Mouse Whole Blood Research Articles

Establishment of a humanized ST6GAL1 mouse model for influenza research.

This study aimed to construct and characterize a humanized influenza mouse model expressing hST6GAL1. Humanized fragments, consisting of the endothelial cell-specific K18 promoter, human ST6GAL1-encoding gene, and luciferase gene, were microinjected into the fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. The offspring were identified using PCR. Mice exhibiting elevated expression of the hST6GAL1 gene were selectively bred for propagation, and invivo analysis was performed for screening. Expression of the humanized gene was tested by performing immunohistochemical (IHC) analysis. Hematologic and biochemical analyses using the whole blood and serum of humanized hST6GAL1 mice were performed. Successful integration of the human ST6GAL1 gene into the mouse genome led to the overexpression of human SiaT ST6GAL1. Seven mice were identified as carrying copies of the humanized gene, and the invivo analysis indicated that hST6GAL1 gene expression in positive mice mirrored influenza virus infection characteristics. The IHC results revealed that hST6GAL1 was expressed in the lungs of humanized mice. Moreover, the hematologic and biochemical parameters of the positive mice were within the normal range. A humanized influenza mouse model expressing the hST6GAL1 gene was successfully established and characterized.

Integrin-linked kinase mRNA expression in circulating mononuclear cells as a biomarker of kidney and vascular damage in experimental chronic kidney disease

BackgroundTraditional biomarkers of chronic kidney disease (CKD) detect the disease in its late stages and hardly predict associated vascular damage. Integrin-linked kinase (ILK) is a scaffolding protein and a serine/threonine protein kinase that plays multiple roles in several pathophysiological processes during renal damage. However, the involvement of ILK as a biomarker of CKD and its associated vascular problems remains to be fully elucidated.MethodsCKD was induced by an adenine-rich diet for 6 weeks in mice. We used an inducible ILK knockdown mice (cKD-ILK) model to decrease ILK expression. ILK content in mice's peripheral blood mononuclear cells (PBMCs) was determined and correlated with renal function parameters and with the expression of ILK and fibrosis and inflammation markers in renal and aortic tissues. Also, the expression of five miRNAs that target ILK was analyzed in whole blood of mice.ResultsThe adenine diet increased ILK expression in PBMCs, renal cortex, and aortas, and creatinine and urea nitrogen concentrations in the plasma of WT mice, while these increases were not observed in cKD-ILK mice. Furthermore, ILK content in PBMCs directly correlated with renal function parameters and with the expression of renal and vascular ILK and fibrosis and inflammation markers. Finally, the expression of the five miRNAs increased in the whole blood of adenine-fed mice, although only four correlated with plasma urea nitrogen, and of those, three were downregulated in cKD-ILK mice.ConclusionsILK, in circulating mononuclear cells, could be a potential biomarker of CKD and CKD-associated renal and vascular damage.Graphical ILK content in circulating mononuclear cells reflects renal and vascular damage in a CKD experimental model.

Pharmacologic Reduction of Platelet Count Attenuates Whole Blood Hypofibrinolysis in a Mouse Model of Sickle Cell Disease

