In-vivo Mouse Research Articles

Estradiol suppresses psoriatic inflammation in mice by regulating neutrophil and macrophage functions

Psoriasis is a common inflammatory skin disease resulting from dysregulation of the IL-23/TH17 immune axis. The prevalence and severity of psoriasis is higher in men than in women, although the underlying reasons for this are unclear. We studied whether estradiol, a female hormone, plays protective roles in imiquimod-induced psoriatic inflammation in mice by regulating neutrophil and macrophage functions. Wild-type mice and conditional knockout mice were ovariectomized, supplemented with placebo or estradiol pellets, and an imiquimod-containing cream applied. Mice without endogenous ovarian hormones exhibited exacerbated psoriatic inflammation including increased production of IL-17A and IL-1β, which was reversed by exogenously added estradiol. The suppressive effect of estradiol on the production of IL-1β and IL-17A was abolished in mice lacking estrogen receptors in neutrophils and macrophages (Esr1f/fEsr2f/fLysM-Cre+ mice). IL-1β, which is required for production of IL-17A in the psoriasis model, was mainly produced by neutrophils and inflammatory macrophages. Estradiol suppressed IL-1β production from neutrophils and macrophages in mice both invivo and invitro and from human neutrophils invitro. Our results suggest a novel mechanism for sex-dependent differences in psoriasis clinical phenotypes that may shed new light on the pathology of psoriasis.

Biodistribution of 99mTc-PLA/PVA/Atezolizumab nanoparticles for non-small cell lung cancer diagnosis

Lung cancer (LC) is a common type of cancer, which is a leading cause of death around the world. There is an urgency for the development of new drugs that could diagnose the LC in the early stages and in a precise manner. In this direction, the development of nanoparticles radiolabeled with the diagnostic radioisotopes represent an important advance in the field of cancer imaging. In this study were developed PLA/PVA/Atezolizumab nanoparticles which were radiolabeled with 99mTc (Technetium-99m). The radiolabeled nanoparticles were evaluated in both: in-vitro (L-929 and A-549) as in-vivo (mice). The results showed no cytotoxicity effect in the healthy cells (L-929) and cytotoxicity effect in the tumor cells (A-549). The biodistribution assay demonstrated that 99mTc-PLA/PVA/Atezolizumab could reach the tumor site 14-folds higher than the nonparticulate atezolizumab. In conclusion, 99mTc-PLA/PVA/Atezolizumab nanoparticles showed to be a new drug which is able to precisely image the lung tumor, and it must be considered for clinical trials.

The Mettl3 epitranscriptomic writer amplifies p53 stress responses

The p53 transcription factor drives anti-proliferative gene expression programs in response to diverse stressors, including DNA damage and oncogenic signaling. Here, we seek to uncover new mechanisms through which p53 regulates gene expression using tandem affinity purification/mass spectrometry to identify p53-interacting proteins. This approach identified METTL3, an m6A RNA-methyltransferase complex (MTC) constituent, as a p53 interactor. We find that METTL3 promotes p53 protein stabilization and target gene expression in response to DNA damage and oncogenic signals, by both catalytic activity-dependent and independent mechanisms. METTL3 also enhances p53 tumor suppressor activity in invivo mouse cancer models and human cancer cells. Notably, METTL3 only promotes tumor suppression in the context of intact p53. Analysis of human cancer genome data further supports the notion that the MTC reinforces p53 function in human cancer. Together, these studies reveal a fundamental role for METTL3 in amplifying p53 signaling in response to cellular stress.

Autocrine P2X4 receptor activation in RBCs drives oxygen‐dependent hyperemic responses in mouse skeletal muscle capillaries

