scholarly journals Diverse Species-Specific Phenotypic Consequences of Loss of Function Sorting Nexin 14 Mutations

2019 ◽  
Author(s):  
Dale Bryant ◽  
Marian Seda ◽  
Emma Peskett ◽  
Constance Maurer ◽  
Gideon Pomeranz ◽  
...  
Keyword(s):  
Neutral Lipids ◽  
Dermal Fibroblasts ◽  
Loss Of Function ◽  
Sorting Nexin ◽  
Species Specific ◽  
The Impact ◽  
Developmental Analysis

AbstractMutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. SCAR20 is understood to involve subcellular disruption to autophagy and lipid metabolism. Previously reported studies on the phenotypic consequences of SNX14 mutations have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. In addition, studies have investigated the biochemical roles of SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) which have demonstrated an important role during lipid biogenesis. This study investigates the impact of constitutive Snx14 mutations in laboratory species: mice and zebrafish. Loss of SNX14 in mice was found to be embryonic lethal around mid-gestation. This is due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. Zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation contrasts with other vertebrates, being both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the overall consequences of loss of function varies considerably between species. New mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.

scholarly journals Liver X Receptor Nuclear Receptors Are Transcriptional Regulators of Dendritic Cell Chemotaxis

2018 ◽  
Vol 38 (10) ◽  
Author(s):  
Susana Beceiro ◽  
Attila Pap ◽  
Zsolt Czimmerer ◽  
Tamer Sallam ◽  
Jose A. Guillén ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Low Density Lipoprotein ◽  
Myeloid Cells ◽  
Density Lipoprotein ◽  
Loss Of Function ◽  
Transcriptional Responses ◽  
Cd38 Expression ◽  
The Impact

ABSTRACTThe liver X receptors (LXRs) are ligand-activated nuclear receptors with established roles in the maintenance of lipid homeostasis in multiple tissues. LXRs exert additional biological functions as negative regulators of inflammation, particularly in macrophages. However, the transcriptional responses controlled by LXRs in other myeloid cells, such as dendritic cells (DCs), are still poorly understood. Here we used gain- and loss-of-function models to characterize the impact of LXR deficiency on DC activation programs. Our results identified an LXR-dependent pathway that is important for DC chemotaxis. LXR-deficient mature DCs are defective in stimulus-induced migrationin vitroandin vivo. Mechanistically, we show that LXRs facilitate DC chemotactic signaling by regulating the expression of CD38, an ectoenzyme important for leukocyte trafficking. Pharmacological or genetic inactivation of CD38 activity abolished the LXR-dependent induction of DC chemotaxis. Using the low-density lipoprotein receptor-deficient (LDLR−/−) LDLR−/−mouse model of atherosclerosis, we also demonstrated that hematopoietic CD38 expression is important for the accumulation of lipid-laden myeloid cells in lesions, suggesting that CD38 is a key factor in leukocyte migration during atherogenesis. Collectively, our results demonstrate that LXRs are required for the efficient emigration of DCs in response to chemotactic signals during inflammation.

scholarly journals Cerebrospinal Fluid Pulsation Stress Promotes the Angiogenesis of Tissue-Engineered Laminae

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Linli Li ◽  
Yiqun He ◽  
Han Tang ◽  
Wei Mao ◽  
Haofei Ni ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Cerebrospinal Fluid ◽  
Engineering Technology ◽  
Expression Levels ◽  
Fluid Pulsation ◽  
Endothelial Growth Factor ◽  
The Impact

Background. Angiogenesis is a prerequisite step to achieve the success of bone regeneration by tissue engineering technology. Previous studies have shown the role of cerebrospinal fluid pulsation (CSFP) stress in the reconstruction of tissue-engineered laminae. In this study, we investigated the role of CSFP stress in the angiogenesis of tissue-engineered laminae. Methods. For the in vitro study, a CSFP bioreactor was used to investigate the impact of CSFP stress on the osteogenic mesenchymal stem cells (MSCs). For the in vivo study, forty-eight New Zealand rabbits were randomly divided into the CSFP group and the Non-CSFP group. Tissue-engineered laminae (TEL) was made by hydroxyapatite-collagen I scaffold and osteogenic MSCs and then implanted into the lamina defect in the two groups. The angiogenic and osteogenic abilities of newborn laminae were examined with histological staining, qRT-PCR, and radiological analysis. Results. The in vitro study showed that CSFP stress could promote the vascular endothelial growth factor A (VEGF-A) expression levels of osteogenic MSCs. In the animal study, the expression levels of angiogenic markers in the CSFP group were higher than those in the Non-CSFP group; moreover, in the CSFP group, their expression levels on the dura mater surface, which are closer to the CSFP stress stimulation, were also higher than those on the paraspinal muscle surface. The expression levels of osteogenic markers in the CSFP group were also higher than those in the Non-CSFP group. Conclusion. CSFP stress could promote the angiogenic ability of osteogenic MSCs and thus promote the angiogenesis of tissue-engineered laminae. The pretreatment of osteogenic MSC with a CSFP bioreactor may have important implications for vertebral lamina reconstruction with a tissue engineering technique.