Increased risk of venous thrombosis is a hallmark of sickle cell disease (SCD). We previously reported that SCD dramatically alters the properties of venous clots, including resistance to tissue plasminogen activator (tPA)-mediated fibrinolysis. Using an internal fibrinolysis assay (tPA added to whole blood before clot formation), clot lysis time (CLT) was significantly (p<0.01) prolonged in clots formed ex vivo from whole blood of SCD patients (SS) (651±34 min) compared to race-matched controls (AA) (370±75). We then investigated how therapeutic RBC exchange affects CLT in SCD patients. Interestingly, despite hemoglobin A levels of approximately 50%, CLT for the pre-exchange SS clots (639±81 min) was similar to CLT recorded for blood clots from SS patients who did not undergo RBC exchange (see above). However, CLT for post-exchange SS clots was significantly shortened (240±34; p<0.05 vs pre-exchange) to the level observed for clots from AA controls. We noticed that the RBC exchange procedure not only changes the proportion of AA and SS RBCs, but also reduces both platelet counts and serum plasminogen activator inhibitor-1 (PAI-1) levels by approximately 50-60%. Importantly, exvivo restoration of the platelet number in post-exchange samples to the level observed in pre-exchange blood resulted in restoration of the hypofibrinolytic phenotype and a partial restoration of serum PAI-1 levels. Changes in platelet count after RBC exchange are quickly normalized. Therefore, in the current study, we investigated the effect of sustained reduction of platelet count on whole blood hypofibrinolysis in SCD mice. Using an external fibrinolysis assay (2.5 nM tPA added after clot formation) as described by Bonnard et al. (Sci Rep, 2017) we determined if hypofibrinolysis is also observed in Townes sickle cell mice (SS). Consistent with human SCD, the CLT was profoundly increased in ex vivo clots formed from whole blood of SS mice (n=13) compared to non-sickle (AA) control mice (n=12) (Figure 1A; first two bars). Next, we demonstrated that the cellular fraction of SS blood was responsible for resistance of the clots to tPA-mediated fibrinolysis, regardless of whether the plasma was from SS or AA mice (Figure 1A; last four bars). We then used a pharmacologic strategy to reduce platelet counts. Thrombopoietin (TPO) is a growth factor that promotes maturation of hematopoietic stem cells into megakaryocytes and thereby increases platelet production. We previously showed that silencing liver TPO expression using a liver-specific antisense oligonucleotide (TPO-ASO) resulted in a sustained 50% reduction in both plasma TPO levels and platelet counts without affecting hemostasis in both baboons and mice. In the present study, TPO-ASO (20 mg/kg subcutaneous) or vehicle control were administered to 3-month old AA and SS mice (n=3-6) on days 0, 3, 5 and 7, followed by weekly maintenance injections for the next 3 weeks. 4 weeks after initiation of treatment, mice were euthanized, and blood was collected to evaluate plasma levels of TPO and external whole blood fibrinolysis. Platelet, but no other cell count was significantly reduced in both AA (by 62%, p<0.05) and SS (by 47%, P<0.05) mice treated with TPO-ASO. Baseline plasma levels of TPO were reduced in SS mice (101±20 pg/ml) compared to AA controls (182±10; p<0.05). TPO-ASO treatment resulted in significant reduction of TPO plasma levels in both AA (21±16; p<0.01) and SS (17±9; p<0.05) mice. Interestingly, TPO-ASO-mediated reduction of platelet count did not affect CLT of ex vivo clots formed from whole blood of AA mice (Figure 1B). In contrast, TPO-ASO treatment dramatically reduced CLT in SS clots (p<0.05) (Figure 1B). In conclusion, we demonstrated that: (i) the resistance of ex vivo formed clots to tPA-mediated fibrinolysis observed in SCD patients can be reproduced in a mouse model of the disease; (i) the external fibrinolysis assay provides a robust and reliable model for studying whole blood hypofibrinolysis in SCD; (iii) hypofibrinolysis in SCD appears to be predominantly driven by the cellular fraction of blood; and (iv) platelets play an important role in the hypofibrinolytic phenotype in SCD, demonstrated by mitigation of the hypofibrinolytic phenotype in SS mice subjected to long-term reduction of platelet count. Further studies investigating the effect of platelet count reduction on venous thrombosis and vascular stasis in SS mice are currently ongoing.

UPLC-MS/MS method for Icariin and metabolites in whole blood of C57 mice: development, validation, and pharmacokinetics study.

Icariin, a Chinese medicinal herb with significant effects on Alzheimer's disease, lacks pharmacokinetic data in mice. To address this, a UPLC-MS/MS method was developed and validated for quantifying Icariin and its metabolites, Icariside I and Icariside II, in the whole blood of mice. The method processed micro-whole blood from serial collections of the same C57 mouse, with well-fitted linearity (0.25-800ngmL-1) and intra- and inter-day precision and accuracy within 15%. Short-time and autosampler stability were verified, with acceptable extraction recoveries and matrix effects over 74.55%. After intravenous administration (15mgkg-1) of Icariin in C57 mice, Icariside I and Icariside II were detected within 2min. However, after the intragastric administration (30, 90, and 150mgkg-1) of Icariin in C57 mice, Icariin and Icariside I were not detected, and Icariin was rapidly converted into Icariside II. Furthermore, the Cmax and AUC0-t of three doses (30, 90, and 150mgkg-1) of Icariside II increased as the dose increased. In conclusion, this method improves the traditional method of collecting only one blood sample from each mouse, detecting Icariin and its metabolites in the whole blood of mice, especially for serial collection of micro-whole blood.