Microvascular blood flow is tightly coupled to tissue function and when disturbed, organ insult ensues. Changes in red blood cell (RBC) oxygenation are thought to be key to the initiation of robust microvascular responses, a process mechanistically linked to ATP release from RBCs and conducted signalling. The RBC signaling cascade governing ATP release was the focus of this study, and of note is the role of ATP‐activated P2X4 receptors. Using an in‐vivo mouse muscle preparation (extensor digitorum longus, EDL) and a focally delivered oxygen stimulus via a rapid gas exchange chamber, we probed capillary hemodynamics, prior to and following purinergic receptor antagonism. Dropping oxygen tension from 7% to 2% reduced RBC oxygen saturation in surface capillaries while increasing RBC supply rate. Incubating the EDL with PPADS (non‐selective purinergic receptor antagonist, 30 µM) or 5‐BDBD (selective P2X4 antagonist, 20 µM) reduced baseline RBC supply rate by ≍ 40% and 47%, respectively, and attenuated the capillary responses to the oxygen stimulus by ≍ 5 and 3 RBC/s, respectively. This blunting predictably enhanced the drop in capillary RBC oxygen saturation, as more oxygen was drawn per RBC to meet tissue demand. Immunofluorescence labeling revealed robust expression of P2X4 receptors in RBCs but not endothelial cells. Together, this data begins to elucidate a role for RBC P2X4 receptors, potentially in a positive feedback loop with pannexin channels, to dynamically regulate demand‐supply coupling in skeletal muscle.

Growth differentiation factor-15 preserves Klotho expression in acute kidney injury and kidney fibrosis

Growth differentiation factor-15 (GDF15) is a member of the GDF subfamily with potential kidney protective functions. Here, we explored the impact of GDF15 on the expression of the kidney protective factor Klotho in models of acute kidney injury and kidney fibrosis in mice. GDF15 was the most upregulated GDF family gene in experimental toxic acute kidney injury and in kidney fibrosis transcriptomics. GDF15 function was explored in toxic acute kidney injury in genetically modified mice and following treatment with GDF15. Gdf15-deficient mice developed more severe toxic acute kidney injury (folic acid or cisplatin) while GDF15 overexpression or GDF15 administration were protective. Kidney expression of Klotho was more severely depressed in Gdf15-deficient mice and was preserved by GDF15 overexpression or GDF15 treatment. Moreover, increased plasma calcitriol levels inversely correlated with kidney Klotho across models with diverse levels of GDF15 availability. Kidney fibrosis induced by unilateral ureteral obstruction was more severe in Gdf15-deficient mice while GDF15 overexpression decreased kidney injury and preserved Klotho expression. GDF15 increased Klotho expression invivo in healthy mice, in cultured tubular cells, and prevented Klotho downregulation by inflammatory factors in tubular cells by preventing transcription factor NF-ĸB activation. Thus, spontaneous increased kidney expression of endogenous GDF15 is not enough to prevent kidney injury, but further increments in GDF15 are kidney protecting and preserve expression of the kidney protective factor Klotho within the kidney in acute and chronic settings.

Soma and Neurite Density MRI (SANDI) of the in-vivo mouse brain and comparison with the Allen Brain Atlas

Diffusion MRI (dMRI) provides unique insights into the neural tissue milieu by probing interactions between diffusing molecules and tissue microstructure. Most dMRI techniques focus on white matter (WM) tissues, nevertheless, interest in gray matter characterizations is growing. The Soma and Neurite Density MRI (SANDI) methodology harnesses a model incorporating water diffusion in spherical objects (assumed to be associated with cell bodies) and in impermeable “sticks” (assumed to represent neurites), which potentially enables the characterization of cellular and neurite densities. Recognising the importance of rodents in animal models of development, aging, plasticity, and disease, we here employ SANDI for in-vivo preclinical imaging and provide a first validation of the methodology by comparing SANDI metrics with cellular density reflected by the Allen mouse brain atlas. SANDI was implemented on a 9.4T scanner equipped with a cryogenic coil, and in-vivo experiments were carried out on N = 6 mice. Pixelwise, ROI-based, and atlas comparisons were performed, magnitude vs. real-valued analyses were compared, and shorter acquisitions with reduced the number of b-value shells were investigated. Our findings reveal good reproducibility of the SANDI parameters, including the sphere and stick fractions, as well as sphere size (CoV < 7%, 12% and 3%, respectively). Additionally, we find a very good rank correlation between SANDI-driven sphere fraction and Allen mouse brain atlas contrast that represents cellular density. We conclude that SANDI is a viable preclinical MRI technique that can greatly contribute to research on brain tissue microstructure.