scholarly journals Role of Hyaluronan in Human Adipogenesis: Evidence from in-Vitro and in-Vivo Studies

2019 ◽  
Vol 20 (11) ◽  
pp. 2675 ◽  
Author(s):  
Nicholas Wilson ◽  
Robert Steadman ◽  
Ilaria Muller ◽  
Mohd Draman ◽  
D. Aled Rees ◽  
...  
Keyword(s):  
Stem Cell Differentiation ◽  
In Vivo Studies ◽  
Peroxisome Proliferator ◽  
Synthesis Inhibitor ◽  
Peroxisome Proliferator Activated Receptor ◽  
Inhibitory Role ◽  
The Impact

Hyaluronan (HA), an extra-cellular matrix glycosaminoglycan, may play a role in mesenchymal stem cell differentiation to fat but results using murine models and cell lines are conflicting. Our previous data, illustrating decreased HA production during human adipogenesis, suggested an inhibitory role. We have investigated the role of HA in adipogenesis and fat accumulation using human primary subcutaneous preadipocyte/fibroblasts (PFs, n = 12) and subjects of varying body mass index (BMI). The impact of HA on peroxisome proliferator-activated receptor gamma (PPARγ) expression was analysed following siRNA knockdown or HA synthase (HAS)1 and HAS2 overexpression. PFs were cultured in complete or adipogenic medium (ADM) with/without 4-methylumbelliferone (4-MU = HA synthesis inhibitor). Adipogenesis was evaluated using oil red O (ORO), counting adipogenic foci, and measurement of a terminal differentiation marker. Modulating HA production by HAS2 knockdown or overexpression increased (16%, p < 0.04) or decreased (30%, p = 0.01) PPARγ transcripts respectively. The inhibition of HA by 4-MU significantly enhanced ADM-induced adipogenesis with 1.52 ± 0.18- (ORO), 4.09 ± 0.63- (foci) and 2.6 ± 0.21-(marker)-fold increases compared with the controls, also increased PPARγ protein expression (40%, (p < 0.04)). In human subjects, circulating HA correlated negatively with BMI and triglycerides (r = −0.396 (p = 0.002), r = −0.269 (p = 0.038), respectively), confirming an inhibitory role of HA in human adipogenesis. Thus, enhancing HA action may provide a therapeutic target in obesity.

scholarly journals The gut microbial metabolite trimethylamine N-oxide aggravates GVHD by inducing M1 macrophage polarization in mice

Blood ◽  
2020 ◽  
Vol 136 (4) ◽  
pp. 501-515 ◽  
Author(s):  
Kunpeng Wu ◽  
Yan Yuan ◽  
Huihui Yu ◽  
Xin Dai ◽  
Shu Wang ◽  
...  
Keyword(s):  
Nlrp3 Inflammasome ◽  
Macrophage Polarization ◽  
Short Chain Fatty Acids ◽  
Inflammasome Activation ◽  
Microbial Metabolites ◽  
M1 Macrophage ◽  
The Impact

Abstract The diversity of the human microbiome heralds the difference of the impact that gut microbial metabolites exert on allogenic graft-versus-host (GVH) disease (GVHD), even though short-chain fatty acids and indole were demonstrated to reduce its severity. In this study, we dissected the role of choline-metabolized trimethylamine N-oxide (TMAO) in the GVHD process. Either TMAO or a high-choline diet enhanced the allogenic GVH reaction, whereas the analog of choline, 3,3-dimethyl-1-butanol reversed TMAO-induced GVHD severity. Interestingly, TMAO-induced alloreactive T-cell proliferation and differentiation into T-helper (Th) subtypes was seen in GVHD mice but not in in vitro cultures. We thus investigated the role of macrophage polarization, which was absent from the in vitro culture system. F4/80+CD11b+CD16/32+ M1 macrophage and signature genes, IL-1β, IL-6, TNF-α, CXCL9, and CXCL10, were increased in TMAO-induced GVHD tissues and in TMAO-cultured bone marrow–derived macrophages (BMDMs). Inhibition of the NLRP3 inflammasome reversed TMAO-stimulated M1 features, indicating that NLRP3 is the key proteolytic activator involved in the macrophage’s response to TMAO stimulation. Consistently, mitochondrial reactive oxygen species and enhanced NF-κB nuclear relocalization were investigated in TMAO-stimulated BMDMs. In vivo depletion of NLRP3 in GVHD recipients not only blocked M1 polarization but also reversed GVHD severity in the presence of TMAO treatment. In conclusion, our data revealed that TMAO-induced GVHD progression resulted from Th1 and Th17 differentiation, which is mediated by the polarized M1 macrophage requiring NLRP3 inflammasome activation. It provides the link among the host choline diet, microbial metabolites, and GVH reaction, shedding light on alleviating GVHD by controlling choline intake.