Characteristics of radiosensitivity of organisms using parameters of redox properties of blood plasma

The dependences of the parameters of the kinetic curve of chemiluminescence of the ABAP+luminol system in the presence of blood plasma after irradiation of whole blood of Wistar rats (10 and 16 months) and mice (4 months) of the BALB/c line with in vitro X-ray radiation at a dose of 0,5-200 Gy were analyzed. The differences in the nature of the dependences of the studied parameters associated with the redox properties of blood plasma on the type of organism and its age are established. The results of the analysis of the data obtained indicate a higher radiosensitivity of mouse blood compared to rat blood, which can serve as confirmation of the radiosensitivity of the BALB/c mouse line.

Chronic treatment with the anti-diabetic drug metformin rescues impaired brain mitochondrial activity and selectively ameliorates defective cognitive flexibility in a female mouse model of Rett syndrome

Metformin is the most common anti-diabetic drug and a promising therapy for disorders beyond diabetes, including Rett syndrome (RTT), a rare neurologic disease characterized by severe intellectual disability. A 10-day-long treatment rescued aberrant mitochondrial activity and restrained oxidative stress in a female RTT mouse model. However, this treatment regimen did not improve the phenotype of RTT mice. In the present study, we demonstrate that a 4-month-long treatment with metformin (150 mg/Kg/day, delivered in drinking bottles) provides a selective normalization of cognitive flexibility defects in RTT female mice at an advanced stage of disease, but it does not affect their impaired general health status and abnormal motor skills. The 4-month-long treatment also rescues the reduced activity of mitochondrial respiratory chain complex activities, the defective brain ATP production and levels as well as the increased production of reactive oxidizing species in the whole blood of RTT mice. A significant boost of PGC-1α-dependent pathways related to mitochondrial biogenesis and antioxidant defense occurs in the brain of RTT mice that received the metformin treatment. Further studies will have to verify whether these effects may underlie its long-lasting beneficial effects on brain energy metabolism.

Design, synthesis, and antibacterial activity of derivatives of Tryptophanyl-tRNA synthetase inhibitor indolmycin

In this study, indlomycin, an inhibitor of tryptophanyl-tRNA synthetase (TrpRS), and 29 racemic indolmycin derivatives were synthesized, their antibacterial activity were evaluated against methicillin-resistant Staphylococcus aureus (S. aureus) NRS384, ATCC29213, and Escherichia coli (E. coli) ATCC25922 strains. Compounds (±)-7a, (±)-7b, (±)-7c and (±)-7e exhibited minimum inhibitory concentration (MIC) values of 1–2 μg/mL against S. aureus NRS384 and ATCC29213, exhibiting significant antibacterial activity, but none of the compounds exhibited antibacterial activity against E. coli. To investigate the effect of conformation on antibacterial activity, seven racemic compounds with good antibacterial activity were separated, and the antibacterial activity of these 14 compounds was evaluated on 25 bacterial strains. This revealed that the isomers with natural conformations (1′R, 5S) had significantly better antibacterial activity than the enantiomeric isomers and racemates. Compounds 7aa, 7ba, 7ca, and 7ea exhibited good antibacterial activity against 21 strains of S. aureus and S. epidermidis with MIC values of 0.125–2 μg/mL, which were superior to that of vancomycin, used in clinical practice. The compounds 7aa, 7ba, 7ca and 7ea were moderately bound to plasma proteins and were stable in the whole blood of CD-1 mice. In conclusion, a series of new indomycin derivatives with stronger antibacterial activity against G+ bacteria were obtained.

Prevention Effect of Protopanaxadiol-Type Saponins Saponins and Protopanaxatriol-Type Saponins on Myelosuppression Mice Induced by Cyclophosphamide.