Saikosaponin D Inhibited IL-1β Induced ATDC 5 Chondrocytes Apoptosis In Vitro and Delayed Articular Cartilage Degeneration in OA Model Mice In Vivo.

Osteoarthritis (OA) is the most common joint disease in the elderly, characterized by cartilage degradation and proliferation of subchondral bone. The pathogenesis of OA involves a variety of inflammatory mediators, including nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β. From the molecular mechanism, the nuclear factor-erythroid 2-related factor (Nrf2)/heme oxygenase-1 (HO-1) pathway and the expression of ROS regulated the production of the above inflammatory mediators. Saikosaponin D (SSD), which is an active ingredient isolated from Bupleurum, has various biological functions. In this study, IL-1β was used as a pro-inflammatory factor to create an in vitro OA model. According to the results of high-density culture, qPCR, ROS measurement, Western blot, and immunofluorescence, SSD activated the Nrf2/HO-1/ROS axis, inhibited the production of inflammatory mediators, and protected against ECM destruction. The DMM mouse model was used as a model of OA in mice. From the results of safranin O/fast green staining, hematoxylin–eosin staining, tartrate-resistant acid phosphatase (TRAP) staining, and OARSI scores, SSD protected against the mice knee articular cartilage degeneration and reduced the number of osteoclasts in the subchondral bone. Experimental results found that SSD suppressed IL-1β–induced differentiated ATDC 5 chondrocytes apoptosis via the Nrf2/HO-1/ROS axis in vitro. SSD delayed the progression of OA in DMMs model mice in vivo. Therefore, SSD has the potential to become a drug for clinical treatment of OA.

Exploring of novel 4-hydroxy-2H-benzo[e][1,2]thiazine-3-carbohydrazide 1,1-dioxide derivative as a dual inhibitor of α-glucosidase and α-amylase: Molecular docking, biochemical, enzyme kinetic and in-vivo mouse model study

Diabetes mellitus (DM) is a metabolic disorder that leads to hyperglycemia due to improper insulin secretion. The study aims to investigate the anti-diabetic potential of benzothiazine derivatives. Molecular docking and Molecular Dynamics simulation study revealed that Compound S6 (4-hydroxy-2H-benzo[e][1,2]thiazine-3-carbohydrazide 1,1-dioxide) and S7 (4-Hydroxy-2-methyl-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide) had less conformational changes during MD simulation analysis at 100 ns. Compound S6 and S7 showed potent activity with IC50 values of 5.93 μM, 6.91 μM and 75.17, 29.10 μM for α-glucosidase and α-amylase respectively and competitive type of inhibition was observed during enzyme kinetic study with a low value of Ki and Ki′ for α-glucosidase and α-amylase, respectively. S6 has the lowest Ki (0.0736) and Ki′ (−0.0982) for α-glucosidase. Furthermore, in vivo studies were carried out to distinguish the effects of the drug on the body. Histology analysis on mice model showed that compound S6 has a low necrosis rate in the liver, kidney, and pancreas compared to S7. Biochemical results of S6 revealed lower sugar level (112 mg/dL), increase insulin secretion (23, 25 μM/L), and low level of cholesterol (80, 85 mg/dL) and creatinine (1.6, 1.4 mg/dL). The results conclude that compound S6 is a new anti-diabetic agent that minimizes hyperglycemia complications.

Background-suppressed tumor-targeted photoacoustic imaging using bacterial carriers

Photoacoustic (PA) imaging offers promise for biomedical applications due to its ability to image deep within biological tissues while providing detailed molecular information; however, its detection sensitivity is limited by high background signals that arise from endogenous chromophores. Genetic reporter proteins with photoswitchable properties enable the removal of background signals through the subtraction of PA images for each light-absorbing form. Unfortunately, the application of photoswitchable chromoproteins for tumor-targeted imaging has been hampered by the lack of an effective targeted delivery scheme; that is, photoswitchable probes must be delivered invivo with high targeting efficiency and specificity. To overcome this limitation, we have developed a tumor-targeting delivery system in which tumor-homing bacteria (Escherichia coli) are exploited as carriers to affect the point-specific delivery of genetically encoded photochromic probes to the tumor area. To improve the efficiency of the desired background suppression, we engineered a phytochrome-based reporter protein (mDrBphP-PCMm/F469W) that displays higher photoswitching contrast than those in the current state of the art. Photoacoustic computed tomography was applied to achieve good depth and resolution in the context of invivo (mice) imaging. The present system effectively integrates a genetically encoded phytochrome-based reporter protein, PA imaging, and synthetic biology (GPS), to achieve essentially background-suppressed tumor-targeted PA monitoring in deep-seated tissues. The ability to image tumors at substantial depths may enable target-specific cancer diagnoses to be made with greater sensitivity, fidelity, and specificity.

