Mouse Blood Samples Research Articles

Deployment of a Reservoir-Targeted Vaccine Against Borrelia burgdorferi Reduces the Prevalence of Babesia microti Coinfection in Ixodes scapularis Ticks.

In the Northeast and upper Midwest of the United States, Babesia microti and Borrelia burgdorferi use Ixodes scapularis ticks as vector and Peromyscus leucopus mice as major reservoir host. We previously established, in a 5-year field trial, that a reservoir-targeted outer surface protein A vaccine reduces the prevalence of B. burgdorferi-infected ticks. We accessed ticks and mouse blood samples collected during the trial, extracted total DNA, and amplified the B. microti 18S rRNA gene. Vaccine deployment reduced the prevalence of ticks coinfected with B. microti and that of mice infected with B. microti. Breaking the enzootic cycle of B. burgdorferi may reduce the incidence of babesiosis.

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.

Vertically Oriented Zinc Oxide Nanorod-Based Electrolyte-Gated Field-Effect Transistor for High-Performance Glucose Sensing.

Nanomaterial-based biosensors are a promising fit for portable and field-deployable diagnosis sensor devices due to their mass production, miniaturization, and integration capabilities. However, the fabrication of highly stable and reproducible biosensor devices is challenging. In this work, we grow a vertically oriented architecture of zinc oxide nanorods onto the active working area (i.e., the channel between the source and drain) of a field-effect transistor (FET) using a low-temperature hydrothermal method. The glucose oxidase enzyme was immobilized on the zinc oxide nanorod surface by a physical adsorption method to fabricate the electrolyte-gated FET-based glucose biosensor. The electrical properties of the electrolyte-gated FET biosensor were measured with different glucose concentrations. We found a linear increase in current up to 80 mM glucose concentration with high sensitivity (74.78 μA/mMcm2) and a low detection limit (∼0.05 mM). We illustrate a highly reproducible fabrication process of zinc oxide nanorod-based FETs, where vertically grown nanorods with a higher surface-to-volume ratio enhance the enzyme immobilization, provide a microenvironment for longer enzyme activity, and translate to better glucose sensing parameters. Additionally, our electrolyte-gated FET biosensor showed promising application in freshly drawn mouse blood samples. These findings suggest a great opportunity to translate into practical high-performance biosensors for a broad range of analytes.

Co-Amorphous Formation of Simvastatin-Ezetimibe: Enhanced Physical Stability, Bioavailability and Cholesterol-Lowering Effects in LDLr-/-Mice.

Hypercholesterolemia is one of the independent risk factors for the development of cardiovascular diseases such as atherosclerosis. The treatment of hypercholesterolemia is of great significance to reduce clinical cardiovascular events and patient mortality. Simvastatin (SIM) and ezetimibe (EZE) are commonly used clinically as cholesterol-lowering drugs; however, their treatment efficacy is severely affected by their poor water solubility and low bioavailability. In this study, SIM and EZE were made into a co-amorphous system to improve their dissolution, oral bioavailability, storage stability, and cholesterol-lowering effects. The SIM-EZE co-amorphous solids (CO) were prepared successfully using the melt-quenched technique, and the physicochemical properties of CO were characterized accordingly, which exhibited improved physical stability and faster dissolution release profiles than their physical mixture (PM). In the pharmacokinetic study, the SIM-EZE CO or PM was given once by oral gavage, and mouse blood samples were collected retro-orbitally at multiple time points to determine the plasma drug concentrations. In the pharmacodynamic study, low-density lipoprotein receptor-deficient (LDLr−/−) mice were fed with a high-fat diet (HFD) for two weeks to establish a mouse model of hypercholesterolemia. Using PM as a control, we investigated the regulation of CO on plasma lipid levels in mice. Furthermore, the mice feces were collected to determine the cholesterol contents. Besides, the effect of EZE on the NPC1L1 mRNA expression level in the mouse intestines was also investigated. The pharmacokinetics results showed that the SIM-EZE CO has improved bioavailability compared to the PM. The pharmacodynamic studies showed that SIM-EZE CO significantly increased the cholesterol-lowering effects of the drugs compared to their PM. The total cholesterol excretion in the mouse feces and inhibitory effect on NCP1L1 gene expression in the mouse intestines after being given the SIM-EZE CO were more dramatic than the PM. Our study shows that the SIM-EZE CO prepared by the melt-quenched method can significantly improve the stability, bioavailability, and cholesterol-lowering efficacy with excellent development potential as a new drug formulation.