Ginsenosides from ginseng are used as a therapeutic agent for various diseases. They enhance the immunomodulatory effect in cyclophosphamide (CP)-treated tumor disease. The structural characteristics of steroidal saponins are mainly divided into protopanaxadiol-type saponin (PDS) and protopanaxatriol-type saponin (PTS). At present, few researchers have studied which kind of saponin plays a more important role, thus, we compared the prevention effect of PDS and PTS on myelosuppression mice induced by CP. The components and contents of saponin and monosaccharide were analyzed by using ultra high performance liquid chromatography-charged aerosol detector (UPLC-CAD) and reversed phase-high performance liquid chromatography (RP-HPLC), respectively. Thirty-two mice were randomly divided into four groups, including control, model (CP), CP+PDS, and CP+PTS. The mice were orally administered with PDS or PTS for 28 days and then injected with CP saline solution on 25, 26, 27, and 28 days at a dose of 50 mg × kg−1. After the end of modeling, the whole blood of mice from the ophthalmic venous plexus was collected to detect routine blood tests, inflammatory cytokines, and hematopoiesis-related cytokines. Cell cycle and the apoptosis of bone marrow in the right femur were detected. The spleen and thymus were used to calculate the organ index and histological examination, and splenocytes were used to detect the percentage of CD4+ and CD25+ T cells. In the saponins analysis, PDS mainly included the Rb1, Rc, Rb2, and Rd of protopanaxadiol-type ginsenosides (accounted for 91.64%), and PTS mainly included the Re, Rg1, and Rf of protopanaxatriol-type ginsenosides (accounted for 75.46%). The animal results showed that both PDS and PTS improved the most indicators of myelosuppression mice induced by CP, including increased weight, blood cell numbers, hematopoiesis-related cytokines, and inflammatory cytokines; promoted the cell cycle of bone marrow and inhibited the apoptosis of bone marrow; elevated the spleen and thymus indexes and CD4+ count of splenocytes. The prevention effect of PDS was better than PTS in some indicators, such as red blood cells, hemoglobin, interleukin (IL)-1β, IL-4, IL-10, tumor necrosis factor-α, CD4+, and thymus index. These results suggest both PDS and PTS can prevent myelosuppression of mice induced by CP. Meanwhile, PDS and its metabolite showed higher bioavailability and bioactivity compared with PTS.

Downregulation of Erythrocyte miR-210 Induces Endothelial Dysfunction in Type 2 Diabetes.

Red blood cells (RBC) act as mediators of vascular injury in type 2 diabetes mellitus (T2DM). miR-210 plays a protective role in cardiovascular homeostasis and is decreased in whole blood of T2DM mice. We hypothesized that downregulation of RBC miR-210 induces endothelial dysfunction in T2DM. RBC were coincubated with arteries and endothelial cells ex vivo and transfused in vivo to identify the role of miR-210 and its target protein tyrosine phosphatase 1B (PTP1B) in endothelial dysfunction. RBC from patients with T2DM and diabetic rodents induced endothelial dysfunction ex vivo and in vivo. miR-210 levels were lower in human RBC from patients with T2DM (T2DM RBC) than in RBC from healthy subjects. Transfection of miR-210 in human T2DM RBC rescued endothelial function, whereas miR-210 inhibition in healthy subjects RBC or RBC from miR-210 knockout mice impaired endothelial function. Human T2DM RBC decreased miR-210 expression in endothelial cells. miR-210 expression in carotid artery plaques was lower in T2DM patients than in patients without diabetes. Endothelial dysfunction induced by downregulated RBC miR-210 involved PTP1B and reactive oxygen species. miR-210 mimic attenuated endothelial dysfunction induced by RBC via downregulating vascular PTP1B and oxidative stress in diabetic mice in vivo. These data reveal that the downregulation of RBC miR-210 is a novel mechanism driving the development of endothelial dysfunction in T2DM.

Licochalcone A improves the cognitive ability of mice by regulating T- and B-cell proliferation.

