelial extracts, and environmental samples using several approaches. These include as follows: two-dimensional electrophoresis and immunoblotting; liquid chromatography-high-resolution mass spectrometry (LC/HRMS); multiple reaction monitoring (MRM) mass spectrometry; and LC/HRMS analysis of glycans at the N-66 residue of MUP3. Albumin is predominant in the extracts, while MUPs are predominant in urine. LC/HRMS of 4 mouse allergen extracts revealed surprising heterogeneity. Of 22 known mouse MUPs, only 6 (MUP3, MUP4, MUP5, MUP13, MUP20, and MUP21) could be identified with MRM using unique peptides. Assessment of MUP content in urine, extracts, and dust samples showed good correlation between MRM and other methods working with different detection principles. All 6 identifiable MUPs were found in electrophoretically separated urine bands, but only MUP3 and MUP20 were above LOQ in unseparated mouse urine, and only MUP3, MUP4, and MUP20 were found in mouse epithelial extracts. Glycan heterogeneity was noted among 4 individual inbred mice: of 13 glycan structures detected, 8 were unique to one mouse, and only 2 glycan modifications were present in all 4 mice. Using mass spectrometry and MRM, mouse allergen extracts and urine samples are shown to be complex and heterogeneous. The efficacy and safety of commercial mouse allergen extracts will be improved with better controls of allergen content.

Evaluation of the Enzymatic Activity of Soluble CD13/APN and CD26/DPP4 in Serum and Urine Samples of Mice with Experimental Autoimmune Encephalomyelitis

Evaluation of the Enzymatic Activity of Soluble CD13/APN and CD26/DPP4 in Serum and Urine Samples of Mice with Experimental Autoimmune Encephalomyelitis

Tissue/Biofluid Specific Molecular Cartography of Leishmania donovani Infected BALB/c Mice: Deciphering Systemic Reprogramming.

Pathophysiology of visceral leishmaniasis (VL) is not fully understood and it has been widely accepted that the parasitic components and host immune response both contribute to the perpetuation of the disease. Host alterations during leishmaniasis is a feebly touched area that needs to be explored more to better understand the VL prognosis and diagnosis, which are vital to reduce mortality and post-infection sequelae. To address this, we performed untargeted metabolomics of Leishmania donovani (Ld) infected, uninfected and treated BALB/c mice’s tissues and biofluids to elucidate the host metabolome changes using gas chromatography–mass spectrometry. Univariate and multivariate data treatments provided numerous significant differential hits in several tissues like the brain, liver, spleen and bone marrow. Differential modulations were also observed in serum, urine and fecal samples of Ld-infected mice, which could be further targeted for biomarker and diagnostic validations. Several metabolic pathways were found to be upregulated/downregulated in infected (TCA, glycolysis, fatty acids, purine and pyrimidine, etcetera) and treated (arginine, fumaric acid, orotic acid, choline succinate, etcetera) samples. Results also illustrated several metabolites with different pattern of modulations in control, infected and treated samples as well as in different tissues/biofluids; for e.g. glutamic acid identified in the serum samples of infected mice. Identified metabolites include a range of amino acids, saccharides, energy-related molecules, etcetera. Furthermore, potential biomarkers have been identified in various tissues—arginine and fumaric acid in brain, choline in liver, 9-(10) EpOME in spleen and bone marrow, N-acetyl putrescine in bone marrow, etcetera. Among biofluids, glutamic acid in serum, hydrazine and deoxyribose in urine and 3-Methyl-2-oxo pentanoic acid in feces are some of the potential biomarkers identified. These metabolites could be further looked into for their role in disease complexity or as a prognostic marker. The presented profiling approach allowed us to attain a metabolic portrait of the individual tissue/biofluid modulations during VL in the host and represent a valuable system readout for further studies. Our outcomes provide an improved understanding of perturbations of the host metabolome interface during VL, including identification of many possible potential diagnostic and therapeutic targets.

Effect of Oxime Encapsulation on Acetylcholinesterase Reactivation: Pharmacokinetic Study of the Asoxime-Cucurbit[7]uril Complex in Mice Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry.

Oxime-based molecules are used for the treatment of patients to reactivate acetylcholinesterase (AChE) function after organophosphate intoxication. However, their efficacy is limited by low penetration through the blood-brain barrier and fast elimination. In this work, the cucurbit[7]uril (CB[7]) carrier was used for the encapsulation of the clinical agent asoxime to enhance brain bioavailability and the treatment window. We present a pharmacokinetic study of asoxime and the asoxime-CB[7] complex in an in vivo mouse model. Ultrahigh-performance liquid chromatography with electrospray ionization-mass spectrometry detection was developed to determine asoxime and CB[7] in biological fluids and tissues after thorough optimization of chromatographic conditions. The dihydroxypropane-silica stationary phase using hydrophilic interaction liquid chromatography conditions provided the best chromatographic performance. The final method was validated and applied for the pharmacokinetic study of mouse plasma, urine, bile, liver, kidney, and brain samples at different times after administration of asoxime and the asoxime-CB[7] complex. The results showed a greater than 3-fold increase in the area under the curve (AUC) in the brain for asoxime administered as a complex with CB[7] relative to that for the administration of asoxime alone. The effectiveness of the treatment strategy was evaluated using a reactivation study and a functional observatory battery. Protection of brain AChE activity is crucial for saving human lives or reducing the consequences of poisoning. The asoxime administered as a complex increased the brain activity by approximately 30% compared to that with atropine alone. CB[7] coadministration improved the AChE activity by 11%, which agrees with the higher asoxime AUC assessed in the pharmacokinetic study.

Simultaneous determination of 12 lipophilic shellfish toxins in plasma and urine by ultra-high performance liquid chromatography-tandem mass spectrometry

生物样品中脂溶性贝类毒素的检测,可为食物中毒等突发公共卫生事件的流行病学调查以及中毒者的临床救治提供技术支持。目前的研究存在目标化合物少,以及方法前处理复杂、灵敏度低等问题。该研究通过优化前处理和色谱分离技术,建立了超高效液相色谱-串联质谱法测定血浆、尿液中12种脂溶性贝类毒素的方法。实验对提取试剂以及流动相的选择进行了优化,采用乙腈对尿液和血浆样品进行提取。采用Phenomenex Kinetex C18色谱柱(50 mm×3 mm, 2.6 μm)进行分离,以0.05%(v/v)氨水水溶液、90%(v/v)乙腈水溶液为流动相,以流速0.40 mL/min梯度洗脱时,12种目标化合物分离效果最好。串联质谱的离子源为电喷雾离子(ESI)源,采用多反应监测(MRM)模式检测。12种目标物的基质效应均在0.8~1.1之间,表明该前处理方法的基质干扰低,采用外标法可对化合物进行准确定量。12种贝类毒素的线性范围为0.03~36.25 μg/L,相关系数均大于0.995。尿液检测的方法定量限为0.23~0.63 μg/L,血浆检测的方法定量限为0.31~0.84 μg/L。3个加标水平的回收率为72.7%~124.1%,日内精密度为2.1%~20.0%,日间精密度为2.1%~15.3%。利用该方法检测健康人尿液和血浆样本,以及经腹腔注射12种贝类毒素的小鼠尿液和血液样本。20份健康人样本中未检出目标物,20份小鼠样本中12种贝类毒素均有检出。该方法操作简便,样品取样量少,方法灵敏高,适用于血浆和尿液中脂溶性贝类毒素的快速检测。