A magnetoimpedance biosensor microfluidic platform for detection of glial fibrillary acidic protein in blood for acute stroke classification

Acute stroke is the third leading cause of mortality and disability worldwide. Administration of appropriate therapy for acute stroke is critically dependent on timely classification into either ischemic or hemorrhagic subtypes, which have divergent treatment pathways. The current classification method is based on neuroimaging, which generally requires the transport of the patient to a hospital-based facility unless a mobile stroke unit is available. Plasma glial fibrillary acidic protein (GFAP) level has been identified as a useful blood-based biomarker to differentiate stroke subtypes. However, its conventional immunoassay methods are laboratory-based and time-consuming. Novel approaches for rapid stroke classification near the patients are urgently needed. Here, we report the development and testing of a microfluidic-based magnetoimpedance biosensor platform for measuring GFAP levels. The platform consists of a microfluidic chip for GFAP extraction from a blood sample and a magnetoimpedance (MI) biosensor that employs Dynabeads as a magnetic label to capture the GFAP molecules. We demonstrated the detection of recombinant GFAP protein in phosphate-buffered saline (PBS) and in mouse blood samples (detection limit 0.01 ng/mL) and of physiological GFAP in blood and plasma samples (detection limit 1.0 ng/mL) obtained from acute stroke patients. This detection level is within the range of cut-off levels required for clinical stroke subtype differentiation. This platform has the potential to be incorporated into a small device with further development to assist in the classification of acute stroke patients and clinical decision-making at the point-of-care.

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.

Haptenation of Macrophage Migration Inhibitory Factor: A Potential Biomarker for Contact Hypersensitivity.

The immunological response in contact hypersensitivity is incited by small electrophilic compounds, known as haptens, that react with endogenous proteins after skin absorption. However, the identity of hapten-modified proteins seen as immunogenic remains as yet largely unknown. In a recent study, we have for the first time identified a hapten-modified protein in the local lymph nodes of mice treated topically with the model hapten tetramethylrhodamine isothiocyanate (TRITC). The TRITC modification was located on the N-terminal proline of the protein macrophage migration inhibitory factor (MIF). The focus of the current study was to investigate the presence of the same hapten-protein conjugate in blood samples from mice treated topically with TRITC. Furthermore, TRITC modifications of the two major blood proteins, namely hemoglobin (Hb) and albumin (Alb), as well as TRITC modifications of MIF other than the N-terminal proline, were examined. Following incubation with different molar ratios of TRITC, a proteomic approach was applied to characterize conjugate formation of the three aforementioned proteins, using high resolution mass spectrometry (HRMS). The targeted screening of the TRITC-treated mice blood and lymph node samples for these sites led to the identification of only the same TRITC-MIF conjugate previously detected in the lymph nodes. No Hb and Alb conjugates were detected. Quantification of both the TRITC-modified and unmodified N-terminal peptide of MIF in blood and lymph node samples gave interesting insights of MIF’s role in murine contact hypersensitivity. Incubation of MIF with four different haptens encompassing different reactivity mechanisms and potencies, showed adduct formation at different amino acid residues, suggesting that MIF can be the preferred target for a wide variety of haptens. The present study provides essential progress toward understanding of hapten-protein conjugate formation in contact hypersensitivity and identifies hapten-modified MIF as a potential biomarker for this condition. Further investigation of MIF as a target protein can be a next step to determine if MIF is a biomarker that can be used to develop better diagnostic tools and targeted therapeutics for individuals with allergic contact dermatitis.