Licochalcone A (LA), a flavonoid found in licorice, has anticancer, antioxidant, anti-inflammatory, and neuroprotective properties. Here, we explored the effect of injecting LA into the tail vein of middle-aged C57BL/6 mice on their cognitive ability as measured by the Morris water maze (MWM) test and cerebral blood flow (CBF). The related mechanisms were assessed via RNA-seq, and T (CD3e+) and B (CD45R/B220+) cells in the spleen and whole blood were quantified via flow cytometry. LA improved the cognitive ability, according to the MWM test results, and upregulated the CBF level of treated mice. The RNA-seq results indicate that LA affected the interleukin (IL)-17 signaling pathway, which is related to T- and B-cell proliferation, and the flow cytometry data suggest that LA promoted T- and B-cell proliferation in the spleen and whole blood. We also performed immune reconstruction via a tail vein injection of lymphocytes into B-NDG (NOD-PrkdcscidIl2rgtm1/Bcge) mice before treating them with LA. We tested cognitive ability by subjecting these animals to new object recognition tests and quantified the splenic and whole blood T and B cells. Cognitive ability improved after immune reconstruction and LA treatment, and LA promoted T- and B-cell proliferation in the spleen and whole blood. This study demonstrates that LA, by activating the IL-17 signaling pathway, promotes T- and B-cell proliferation in the spleen and whole blood of mice and improves cognitive ability. Thus, LA may have immune-modulating therapeutic potential for improving cognition.

NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein.

Background: The superoxide-generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2 or gp91phox, the phagocytic isoform) was reported as a major source of oxidative stress in various human diseases. Genetic deletion is widely used to study the impact of NOX2-derived reactive oxygen species (ROS) on disease development and progression in various animal models. Here, we investigate why NOX2 knockout mice show no NOX2 activity but express NOX2 mRNA and protein. Methods and Results: Oxidative burst (NOX2-dependent formation of ROS) was measured by L-012-based chemiluminescence and was largely absent in whole blood of NOX2 knockout mice. Protein expression was still detectable in different tissues of the NOX2 knockout mice, at the expected and a slightly lower molecular weight (determined by Western blot). The NOX2 gene was even largely enhanced at its expressional level in NOX2 knockout mice. RNA sequencing revealed a modified NOX2 mRNA in the knockout mice that is obviously translated to a truncated inactive mutant enzyme. Conclusion: Although the commercial NOX2 knockout mice display no considerable enzymatic NOX2 activity, expression of the NOX2 gene (when using standard primers) and protein (when using antibodies binding to the carboxy-terminal end) can still be detected, which may lead to confusion among investigators.

Quantitative Optical Diffraction Tomography Imaging of Mouse Platelets.

Platelets are specialized anucleate cells that play a major role in hemostasis following vessel injury. More recently, platelets have also been implicated in innate immunity and inflammation by directly interacting with immune cells and releasing proinflammatory signals. It is likely therefore that in certain pathologies, such as chronic parasitic infections and myeloid malignancies, platelets can act as mediators for hemostatic and proinflammatory responses. Fortunately, murine platelet function ex vivo is highly analogous to human, providing a robust model for functional comparison. However, traditional methods of studying platelet phenotype, function and activation status often rely on using large numbers of whole isolated platelet populations, which severely limits the number and type of assays that can be performed with mouse blood. Here, using cutting edge 3D quantitative phase imaging, holotomography, that uses optical diffraction tomography (ODT), we were able to identify and quantify differences in single unlabeled, live platelets with minimal experimental interference. We analyzed platelets directly isolated from whole blood of mice with either a JAK2V617F-positive myeloproliferative neoplasm (MPN) or Leishmania donovani infection. Image analysis of the platelets indicates previously uncharacterized differences in platelet morphology, including altered cell volume and sphericity, as well as changes in biophysical parameters such as refractive index (RI) and dry mass. Together, these data indicate that, by using holotomography, we were able to identify clear disparities in activation status and potential functional ability in disease states compared to control at the level of single platelets.