Abstract P1-06-09: The role of miR-155-5p in MSI2 induced metastasis of prolonged DEHP exposed triple-negative breast cancer cells

Abstract Phthalate leaching, environmental pollution,and human exposure is major issue. Endocrine disrupting effects of phthalate exposure on human health are well documented affecting hormonal balance, reproductive system, endometriosis, and is a suspected carcinogen. Continuous and prolonged use of plastic products leads to direct human exposure to phthalates, a problem observed in developing countries. In an attempt to mimic continuous human exposure to phthalates, we investigated the effects of long term DEHP exposure at physiological concentration on breast cancer cells. Long-term DEHP exposure enhanced migration and invasion in triple negative breast cancer cells in vitro and in vivo zebrafish xenograft assay indicating ER independent activity of DEHP. NGS identified oncogenic protein, Musashi RNA binding protein2 (MSI2) as metastasis regulator in DEHP exposed breast cancer cells. Elevated MSI2 expression induced metastatic potential through MSI2/Vimentin interaction, EMT and PI3K/Akt/NFκB/MMP9 axis activation. Besides metastatic properties MSI2 overexpression increased stem cell potential in triple negative breast cancer cells. Novel MSI2-Vimentin protein-protein interaction was evidenced in DEHP exposed breast cancer cells highlighting new function of MSI2 apart from RNA-binding and translation regulation. Reduced expression of miR-155-5p was found to negatively regulate MSI2 expression in DEHP exposed breast cancercells. miR-155-5p mimic treatment reduced expression of MSI2 and MSI2 mediated metastasis. MSI2 knockdown reversed EMT and PI3K/Akt/NFκB/MMP9 axis induced metastasis in vitro and in vivo zebrafish xenograft in DEHP exposed breast cancer cells, showing no effect non DEHP exposed breast cancer cells. Organ specific metastasis activity was observed in invivo mouse breast cancer metastasis model showing lung metastasis in DEHP exposed breast cancer cell implanted mice. MSI2 depletion reduced lung metastasis in vivo, however no effectof MSI2 depletion observed on proliferation and tumor size. MSI2 targeting might serve as a promising strategy to overcome highly metastatic breast cancer and to limit stem cell like cancer progression. Keywords: Breast cancer;DEHP; long phthalate exposure; ATP-binding transporter proteins; drug resistance; tariquidar; NGS; MSI2; metastasis; EMT; Vimentin; miR-155-5p;stemness Citation Format: Mahendra Gunvantrao Jadhao, Chien Chih Chiu. The role of miR-155-5p in MSI2 induced metastasis of prolonged DEHP exposed triple-negative breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-06-09.

Helicobacter pylori–induced RASAL2 Through Activation of Nuclear Factor-κB Promotes Gastric Tumorigenesis via β-catenin Signaling Axis