STING agonist enhances the efficacy of programmed death-ligand 1 monoclonal antibody in breast cancer immunotherapy by activating the interferon-β signalling pathway

ABSTRACT This study aimed to explore the role of a stimulator of interferon (IFN) gene (STING) agonist in breast cancer (BCa) immunotherapy. Clinical samples were collected from 37 patients with BCa. A tumor-bearing mouse model was established by injecting 4T1 cells into the mammary fat pad of mice. STING agonist and atezolizumab were injected in the mice twice a week for 2 weeks. Peripheral blood, tumor mass, lung, liver, brain cortex and kidney samples of the tumor-bearing mice were collected. Anti-IFN alpha receptor subunit 1 (IFNAR1) was used to treat 4T1 cells. Tumor tissues of patients with BCa exhibited lower STING and high programmed cell death protein 1 and programmed death-ligand 1 protein expressions. The STING agonist inhibited 4T1 cell growth in mice (P < 0.001) and increased the IFN-β level and phosphorylation of STING, TBK1, IRF3 and STAT1 in tumor mass of tumor-bearing mice (P < 0.001). It synergized with atezolizumab to inhibit 4T1 cell growth in mice and increased tumor necrosis factor-α, IFN-β, interleukin-10 and IFN-γ levels in the peripheral blood and tumor mass (P < 0.01). It synergized with atezolizumab to increase CD8+ cytotoxic T cells and decrease FOXP3+ Treg cells in the tumor-bearing mouse model. The STING agonist was nontoxic to the lung, liver, brain cortex and kidney. Anti-IFNAR1 reversed the STING agonist promotion on TBK1, IRF3 and STAT1 phosphorylation in 4T1 cells (P < 0.01). STING agonists enhance the efficacy of atezolizumab in BCa immunotherapy by activating the IFN-β signaling pathway.

Extracellular vesicles from helicobacter pylori-infected cells and helicobacter pylori outer membrane vesicles in atherosclerosis.

The role of H.pylori infection has been reported in various extragastric diseases, particularly, the correlation between H.pylori and atherosclerosis (AS) have received lots of attention. Some scholars demonstrated that the presence of H.pylori-specific DNA in the sclerotic plaques of atheromatous patients provides biological evidences, with indicating that H.pylori infection is a potential factor of AS. However, the underlying mechanism of H.pylori or their products cross the epithelial barriers to enter the blood circulation remains unclear. Recent studies have shown that the extracellular vesicles (EVs) derived from H.pylori-infected gastric epithelial cells encapsulated H.pylori virulence factor cytotoxin-associated gene A (CagA) and existed in the blood samples of patients or mice, which indicating that they can carry CagA into the blood circulation. Based on these findings, some researchers proposed a hypothesis that H.pylori is involved in the pathogenesis of AS via EVs-based mechanisms. In addition, outer membrane vesicles (OMVs) serve as transport vehicles to deliver H.pylori virulence factors to epithelial cells. It is necessary to discuss the role of H.pyloriOMVs in the development of AS. This review will focus on the correlation between H.pyloriinfection and AS and tried to unveil the possible role of EVs from H.pylori-infected cells and H.pyloriOMVs in the pathogenesis of AS, with a view to providing help in refining our knowledge in this aspect. All of information included in this review was retrieved from published studies on H.pyloriinfection in AS. H.pyloriinfection may be an atherosclerotic risk factor and drives researchers to reevaluate the role of H.pyloriin the pathogenesis of AS. Some findings proposed a new hypothesis that H.pylorimay be involved in the pathogenesis of AS through EVs-based mechanisms. Besides EVs from H.pylori-infected cells, whether H.pylori OMVs may play some role in the pathogenesis of AS is still remain unclear. Existing epidemiological and clinical evidence had shown that there is a possible association between H.pyloriand AS. However, except for the larger randomized controlled trials, more basic research about EVs from H.pylori-infected cells and H.pyloriOMVs is the need of the hour to unveil the possible role of H.pyloriinfection in the pathogenesis of AS.

A Joint Technology Combining the Advantages of Capillary Microsampling with Mass Spectrometry Applied to the Trans-Resveratrol Pharmacokinetic Study in Mice.