Establishment of mouse lung cancer model induced by benzo[a]pyrene dynamic inhalation exposure

To establish a mouse lung cancer model induced by benzo[a]pyrene(B[a]P) dynamic inhalation exposure. A total of 96 C57 BL/6 J mice weighing 18 to 20 g were randomly divided into the control group(n=48)and the experimental group(n=48), male and female in half. The experimental group was treated with 10. 0 μg/m~3 BaP for 13 weeks or 25 weeks(6 h per day and 5 days per week) by dynamic inhalation exposure, while the control group was given dimethyl sulfoxide(DMSO). The 13 weeks or 25 weeks after B[a]P exposure, 24 mice were sacrificed and their lung tissues dissected and observed for tumor formation. During B[a]P exposure, the gas in the poisoning cabinet was collected by the active carbon tube method regularly, and the concentration of B[a]P in the poisoning cabinet was analyzed by gas chromatography mass spectrometer, and the(7 R, 8 S)-dihydroxy-(9 S, 10 R)-epoxy-7, 8, 9, 10-tetrahydrobenzo [a] pyrene(BPDE)-DNA adduct content in the whole blood of mice was determined by ELISA. Concentration of B[a]P in the dynamic inhalation cabinet was stable at(12. 794±0. 518)μg/m~3. There was no significant difference in the BPDE-DNA content in the experimental objects between 13 weeks and 25 weeks after B[a]P exposure(P>0. 05). After 25 weeks of B[a]P exposure, the lung cancer formation rate in the experimental group was significantly higher than that in the control group(P<0. 05), the tumor formation rate of female mice was up to 100%. The mice lung cancer model induced by B[a]P dynamic inhalation exposure was established successfully.

Arsenic Accumulation of Realgar Altered by Disruption of Gut Microbiota in Mice.

Objective To investigate the influence of gut microbiota on arsenic accumulation of realgar in mice. Methods Mice were treated with antibiotics to form a mouse model of gut microbial disruption. Antibiotic-treated and normally raised mice were given 15 mg/kg, 150 mg/kg, and 750 mg/kg realgar by gavage and 0.2 mg/kg and 1 mg/kg arsenic solution by subcutaneous injection for 7 days. The concentration of arsenic in mice whole blood was determined by inductively coupled plasma mass spectrometry (ICP-MS). Arsenic accumulation in antibiotic-treated mice and normally raised mice was compared. Results After exposure to low dose (15 mg/kg) and middle dose (150 mg/kg) of realgar, significantly, more arsenic was accumulated in the whole blood of antibiotic-treated mice compared to normally raised counterparts, which indicated that the disruption of gut microbiota could lead to higher arsenic load of realgar in mice. The homeostasis of gut microbiota was supposed to be disrupted by high dose (750 mg/kg) of realgar because after exposure to high dose of realgar, there was no significant difference in arsenic accumulation between antibiotic-treated and normally raised mice. Furthermore, arsenic solution was administered by subcutaneous injection to mice to investigate the influence of gut microbial differences on arsenic accumulation in addition to the absorption process, and there was no significant difference in arsenic accumulation between mice with these two different statuses of gut microbiota. Conclusions Gut microbiota disruption could increase arsenic accumulation of realgar in mice.

Novel method to detect, isolate, and culture prostate culturing circulating tumor cells.

Effectively detecting and culturing circulating tumor cells (CTCs), is critical for diagnosis of early metastasis, monitoring anti-cancer therapeutic efficacy, and drug screening. However, most current FDA approved CTC detection methods are based on antibody binding, which has limitations due to the nature of variations in antibody preparation and antibody-CTC size mismatches. Thus, searching for alternative and advanced methods is urgent and necessary. Prostate cancer tissue was digested by collagenase and cultured. Cancer stromal cells were identified and labelled with 4',6-diamidino-2-phenylindole (DAPI) before incubation with whole blood of cancer mice (bearing a later stage of prostate cancer). The attached blood CTCs on the DAPI-labeled cancer stromal cells were detected, isolated, cultured and produced into individual cancer cell lines. Five clones of prostate cancer cells isolated from cancer tissue were successfully cultured. One (Clone-1) of the five clones showed positive staining for all three cancer stromal cell markers (CD133, α2β1 integrin and CD44). Clone-1 cells rich with epithelial cell adhesion molecule (EpCAM) on the cell surface were further identified. The Clone-1 stromal cells labeled as "bait" attracted and caught a trace number of CTCs from the whole blood of mice with advanced stage cancer. Efficient culturing of the caught CTCs from single cell to forming of individual cancer cell line(s) were established. We present a fundamental advancement of CTC detection and culturing using a different mechanism (cell-cell interaction) rather than the traditional antibody-based immune-binding, such as CellSearchTM system. This study has potential to be fully developed into a novel approach for early cancer metastasis detection, and chemotherapy efficacy monitoring. The efficiently cultured CTCs could be used for single-clone CTC analysis and anti-cancer drug screening to further advance the development of individualized medicine.