scholarly journals MicroRNA-28-5p Regulates Liver Cancer Stem Cell Expansion via IGF-1 Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Qing Xia ◽  
Tao Han ◽  
Pinghua Yang ◽  
Ruoyu Wang ◽  
Hengyu Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Liver Cancer ◽  
Critical Role ◽  
Self Renewal ◽  
Sorafenib Treatment ◽  
The Impact

Background. MicroRNAs (miRNAs) play a critical role in the regulation of cancer stem cells (CSCs). However, the role of miRNAs in liver CSCs has not been fully elucidated. Methods. Real-time PCR was used to detect the expression of miR-miR-28-5p in liver cancer stem cells (CSCs). The impact of miR-28-5p on liver CSC expansion was investigated both in vivo and in vitro. The correlation between miR-28-5p expression and sorafenib benefits in HCC was further evaluated in patient-derived xenografts (PDXs). Results. Our data showed that miR-28-5p was downregulated in sorted EpCAM- and CD24-positive liver CSCs. Biofunctional investigations revealed that knockdown miR-28-5p promoted liver CSC self-renewal and tumorigenesis. Consistently, miR-28-5p overexpression inhibited liver CSC’s self-renewal and tumorigenesis. Mechanistically, we found that insulin-like growth factor-1 (IGF-1) was a direct target of miR-28-5p in liver CSCs, and the effects of miR-28-5p on liver CSC’s self-renewal and tumorigenesis were dependent on IGF-1. The correlation between miR-28-5p and IGF-1 was confirmed in human HCC tissues. Furthermore, the miR-28-5p knockdown HCC cells were more sensitive to sorafenib treatment. Analysis of patient-derived xenografts (PDXs) further demonstrated that the miR-28-5p may predict sorafenib benefits in HCC patients. Conclusion. Our findings revealed the crucial role of the miR-28-5p in liver CSC expansion and sorafenib response, rendering miR-28-5p an optimal therapeutic target for HCC.

scholarly journals Alpha 1-antichymotrypsin contributes to stem cell characteristics and enhances tumorigenicity of Glioblastoma

2020 ◽  
Author(s):  
Montserrat Lara-Velazquez ◽  
Natanael Zarco ◽  
Anna Carrano ◽  
Jordan Phillipps ◽  
Emily S Norton ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Migration ◽  
Brain Tumor ◽  
Primary Cultures ◽  
Cellular Migration ◽  
Tumor Initiating Cells ◽  
The Impact

Abstract Background Glioblastomas (GBMs) are the most common primary brains tumors in adults with almost 100% recurrence rate. Patients with lateral ventricle proximal GBMs (LV-GBMs) exhibit worse survival compared to distal locations for reasons that remain unknown. One potential explanation is the proximity of these tumors to the cerebrospinal fluid (CSF) and its contained chemical cues that can regulate cellular migration and differentiation. We therefore investigated the role of CSF on GBM gene expression and the role of a CSF-induced gene, SERPINA3, in GBM malignancy in vitro and in vivo. Methods We utilized patient-derived CSF and primary cultures of GBM brain tumor initiating cells (BTICs). We determined the impact of SERPINA3 expression in glioma patients using TCGA database. SERPINA3 expression changes were evaluated at both the mRNA and protein levels. The effects of knockdown (KD) and overexpression (OE) of SERPINA3 on cell behavior were evaluated by transwell assay (for cell migration), and alamar blue and Ki67 (for viability and proliferation respectively). Stem cell characteristics on KD cells were evaluated by differentiation and colony formation experiments. Tumor growth was studied by intracranial and flank injections. Results GBM CSF induced a significant increase in BTIC migration accompanied by upregulation of the SERPINA3 gene. In patient samples and TCGA data we observed SERPINA3 to correlate directly with brain tumor grade and indirectly with GBM patient survival. Silencing of SERPINA3 induced a decrease in cell proliferation, migration, invasion, and stem cell characteristics, while SERPINA3 overexpression increased cell migration. In vivo, mice orthotopically-injected with SERPINA3 KD BTICs showed increased survival. Conclusions SERPINA3 plays a key role in GBM malignancy and its inhibition results in a better outcome using GBM preclinical models.