Mice are the most frequently used animals in pharmacokinetic studies; however, collecting series of blood samples from mice is difficult because of their small sizes and tiny vessels. In addition, due to the small sample size, it is problematic to perform high required quantification. Thus, present work aims to find an effective strategy for overcoming these challenges using trans-resveratrol as a tool drug. Based on the idea of a joint technology, the capillary microsampling (CMS) was chosen for blood sample collection from mice after delivery of trans-resveratrol (150 mg/kg) by gavage, and a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of trans-resveratrol and its main metabolites. All the mouse blood samples were exactly collected by CMS without obvious deviation. This provided credible samples for subsequent quantitative analysis. The HPLC-MS/MS method was found to be sensitive, accurate, and repeatable, and the pharmacokinetic parameters for all analytes were comparable with those reported in previous studies. However, the present joint technology offers the advantages of less animal damage, easy for sample preparation, and improved reliability. It has overcome some of the major limitations revealed in previous pharmacokinetic studies in mice and therefore provides a more effective option for future studies.

MicroRNA-363-3p/sphingosine-1-phosphate receptor 1 axis inhibits sepsis-induced acute lung injury via the inactivation of nuclear factor kappa-B ligand signaling.

Infection-associated inflammation and coagulation are critical pathologies in sepsis-induced acute lung injury (ALI). This study aimed to investigate the effects of microRNA-363-3p (miR-363-3p) on sepsis-induced ALI and explore the underlying mechanisms. A cecal ligation and puncture-induced septic mouse model was established. The results of this study suggested that miR-363-3p was highly expressed in lung tissues of septic mice. Knockdown of miR-363-3p attenuated sepsis-induced histopathological damage, the inflammation response and oxidative stress in lung tissues. Furthermore, knockdown of miR-363-3p reduced the formation of platelet-derived microparticles and thrombin generation in blood samples of septic mice. Downregulation of miR-363-3p suppressed sphingosine-1-phosphate receptor 1 (S1PR1) expression in lung tissues and subsequently inactivated the nuclear factor kappa-B ligand (NF-κB) signaling. A luciferase reporter assay confirmed that miR-363-3p directly targeted the 3’-untranslated region of the mouse S1pr1 mRNA. Collectively, our study suggests that inactivation of NF-κB signaling is involved in the miR-363-3p/S1PR1 axis-mediated protective effect on septic ALI.

Development of a highly efficient in-tube solid-phase microextraction system coupled with UHPLC-MS/MS for analyzing trace hydroxyl polycyclic aromatic hydrocarbons in biological samples.

Hydroxyl polycyclic aromatic hydrocarbons are considered active mutagenic and carcinogenic substances and are found in extremely low levels (ng/g) in biological samples. As a result, their determination in urine and blood samples is challenging, and a sensitive and effective method for the analysis of trace hydroxyl polycyclic aromatic hydrocarbons in complex biological matrices is required. In this work, a novel macroporous in-tube solid-phase microextraction monolith was prepared via a thiol-yne click reaction, and a highly efficient analytical method based on in-tube solid-phase microextraction coupled with UHPLC-MS/MS was developed to determine hydroxyl polycyclic aromatic hydrocarbons with low detection limits of 0.137-11.0ng/L in complex biological samples. Four hydroxyl polycyclic aromatic hydrocarbons, namely, 2-hydroxyanthraquinone, 1-hydroxypyrene, 1,8-dihydroxyanthraquinone, and 6-hydroxychrysene, were determined in the urine samples of smokers, non-smokers, and whole blood samples of mice. Satisfactory recoveries were achieved in the range of 83.1-113% with relative standard deviations of 3.2-9.7%. It was found that implementation of the macroporous monolith gave a highly efficient approach for enriching trace hydroxyl polycyclic aromatic hydrocarbons in biological samples.