Helicobacter pylori infection is the predominant risk factor for gastric cancer. RAS protein activator like 2 (RASAL2) is considered a double-edged sword in carcinogenesis. Herein, we investigated the role of RASAL2 in response to H pylori infection and gastric tumorigenesis. Bioinformatics analyses of local and public databases were applied to analyze RASAL2 expression, signaling pathways, and clinical significance. Invitro cell culture, spheroids, patient-derived organoids, and invivo mouse models wereused. Molecular assays included chromatin immunoprecipitation, co-immunoprecipitation, Western blotting, quantitative polymerase chain reaction, and immunocyto/histochemistry. H pylori infection induced RASAL2expression via a nuclear factor-κB (NF-κB)-dependent mechanism whereby NF-κB was directly bound to the RASAL2 promoter activating its transcription. By gene silencing and ectopic overexpression, we found that RASAL2 upregulated β-catenin transcriptional activity. RASAL2 inhibited protein phosphatase 2A activity through direct binding with subsequent activation of the AKT/β-catenin signaling axis. Functionally, RASAL2 silencing decreased nuclear β-catenin levels and impaired tumor spheroids and organoids formation. Furthermore, the depletion of RASAL2 impaired tumor growth in gastric tumor xenograft mouse models. Clinicopathological analysis indicated that abnormal overexpression of RASAL2 correlated with poor prognosis and chemoresistance in human gastric tumors. These studies uncovered a novel signaling axis of NF-κB/RASAL2/β-catenin, providing a novellink between infection, inflammation and gastric tumorigenesis.

Integrated deep learning framework for accelerated optical coherence tomography angiography

Label-free optical coherence tomography angiography (OCTA) has become a premium imaging tool in clinics to obtain structural and functional information of microvasculatures. One primary technical drawback for OCTA, however, is its imaging speed. The current protocols require high sampling density and multiple acquisitions of cross-sectional B-scans to form one image frame, resulting in low acquisition speed. Recently, deep learning (DL)-based methods have gained attention in accelerating the OCTA acquisition process. They achieve faster acquisition using two independent reconstructing approaches: high-quality angiograms from a few repeated B-scans and high-resolution angiograms from undersampled data. While these approaches have shown promising results, they provide limited solutions that only partially account for the OCTA scanning mechanism. Herein, we propose an integrated DL method to simultaneously tackle both factors and further enhance the reconstruction performance in speed and quality. We designed an end-to-end deep neural network (DNN) framework with a two-staged adversarial training scheme to reconstruct fully-sampled, high-quality (8 repeated B-scans) angiograms from their corresponding undersampled, low-quality (2 repeated B-scans) counterparts by successively enhancing the pixel resolution and the image quality. Using an in-vivo mouse brain vasculature dataset, we evaluate our proposed framework through quantitative and qualitative assessments and demonstrate that our method can achieve superior reconstruction performance compared to the conventional means. Our DL-based framework can accelerate the OCTA imaging speed from 16 to 256times while preserving the image quality, thus enabling a convenient software-only solution to enhance preclinical and clinical studies.

Skin codelivery of contact sensitizers and neurokinin-1 receptor antagonists integrated in microneedle arrays suppresses allergic contact dermatitis

Allergic contact dermatitis (CD) is a chronic inflammatory skin disease caused by type 1 biased adaptive immunity for which there is an unmet need for antigen (Ag)-specific immunotherapies. Exposure to skin sensitizers stimulates secretion of the proinflammatory neuropeptides substance P and hemokinin 1, which signal via the neurokinin-1 receptor (NK1R) to promote the innate and adaptive immune responses of CD. Accordingly, mice lacking the NK1R develop impaired CD. Nonetheless, the role and therapeutic opportunities of targeting the NK1R in CD remain to be elucidated. We sought to develop an Ag-specific immunosuppressive approach to treat CD by skin codelivery of hapten and NK1R antagonists integrated in dissolvable microneedle arrays (MNA). Invivo mouse models of contact hypersensitivity and exvivo models of human skin were used to delineate the effects and mechanisms of NK1R signaling and the immunosuppressive effects of the contact sensitizer NK1R antagonist MNA in CD. We demonstrated in mice that CD requires NK1R signaling by substance P and hemokinin 1. Specific deletion of the NK1R in keratinocytes and dendritic cells, but not in mast cells, prevented CD. Skin codelivery of hapten or Ag MNA inhibited neuropeptide-mediated skin inflammation in mouse and human skin, promoted deletion of Ag-specific effector T cells, and increased regulatory T cells, which prevented CD onset and relapses locally and systemically in an Ag-specific manner. Immunoregulation by engineering localized skin neuroimmune networks can be used to treat cutaneous diseases that like CD are caused by type 1 immunity.