scholarly journals Transcriptome Analysis of the Barley–Deoxynivalenol Interaction: Evidence for a Role of Glutathione in Deoxynivalenol Detoxification

2010 ◽  
Vol 23 (7) ◽  
pp. 962-976 ◽  
Author(s):  
Stephanie A. Gardiner ◽  
Jayanand Boddu ◽  
Franz Berthiller ◽  
Christian Hametner ◽  
Robert M. Stupar ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
Phytopathogenic Fungi ◽  
Glutathione Depletion ◽  
Host Responses ◽  
Application Site ◽  
Nuclear Magnetic Resonance Analysis ◽  
The Impact

Trichothecenes are a major group of toxins produced by phytopathogenic fungi, including Fusarium graminearum. Trichothecenes inhibit protein synthesis in eukaryotic cells and are toxicologically relevant mycotoxins for humans and animals. Because they promote plant disease, the role of host responses to trichothecene accumulation is considered to be an important aspect of plant defense and resistance to fungal infection. Our overall objective was to examine the barley response to application of the type B trichothecene deoxynivalenol (DON). We found that DON is diluted by movement from the application site to acropetal and basipetal florets. A susceptible barley genotype converted DON to DON-3-O-glucoside, indicating that UDP-glucosyltransferases capable of detoxifying DON must exist in barley. RNA profiling of DON-treated barley spikes revealed strong upregulation of gene transcripts encoding ABC transporters, UDP-glucosyltransferases, cytochrome P450s, and glutathione-S-transferases. We noted that transcripts encoding cysteine synthases were dramatically induced by DON, and that toxin-sensitive yeast on glutathione- or cysteine-supplemented media or carrying a gene that encodes a cysteine biosynthetic enzyme exhibit DON resistance, suggesting that preventing glutathione depletion by increasing cysteine supply could play a role in ameliorating the impact of DON. Evidence for nonenzymatic formation of DON-glutathione adducts in vitro was found using both liquid chromatography–mass spectrometry and nuclear magnetic resonance analysis, indicating that the formation of DON-glutathione conjugates in vivo may reduce the impact of trichothecenes. Our results indicate that barley exhibits multiple defense mechanisms against trichothecenes.

scholarly journals The Spectrum of Genetic Variants Associated with the Development of Monogenic Obesity in Qatar

Obesity Facts ◽  
2022 ◽  
Author(s):  
Nadien AbouHashem ◽  
Roan E. Zaied ◽  
Kholoud Al-Shafai ◽  
Mariam Nofal ◽  
Najeeb Syed ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Rare Variants ◽  
Normal Weight ◽  
Routine Testing ◽  
Monogenic Obesity ◽  
Syndromic Obesity ◽  
The Impact

Introduction: Monogenic obesity (MO) is a rare genetic disease characterized by severe early-onset obesity in affected individuals. Previous genetic studies revealed 8 definitive genes for monogenic non-syndromic obesity; many were discovered in consanguineous populations. Here, we examined MO in the Qatari population, whose population is largely consanguineous (54%) and characterized by extensive obesity (45%). Methods: Whole genome sequences of Qatar Biobank samples from 250 subjects with obesity and 250 subjects with normal weight, obtained in association with the Qatar Genome Programme, were searched for genetic variants in the genes known to be associated with MO (i.e., LEP, LEPR, POMC, PCSK1, MC3R, MC4R, MRAP2 and ADCY3). The impact of the variants identified was investigated utilizing in silico tools for prediction in combination with protein visualization by PyMOL. Results: We identified potential MO variants in more than 5% of the cases in our cohort. We revealed 11 rare variants in 6 of the genes targeted, including two disease-causing variants in MC4R and MRAP2, all of which were heterozygous. Moreover, enrichment of a heterozygous ADCY3 variant (c.1658C>T; p.A553V) appeared to cause severe obesity in an autosomal dominant manner. Conclusion: These findings highlight the importance of implementing routine testing for genetic variants that predispose for MO in Qatar. Clearly, additional studies of this nature on populations not yet examined are required. At the same time, functional investigations, both in vitro and in vivo, are necessary in order to better understand the role of the variants identified in the pathogenesis of obesity.