Preclinical evaluation of strasseriolides A\u2013D, potent antiplasmodial macrolides isolated from Strasseria geniculata CF-247,251

BackgroundMalaria is a global health problem for which novel therapeutic compounds are needed. To this end, a recently published novel family of antiplasmodial macrolides, strasseriolides A–D, was herein subjected to in vivo efficacy studies and preclinical evaluation in order to identify the most promising candidate(s) for further development.MethodsPreclinical evaluation of strasseriolides A–D was performed by MTT-based cytotoxicity assay in THLE-2 (CRL-2706) liver cells, cardiotoxicity screening using the FluxOR™ potassium assay in hERG expressed HEK cells, LC–MS-based analysis of drug-drug interaction involving CYP3A4, CYP2D6 and CYP2C9 isoforms inhibition and metabolic stability assays in human liver microsomes. Mice in vivo toxicity studies were also accomplished by i.v. administration of the compounds (vehicle: 0.5% HPMC, 0.5% Tween 80, 0.5% Benzyl alcohol) in mice at 25 mg/kg dosage. Plasma were prepared from mice blood samples obtained at different time points (over a 24-h period), and analysed by LC-MS to quantify compounds. The most promising compounds, strasseriolides C and D, were subjected to a preliminary in vivo efficacy study in which transgenic GFP-luciferase expressing Plasmodium berghei strain ANKA-infected Swiss Webster female mice (n = 4–5) were treated 48 h post-infection with an i.p. dosage of strasseriolide C at 50 mg/kg and strasseriolide D at 22 mg/kg for four days after which luciferase activity was quantified on day 5 in an IVIS® Lumina II imager.ResultsStrasseriolides A–D showed no cytotoxicity, no carditoxicity and no drug-drug interaction problems in vitro with varying intrinsic clearance (CLint). Only strasseriolide B was highly toxic to mice in vivo (even at 1 mg/kg i.v. dosage) and, therefore, discontinued in further in vivo studies. Strasseriolide D showed statistically significant activity in vivo giving rise to lower parasitaemia levels (70% lower) compared to the controls treated with vehicle.ConclusionsAnimal efficacy and preclinical evaluation of the recently discovered potent antiplasmodial macrolides, strasseriolides A–D, led to the identification of strasseriolide D as the most promising compound for further development. Future studies dealing on structure optimization, formulation and establishment of optimal in vivo dosage explorations of this novel compound class could enhance their clinical potency and allow for progress to later stages of the developmental pipeline.

Detection of a Hemoglobin Adduct ofthe Food ContaminantFurfuryl Alcohol in Humans: Levels ofN-((Furan-2-yl)methyl)-valine in Two EpidemiologicalStudies.

Furfuryl alcohol is a heat-induced food contaminant, classified as possibly carcinogenic to humans. The proximal carcinogen 2-sulfoxymethylfuran leads to adduct formation in DNA and proteins (e.g., N-((furan-2-yl)methyl)-Val (FFA-Val) in hemoglobin). This study analyzed human erythrocyte samples from two studies for the presence of FFA-Val: the Risks and Benefits of a Vegan Diet study (RBVD; 72 adults) and the ENVIRonmental influence ON early AGEing birth cohort study (ENVIRONAGE; 100 mother-newborn pairs). In the RBVD study, FFA-Val levels are lower in vegans compared to omnivores (median 13.0 vs 15.8pmol g-1 hemoglobin, p = 0.008), and lower in non-smokers compared to smokers (median 14.1 vs 17.0pmol g-1 hemoglobin, p = 0.003). In the birth cohort, FFA-Val levels are distinctly higher in maternal compared to newborn samples (median 15.2 vs 2.2pmol g-1 hemoglobin, p < 0.001). FFA-Val, hitherto detected only in blood samples of mice, is quantifiable in all human samples, indicating a general exposure to furfuryl alcohol. The low adduct levels in blood samples from newborn children suggested that the placenta is a barrier to furfuryl alcohol. Dietary habits and tobacco smoking are two main influencing factors on the formation of FFA-Val, which may be of use as a biomarker of exposure to furfuryl alcohol.