Erythrocyte Adenosine A2B Receptor-Mediated AMPK Activation: A Missing Component Counteracting CKD by Promoting Oxygen Delivery.

Oxygen deprivation or hypoxia in the kidney drives CKD and contributes to end organ damage. The erythrocyte's role in delivery of oxygen (O2) is regulated by hypoxia, but the effects of CKD are unknown. We screened all of the metabolites in the whole blood of mice infused with angiotensin II (Ang II) at 140 ng/kg per minute up to 14 days to simulate CKD and compared their metabolites with those from untreated mice. Mice lacking a receptor on their erythrocytes called ADORA2B, which increases O2 delivery, and patients with CKD were studied to assess the role of ADORA2B-mediated O2 delivery in CKD. Untargeted metabolomics showed increased production of 2,3-biphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite promoting O2 delivery, in mice given Ang II to induce CKD. Genetic studies in mice revealed that erythrocyte ADORA2B signaling leads to AMPK-stimulated activation of BPG mutase, promoting 2,3-BPG production and O2 delivery to counteract kidney hypoxia, tissue damage, and disease progression in Ang II-induced CKD. Enhancing AMPK activation in mice offset kidney hypoxia by triggering 2,3-BPG production and O2 delivery. Patients with CKD had higher 2,3-BPG levels, AMPK activity, and O2 delivery in their erythrocytes compared with controls. Changes were proportional to disease severity, suggesting a protective effect. Mouse and human evidence reveals that ADORA2B-AMPK signaling cascade-induced 2,3-BPG production promotes O2 delivery by erythrocytes to counteract kidney hypoxia and progression of CKD. These findings pave a way to novel therapeutic avenues in CKD targeting this pathway.

Screening of microRNA and the targets prediction in mice with iron-induced osteopenia

Objective To explore the correlation between bone loss and the differential expression of microRNA (miRNA, miR) in mice with iron accumulation. Methods 12-week-old male ICR mice (n=12) were randomly divided into two groups: FAC group and Control group. Intraperitoneal injections of iron citrate amine (FAC) and equivalent saline were conducted in these two groups. Serum ferritin level was detected after 8 weeks of intervention. Micro-CT was used to detect bone volume and bone structure parameters in mice. The whole blood of mice were collected and RNA from white blood cells were extracted. Differential expression profiles of miRNA chips was conducted, and the result was verified by real-time fluorescence quantitative polymerase chain reaction(FQ- PCR) . Targetscan and miRDB were used to predict the downstream target genes of miRNA. Bioinformatics analysis of target gene was conducted, including celluar component, biologic process, molecular function and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Results Serum ferritin (218.2±29.5) μg/L was significantly higher than that in the control group (30.6±9.9) μg/L after 8 weeks. Micro-CT analysis showed a destruction of bone structure in iron accumulation mice. Bone mass, trabecular thickness (Tb. Th) and bone volume fraction (BV/TV) decreased significantly (P=0.001, 0.005, 0.021) in iron accumulation mice, while trabecular separation/spacing (Tb. Sp) increased. miRNA chip results: 20 miRNAs up regulated: mmu-miR-29b-3p, mmu-miR-324-3p, mmu-miR-133a-5p, mmu-miR-214-5p, mmu-miR-22-3p, mmu-miR-34a-5p, mmu-miR-31-5p, mmu-miR-143-5p, mmu-miR-423-3p, mmu-miR-223, mmu-miR-155, mmu-miR-106a, mmu-miR-2861, mmu-miR-148a, mmu-miR-96, mmu-miR-449a-5p, mmu-miR-423-5p, mmu-miR-204-5p, mmu-miR-211, mmu-miR-23b and 7 miRNAs down regulated: mmu-miR-18a-3p, mmu-miR-223-3p, mmu-miR-199a-5p, mmu-miR-196a-5p, mmu-miR-30c-5p, mmu-miR-15b, mmu-miR-130b were screened out. The difference of mmu-miR-423-5p is the most significant.The result of FQ-PCR on miR-423-5P had a high concordance with the result of microarray.91 downstream target genes of miR-423-5p were predict by Targetscan and miRDB tools. Bioinformatics analysis showed that target genes are clustered on phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt), Rap1, Ras and mitogen-activated protein kinase (MAPK) signaling pathways. Conclusion The iron-induced bone loss may be related to the differential expression of miR-423-5p. Key words: Iron accumulation; Osteoporosis; MicroRNA