Antitumor effect of the selective hypoxia-inducible factor-1 inhibitors echinomycin and PX-478 on uterine fibroids

To investigate the antitumor effects of the selective hypoxia-inducible factor-1 (HIF-1) inhibitors echinomycin and PX-478 on uterine fibroids. Experimental study using invitro primary culture systems and an invivo mouse xenograft model. Academic university center. Women with uterine fibroids who underwent hysterectomy or myomectomy. Administration of the selective HIF-1 inhibitors echinomycin and PX-478 to the media of the primary cultured uterine fibroid cells and to nonobese diabetic/severe combined immunodeficient mice bearing fibroid xenografts consisting of the primary cultured fibroid cells and type Ⅰ collagen gels beneath the kidney capsule. Cell proliferation was measured by Cell Counting Kit-8 assay. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and by measuring caspase 3 and 7 activities. The xenografts were evaluated by gross appearance, surface area, and histology. The Ki-67 index was measured to evaluate proliferation of the xenografts. Both echinomycin and PX-478 inhibited cell proliferation and induced apoptosis in fibroid cells cultured under hypoxia and normoxia. Enlargement of the fibroid xenografts was significantly attenuated. The Ki-67 index significantly decreased after the administration of the HIF-1 inhibitors in the xenograft model. Eight of 27 xenografts treated with the HIF-1 inhibitors contained calcification and hyalinizing components from 3 days after the grafting to 2 weeks, suggesting that the HIF-inhibitors induce degeneration of the fibroid xenografts. The selective HIF-1 inhibitors echinomycin and PX-478 show antitumor effects against uterine fibroids both invitro and invivo. These findings support the potential use of HIF-1 inhibitors for the treatment of uterine fibroids.

Non-invasive and low-artifact in vivo brain imaging by using a scanning acoustic-photoacoustic dual mode microscopy

Photoacoustic imaging is a potential candidate for in vivo brain imaging, whereas, its imaging performance could be degraded by inhomogeneous multi-layered media, consisted of scalp and skull. In this work, we propose a low-artifact photoacoustic microscopy (LAPAM) scheme, which combines conventional acoustic-resolution photoacoustic microscopy with scanning acoustic microscopy to suppress the reflection artifacts induced by multi-layers. Based on similar propagation characteristics of photoacoustic signals and ultrasonic echoes, the ultrasonic echoes can be employed as the filters to suppress the reflection artifacts to obtain low-artifact photoacoustic images. Phantom experiment is used to validate the effectiveness of this method. Furthermore, LAPAM is applied for in-vivo imaging mouse brain without removing the scalp and the skull. Experimental results show that the proposed method successfully achieves the low-artifact brain image, which demonstrates the practical applicability of LAPAM. This work might improve the photoacoustic imaging quality in many biomedical applications which involve tissues with complex acoustic properties, such as brain imaging through scalp and skull.

Sorghum Protein Extract Protects RBC from Sodium Nitrite-Induced Oxidative Stress and Exhibits Anticoagulant and Antiplatelet Activity

Oxidative stress plays a critical role in the progression of diabetes, arthritis, cancer, eryptosis, cardiovascular disease, and thrombosis. Currently, antioxidants from natural sources are in high demand due to their beneficial role in the management of said diseases. The purpose of the study was to evaluate the protective effect of sorghum protein buffer extract (SBE) on sodium nitrite-induced oxidative stress and thrombosis. Protein characterization of SBE was done using SDS-PAGE. Oxidative stress in RBC was induced using sodium nitrite (NaNO2) and the key stress markers such as lipid peroxidation (LPO), protein carbonyl content (PCC), and the level of antioxidant enzymes (SOD and CAT) were measured. The anticoagulant effect of SBE was identified by employing in-vitro plasma recalcification time, activated partial thromboplastin time (APTT), prothrombin time (PT), and in-vivo mouse tail bleeding time. SBE antiplatelet activity was examined using agonist adenosine diphosphate (ADP) and epinephrine-induced platelet aggregation. Non-toxic property of SBE was identified using in-vitro direct haemolytic, haemorrhagic, and edema forming activities using experimental mice. SBE revealed similar protein banding pattern under both reduced and non-reduced conditions on SDS-PAGE. Interestingly, SBE normalized the level of LPO, PCC, SOD, and CAT in stress-inducedRBCs. Furthermore, SBE showed anticoagulant effect in platelet rich plasma by enhancing the clotting time from the control 250 s to 610 s and bleeding time from the control 200 s to more than 500 s (p<0.01) in a dose dependent manner. In addition, SBE prolonged the clot formation process of only APTT but not PT. SBE inhibited the agonists ADP and epinephrine induced platelet aggregation. SBE did not hydrolyze RBC cells, devoid of edema and haemorrhage properties. This study demonstrates for the first time the anticoagulant, antiplatelet, and antioxidant properties of SBE. Thus, the observed results validate consumption of sorghum as good for health and well-being.