The effect of traumatic brain injury on bone healing from a novel exosome centered perspective in a mice model

BackgroundIn patients with traumatic brain injury (TBI) combined with long bone fracture, the fracture healing is always faster than that of patients with single fracture, which is characterized by more callus growth at the fracture site and even ectopic ossification. Exosomes are nanoscale membrane vesicles secreted by cells, which contain cell-specific proteins, miRNAs, and mRNAs.MethodsIn this study, we used exosomes as the entry point to explore the mechanism of brain trauma promoting fracture healing. We established a model of tibia fracture with TBI in mice to observe the callus growth and expression of osteogenic factors at the fracture site. Blood samples of model mice were further collected, exosomes in plasma were extracted by ultra-centrifugation method, and then identified and acted on osteoblasts cultured in vitro. The effects of exosomes on osteoblast differentiation at the cell, protein and gene levels were investigated by Western Blot and q-PCR, respectively. Furthermore, miRNA sequencing of exosomes was performed to identify a pattern of miRNAs that were present at increased or decreased levels.ResultsThe results suggested that plasma exosomes after TBI had the ability to promote the proliferation and differentiation of osteoblasts, which might be due to the increased expression of osteoblast-related miRNA in exosomes. They were transmitted to the osteoblasts at the fracture site, so as to achieve the role of promoting osteogenic differentiation.ConclusionThe TBI-derived exosomes may have potential applications for promoting fracture healing in future.The Translational Potential of this ArticlePlasma exosomes early after TBI have the ability to promote osteoblast proliferation and differentiation. The mechanism may be achieved by miRNA in exosomes. Plasma exosomes may be used as breakthrough clinical treatment for delayed or non-union fractures.

Long-circulating XTEN864-annexin A5 fusion protein for phosphatidylserine-related therapeutic applications

Annexin A5 (anxA5) is a marker for apoptosis, but has also therapeutic potential in cardiovascular diseases, cancer, and, due to apoptotic mimicry, against dangerous viruses, which is limited by the short blood circulation. An 864-amino-acid XTEN polypeptide was fused to anxA5. XTEN864-anxA5 was expressed in Escherichia coli and purified using XTEN as tag. XTEN864-anxA5 was coupled with DTPA and indium-111. After intravenous or subcutaneous injection of 111In-XTEN864-anxA5, mouse blood samples were collected for blood half-life determination and organ samples for biodistribution using a gamma counter. XTEN864-anxA5 was labeled with 6S-IDCC to confirm binding to apoptotic cells using flow cytometry. To demonstrate targeting of atherosclerotic plaques, XTEN864-anxA5 was labeled with MeCAT(Ho) and administered intravenously to atherosclerotic ApoE−/− mice. MeCAT(Ho)-XTEN864-anxA5 was detected together with MeCAT(Tm)-MAC-2 macrophage antibodies by imaging mass cytometry (CyTOF) of aortic root sections. The ability of anxA5 to bind apoptotic cells was not affected by XTEN864. The blood half-life of XTEN864-anxA5 was 13 h in mice after IV injection, markedly longer than the 7-min half-life of anxA5. 96 h after injection, highest amounts of XTEN864-anxA5 were found in liver, spleen, and kidney. XTEN864-anxA5 was found to target the adventitia adjacent to atherosclerotic plaques. XTEN864-anxA5 is a long-circulating fusion protein that can be efficiently produced in E. coli and potentially circulates in humans for several days, making it a promising therapeutic drug.

Circulating myomiRs in Muscle Denervation: From Surgical to ALS Pathological Condition.