scholarly journals L1CAM promotes ovarian cancer stemness and tumor initiation via FGFR1/SRC/STAT3 signaling

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Marco Giordano ◽  
Alessandra Decio ◽  
Chiara Battistini ◽  
Micol Baronio ◽  
Fabrizio Bianchi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Manipulation ◽  
Tumor Formation ◽  
In Vitro Assays ◽  
Tumor Initiation ◽  
Loss Of Function ◽  
Stat3 Signaling

Abstract Background Cancer stem cells (CSC) have been implicated in tumor progression. In ovarian carcinoma (OC), CSC drive tumor formation, dissemination and recurrence, as well as drug resistance, thus contributing to the high death-to-incidence ratio of this disease. However, the molecular basis of such a pathogenic role of ovarian CSC (OCSC) has been elucidated only to a limited extent. In this context, the functional contribution of the L1 cell adhesion molecule (L1CAM) to OC stemness remains elusive. Methods The expression of L1CAM was investigated in patient-derived OCSC. The genetic manipulation of L1CAM in OC cells provided gain and loss-of-function models that were then employed in cell biological assays as well as in vivo tumorigenesis experiments to assess the role of L1CAM in OC cell stemness and in OCSC-driven tumor initiation. We applied antibody-mediated neutralization to investigate L1CAM druggability. Biochemical approaches were then combined with functional in vitro assays to study the molecular mechanisms underlying the functional role of L1CAM in OCSC. Results We report that L1CAM is upregulated in patient-derived OCSC. Functional studies showed that L1CAM promotes several stemness-related properties in OC cells, including sphere formation, tumor initiation and chemoresistance. These activities were repressed by an L1CAM-neutralizing antibody, pointing to L1CAM as a druggable target. Mechanistically, L1CAM interacted with and activated fibroblast growth factor receptor-1 (FGFR1), which in turn induced the SRC-mediated activation of STAT3. The inhibition of STAT3 prevented L1CAM-dependent OC stemness and tumor initiation. Conclusions Our study implicate L1CAM in the tumorigenic function of OCSC and point to the L1CAM/FGFR1/SRC/STAT3 signaling pathway as a novel driver of OC stemness. We also provide evidence that targeting this pathway can contribute to OC eradication.

scholarly journals The Role of Methionine Aminopeptidase 2 in Lymphangiogenesis

2020 ◽  
Vol 21 (14) ◽  
pp. 5148
Author(s):  
Rawnaq Esa ◽  
Eliana Steinberg ◽  
Dvir Dror ◽  
Ouri Schwob ◽  
Mehrdad Khajavi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Function ◽  
Loss Of Function ◽  
Methionine Aminopeptidase ◽  
Zebrafish Model ◽  
Lymphatic Capillary ◽  
Intracellular Enzyme

During the metastasis process, tumor cells invade the blood circulatory system directly from venous capillaries or indirectly via lymphatic vessels. Understanding the relative contribution of each pathway and identifying the molecular targets that affect both processes is critical for reducing cancer spread. Methionine aminopeptidase 2 (MetAp2) is an intracellular enzyme known to modulate angiogenesis. In this study, we investigated the additional role of MetAp2 in lymphangiogenesis. A histological staining of tumors from human breast-cancer donors was performed in order to detect the level and the localization of MetAp2 and lymphatic capillaries. The basal enzymatic level and activity in vascular and lymphatic endothelial cells were compared, followed by loss of function studies determining the role of MetAp2 in lymphangiogenesis in vitro and in vivo. The results from the histological analyses of the tumor tissues revealed a high MetAp2 expression, with detectable sites of co-localization with lymphatic capillaries. We showed slightly reduced levels of the MetAp2 enzyme and MetAp2 mRNA expression and activity in primary lymphatic cells when compared to the vascular endothelial cells. The genetic and biochemical manipulation of MetAp2 confirmed the dual activity of the enzyme in both vascular and lymphatic remodulation in cell function assays and in a zebrafish model. We found that cancer-related lymphangiogenesis is inhibited in murine models following MetAp2 inhibition treatment. Taken together, our study provides an indication that MetAp2 is a significant contributor to lymphangiogenesis and carries a dual role in both vascular and lymphatic capillary formation. Our data suggests that MetAp2 inhibitors can be effectively used as anti-metastatic broad-spectrum drugs.

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