Loss of Endothelial Cell Matrix Metalloproteinase 14 Reduces Melanoma Growth and Metastasis by Increasing Tumor Vessel Stability

Matrix metalloproteinase (MMP) 14 belongs to a large family of zinc-dependent endopeptidases and plays a critical role in skin physiological and pathological processes. Complete loss of the protease resulted in severe developmental defects leading to early death. However, because of the premature death of the mice, the functional significance for endothelial cell (EC) expression of MMP14 in skin physiology and pathology invivo after birth is yet unknown. Using a mouse model with constitutive EC-specific deletion of Mmp14 (Mmp14EC‒/‒), we showed that mice developed and bred normal, but melanoma growth and metastasis were reduced. Although vascularity was unaltered, vessel permeability was decreased. Deletion of MMP14 in ECs led to increased vessel coverage by pericytes and vascular endothelial-cadherin expression in mice invivo and invitro but not in human ECs. Endothelial nitric oxide synthase expression and nitric oxide production were significantly reduced in Mmp14EC‒/‒ ECs and MMP14-silenced human umbilical vein ECs. A direct correlation between endothelial nitric oxide synthase and MMP14 expression was detected in intratumoral vessels of human malignant melanomas. Altogether, we show that endothelial MMP14 controls tumor vessel function during melanoma growth. These data suggest that EC-derived MMP14 direct targeting alone or with vascular stabilizing agents may be therapeutically crucial in inhibiting melanoma growth and metastasis.

AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

ABSTRACT The intestinal epithelial tight junctions (TJs) provide barrier against paracellular permeation of lumenal antigens. Defects in TJ barrier such as increased levels of pore-forming TJ protein CLDN2 (claudin-2) is associated with inflammatory bowel disease. We have previously reported that starvation-induced macroautophagy/autophagy enhances the TJ barrier by degrading pore-forming CLDN2. In this study, we examined the molecular mechanism underlying autophagy-induced CLDN2 degradation. CLDN2 degradation was persistent in multiple modes of autophagy induction. Immunolocalization, membrane fractionation, and pharmacological inhibition studies showed increased clathrin-mediated CLDN2 endocytosis upon starvation. Inhibition of clathrin-mediated endocytosis negated autophagy-induced CLDN2 degradation and enhancement of the TJ barrier. The co-immunoprecipitation studies showed increased association of CLDN2 with clathrin and adaptor protein AP2 (AP2A1 and AP2M1 subunits) as well as LC3 and lysosomes upon starvation, signifying the role of clathrin-mediated endocytosis in autophagy-induced CLDN2 degradation. The expression and phosphorylation of AP2M1 was increased upon starvation. In-vitro, in-vivo (mouse colon), and ex-vivo (human colon) inhibition of AP2M1 activation prevented CLDN2 degradation. AP2M1 knockout prevented autophagy-induced CLDN2 degradation via reduced CLDN2-LC3 interaction. Site-directed mutagenesis revealed that AP2M1 binds to CLDN2 tyrosine motifs (YXXФ) (67–70 and 148–151). Increased baseline expression of CLDN2 and TJ permeability along with reduced CLDN2-AP2M1-LC3 interactions in ATG7 knockout cells validated the role of autophagy in modulation of CLDN2 levels. Acute deletion of Atg7 in mice increased CLDN2 levels and the susceptibility to experimental colitis. The autophagy-regulated molecular mechanisms linking CLDN2, AP2M1, and LC3 may provide therapeutic tools against intestinal inflammation. Abbreviations: Amil: amiloride; AP2: adaptor protein complex 2; AP2A1: adaptor related protein complex 2 subunit alpha 1; AP2M1: adaptor related protein complex 2 subunit mu 1; ATG7: autophagy related 7; CAL: calcitriol; Cas9: CRISPR-associated protein 9; Con: control; CPZ: chlorpromazine; DSS: dextran sodium sulfate; EBSS: Earle’s balanced salt solution; IBD: inflammatory bowel disease; TER: trans-epithelial resistance; KD: knockdown; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MβCD: Methyl-β-cyclodextrin; MET: metformin; MG132: carbobenzoxy-Leu-Leu-leucinal; MTOR: mechanistic target of rapamycin kinase; NT: non target; RAPA: rapamycin; RES: resveratrol; SMER: small-molecule enhancer 28; SQSTM1: sequestosome 1; ST: starvation; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.