ALS is a fatal neurodegenerative disease that is associated with muscle atrophy, motoneuron degeneration and denervation. Different mechanisms have been proposed to explain the pathogenesis of the disease; in this context, microRNAs have been described as biomarkers and potential pathogenetic factors for ALS. MyomiRs are microRNAs produced by skeletal muscle, and they play an important role in tissue homeostasis; moreover, they can be released in blood circulation in pathological conditions, including ALS. However, the functional role of myomiRs in muscle denervation has not yet been fully clarified. In this study, we analyze the levels of two myomiRs, namely miR-206 and miR-133a, in skeletal muscle and blood samples of denervated mice, and we demonstrate that surgical denervation reduces the expression of both miR-206 and miR-133a, while miR-206 but not miR-133a is upregulated during the re-innervation process. Furthermore, we quantify the levels of miR-206 and miR-133a in serum samples of two ALS mouse models, characterized by different disease velocities, and we demonstrate a different modulation of circulating myomiRs during ALS disease, according to the velocity of disease progression. Moreover, taking into account surgical and pathological denervation, we describe a different response to increasing amounts of circulating miR-206, suggesting a hormetic effect of miR-206 in relation to changes in neuromuscular communication.

Development of a UPLC-MS/MS method for the determination of narciclasine and 7-deoxynarciclasine in mouse blood and its application in pharmacokinetics

In this study, we used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to measure the concentration of narciclasine and 7-deoxynarciclasine in mouse blood after intravenous (i.v.) and oral administration (p.o.), and we used this method to investigate their pharmacokinetics profiles in mice. Chromatographic separation of the analytes was achieved using a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) with a mobile phase consisting of acetonitrile–water (0.1% formic acid) by gradient elution. Electrospray ionization (ESI positive-ion mode)-tandem mass spectrometry in multiple reaction monitoring (MRM) mode was employed for quantitative analysis of the analytes in mouse blood samples. Twelve mice were administered narciclasine and 7-deoxynarciclasine (2 mg/kg) intravenously (iv), while the other twelve mice were administered narciclasine and 7-deoxynarciclasine (10 mg/kg) orally. The mouse blood was withdrawn from the caudal vein to be processed, after which the blood was analyzed by UPLC-MS/MS, and the corresponding data were fitted using the Drug and Statistics (DAS) software. Standard curves of narciclasine and 7-deoxynarciclasine were generated over the concentration range of 5–5000 ng/mL. The intra-day accuracy of narciclasine and 7-deoxynarciclasine was 90–105%, and the corresponding inter-day accuracy was 87–108%. The intra-day precision was less than 13%, while the inter-day precision was less than 14%. Matrix effects were also observed (between 94% and 104%), and the recovery calculated was higher than 70%. The developed and validated UPLC-MS/MS method was then successfully applied in determining the mouse pharmacokinetics of narciclasine and 7-deoxynarciclasine. From this, thebioavailabilityofnarciclasine and 7-deoxynarciclasinewasdetermined to be 10.3%and35.4%, respectively.

Protective Effect of Luteolin on D-Galactosamine (D-Gal)/Lipopolysaccharide (LPS) Induced Hepatic Injury by in Mice.

To observe the effects of luteolin on galactosamine (D-Gal)/lipopolysaccharide (LPS) induced liver injury in mice. Male C57BL/6 mice were randomly divided into 4 groups: normal control group, D-GaI/LPS group, D-GaI/LPS + luteolin (Lu, 20 mg/kg), and D-GaI/LPS + luteolin (Lu, 40 mg/kg). Mice in the normal control group and D-GaI/LPS group were given distilled water while other groups were given drugs in 7 days by gavage. 4 hours after the continuous administration, Gal (700 mg/kg) and LPS (10 mg/kg) were injected intraperitoneally. Mice in the normal control group were given the same volume of vegetable oil solution. 24 h after the establishment of the mice model, blood and liver samples were collected. Hematoxylin (HE) staining was used to observe the changes of hepatic histopathology. Alanine aminotransferase (ALT) and glutamic oxalacetic transaminase (AST) in serum, interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor (TNF-α) were measured by related kits. Western blotting was used to demonstrate the expression levels of related inflammation proteins. Lu significantly reduced levels of proinflammatory cytokines including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in serum and liver. Lu restored the pathological changes after galactosamine (D-Gal)/lipopolysaccharide (LPS) treatment. In addition, Lu regulated proteins levels of the NLRP3/NF-κB pathway in liver. Lu exhibited therapeutical effects on D-GaI/LPS induced liver injury in mice which might be related to the regulation of the NLRP3/NF-κB pathway.