Radiation-Induced Long Noncoding RNAs in a Mouse Model after Whole-Body Irradiation.

Long noncoding RNAs (lncRNAs) are emerging as key molecules in regulating many biological processes and have been implicated in development and disease pathogenesis. Biomarkers of cancer and normal tissue response to treatment are of great interest in precision medicine, as well as in public health and medical management, such as for assessment of radiation injury after an accidental or intentional exposure. Circulating and functional RNAs, including microRNAs (miRNAs) and lncRNAs, in whole blood and other body fluids are potential valuable candidates as biomarkers. Early prediction of possible acute, intermediate and delayed effects of radiation exposure enables timely therapeutic interventions. To address whether long noncoding RNAs (lncRNAs) could serve as biomarkers for radiation biodosimetry we performed whole genome transcriptome analysis in a mouse model after whole-body irradiation. Differential lncRNA expression patterns were evaluated at 16, 24 and 48 h postirradiation in total RNA isolated from whole blood of mice exposed to 1, 2, 4, 8 and 12 Gy of X rays. Sham-irradiated animals served as controls. Significant alterations in the expression patterns of lncRNAs were observed after different radiation doses at the various time points. We identified several radiation-induced lncRNAs known for DNA damage response as well as immune response. Long noncoding RNA targets of tumor protein 53 (P53), Trp53cor1, Dino, Pvt1 and Tug1 and an upstream regulator of p53, Meg3, were altered in response to radiation. Gm14005 ( Morrbid) and Tmevpg1 were regulated by radiation across all time points and doses. These two lncRNAs have important potential as blood-based radiation biomarkers; Gm14005 ( Morrbid) has recently been shown to play a key role in inflammatory response, while Tmevpg1 has been implicated in the regulation of interferon gamma. Precise molecular biomarkers, likely involving a diverse group of inducible molecules, will not only enable the development and effective use of medical countermeasures but may also be used to detect and circumvent or mitigate normal tissue injury in cancer radiotherapy.

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Effects of meso-2,3-dimercaptosuccinic acid, potassium iodide and chlorophyll on lead accumulation in male mice.

Lead (Pb) pollution is a serious public health problem all over the world, it especially plays severe damage role in children's health. Apart from reducing lead-induced damages, the decrease of lead accumulation is also critical. This study has been the first attempt to investigate effects of meso-2,3-dimercaptosuccinic acid (DMSA), potassium iodide (KI) and chlorophyll (Chl) on lead accumulation in male mice. Eighty healthy Kunming male mice were selected and divided randomly into 8 groups. They were treated with lead acetate (PbAc) intraperitoneally, individually and in combination with the DMSA, KI or Chl once daily for 5 days. Meanwhile, the control group was treated with normal saline during the whole exposure period. On 30th day, mice were sacrificed and lead concentrations were detected in the whole blood, livers, kidneys, and testicles of mice by means of the graphite furnace atomic absorption spectrometry. In comparison with the control group, lead concentrations increased in mice treated with the PbAc and DMSA, KI and Chl diminished lead accumulation in the whole blood, livers, and kidneys. Chl had specifically the same effects on lead concentrations in the testicles of male mice. Potassium iodide and Chl, as food additives, had the same effects as the DMSA to reduce lead accumulation in male mice effectively. Our results provided experimental evidence in vivo for the preventive measures of lead poisoning. Int J Occup Med Environ Health 2017;30(1):87-93.