Rosiglitazone induces adipogenesis of both marrow and periosteum derived mesenchymal stem cells during endochondral fracture healing

BackgroundType 2 diabetes mellitus (T2DM) afflicts about six percent of the global population, and these patients suffer from a two-fold increased fracture risk. Thiazolidinediones (TZDs), including rosiglitazone, are commonly used medications in T2DM because they have a low incidence of monotherapy failure. It is known that rosiglitazone is associated with secondary osteoporosis, further increasing the fracture risk in an already susceptible population. However, it is not yet understood how rosiglitazone impacts endochondral bone healing after fracture. The aim of this study is to elucidate how rosiglitazone treatment impacts endochondral fracture healing, and how rosiglitazone influences the differentiation of skeletal stem and progenitor cells from the bone marrow and the periosteum. MethodsAn in-vivo mouse femur fracture model was employed to evaluate differences in fracture healing between mice treated with and without rosiglitazone chow. Fracture healing was assessed with histology and micro computed tomography (μCT). In-vitro assays utilized isolated mouse bone marrow stromal cells and periosteal cells to investigate how rosiglitazone impacts the osteogenic capability and adipogenicity of these cells. ResultsThe in-vivo mouse femur fracture model showed that fracture callus in mice treated with rosiglitazone had significantly more adipose content than those of control mice that did not receive rosiglitazone. In addition, μCT analysis showed that rosiglitazone treated mice had significantly greater bone volume, but overall greater porosity when compared to control mice. In-vitro experimentation showed significantly less osteogenesis and more adipogenesis in bone marrow derived progenitor cells that were cultured in osteogenic media. In addition, rosiglitazone treatment alone caused significant increases in adipogenesis in both bone marrow and periosteum derived cells. ConclusionRosiglitazone impairs endochondral fracture healing in mice by increasing adipogenesis and decreasing osteogenesis of both bone marrow and periosteum derived skeletal progenitor cells.

L1-L2 norm regularization via forward-backward splitting for fluorescence molecular tomography.

Fluorescent molecular tomography (FMT) is a highly sensitive and noninvasive imaging approach for providing three-dimensional distribution of fluorescent marker probes. However, owing to its light scattering effect and the ill-posedness of inverse problems, it is challenging to develop an efficient reconstruction algorithm that can achieve the exact location and morphology of the fluorescence source. In this study, therefore, in order to satisfy the need for early tumor detection and improve the sparsity of solution, we proposed a novel L 1-L 2 norm regularization via the forward-backward splitting method for enhancing the FMT reconstruction accuracy and the robustness. By fully considering the highly coherent nature of the system matrix of FMT, it operates by splitting the objective to be minimized into simpler functions, which are dealt with individually to obtain a sparser solution. An analytic solution of L 1-L 2 norm proximal operators and a forward-backward splitting algorithm were employed to efficiently solve the nonconvex L 1-L 2 norm minimization problem. Numerical simulations and an in-vivo glioma mouse model experiment were conducted to evaluate the performance of our algorithm. The comparative results of these experiments demonstrated that the proposed algorithm obtained superior reconstruction performance in terms of spatial location, dual-source resolution, and in-vivo practicability. It was believed that this study would promote the preclinical and clinical applications of FMT in early tumor detection.