Mesenchymal stem cells (MSCs) are considered as the developmental origin of multiple lineage cells including osteocytes, adipocytes, and muscle cells. Previous studies demonstrated that the PH domain-containing protein CKIP-1 plays an important role in the development of osteoblasts and cardiomyocytes. However, whether CKIP-1 is involved in the generation of adipocytes as well as the MSC differentiation remains unknown. Here we show that CKIP-1 is a novel regulator of MSCs differentiating into adipocytes. MSCs derived from CKIP-1-deficient mice display enhanced adipogenesis upon induction. Further analysis showed that CKIP-1 interacts with the histone deacetylase HDAC1 in the nucleus and inhibits the transcription of CCAAT/enhancer-binding protein ? (C/EBP?), which is a crucial adipogenic transcription factor. Ectopic expression of CKIP-1 in a MSC-like cell line C3H/10T1/2 reduced the generation of adipocytes due to suppression of adipogenic factors, including C/EBP?. Moreover, CKIP-1-deficient mice showed an increase in body weight and white adipose tissue gains when fed on a high-fat diet. Collectively, these results suggest that CKIP-1 is a novel inhibitor of MSC-originated adipogenesis by enhancing HDAC1-associated repression of C/EBP?.
The ubiquitin ligase Smad ubiquitination regulatory factor-1 (Smurf1) negatively regulates bone morphogenetic protein (BMP) pathway by ubiquitinating certain signal components for degradation. Thus, it can be an eligible pharmacological target for increasing BMP signal responsiveness. We established a strategy to discover small molecule compounds that block the WW1 domain of Smurf1 from interacting with Smad1/5 by structure based virtual screening, molecular experimental examination and cytological efficacy evaluation. Our selected hits could reserve the protein level of Smad1/5 from degradation by interrupting Smurf1-Smad1/5 interaction and inhibiting Smurf1 mediated ubiquitination of Smad1/5. Further, these compounds increased BMP-2 signal responsiveness and the expression of certain downstream genes, enhanced the osteoblastic activity of myoblasts and osteoblasts. Our work indicates targeting Smurf1 for inhibition could be an accessible strategy to discover BMP-sensitizers that might be applied in future clinical treatments of bone disorders such as osteopenia.
Neddylation, the covalent attachment of ubiquitin-like protein Nedd8, of the Cullin-RING E3 ligase family regulates their ubiquitylation activity. However, regulation of HECT ligases by neddylation has not been reported to date. Here we show that the C2-WW-HECT ligase Smurf1 is activated by neddylation. Smurf1 physically interacts with Nedd8 and Ubc12, forms a Nedd8-thioester intermediate, and then catalyses its own neddylation on multiple lysine residues. Intriguingly, this autoneddylation needs an active site at C426 in the HECT N-lobe. Neddylation of Smurf1 potently enhances ubiquitin E2 recruitment and augments the ubiquitin ligase activity of Smurf1. The regulatory role of neddylation is conserved in human Smurf1 and yeast Rsp5. Furthermore, in human colorectal cancers, the elevated expression of Smurf1, Nedd8, NAE1 and Ubc12 correlates with cancer progression and poor prognosis. These findings provide evidence that neddylation is important in HECT ubiquitin ligase activation and shed new light on the tumour-promoting role of Smurf1.
Macrophages play pivotal roles in development, homeostasis, tissue repair and immunity. Macrophage proliferation is promoted by macrophage colony-stimulating factor (M-CSF)-induced Akt signaling; yet, how this process is terminated remains unclear. Here, we identify casein kinase 2-interacting protein-1 (CKIP-1) as a novel inhibitor of macrophage proliferation. In resting macrophages, CKIP-1 was phosphorylated at Serine 342 by constitutively active GSK3?, the downstream target of Akt. This phosphorylation triggers the polyubiquitination and proteasomal degradation of CKIP-1. Upon M-CSF stimulation, Akt is activated by CSF-1R-PI3K and then inactivates GSK3?, leading to the stabilization of CKIP-1 and ?-catenin proteins. ?-catenin promotes the expression of proliferation genes including cyclin D and c-Myc. CKIP-1 interacts with TRAF6, a ubiquitin ligase required for K63-linked ubiquitination and plasma membrane recruitment of Akt, and terminates TRAF6-mediated Akt activation. By this means, CKIP-1 inhibits macrophage proliferation specifically at the late stage after M-CSF stimulation. Furthermore, CKIP-1 deficiency results in increased proliferation and decreased apoptosis of macrophages in vitro and CKIP-1(-/-) mice spontaneously develop a macrophage-dominated splenomegaly and myeloproliferation. Together, these data demonstrate that CKIP-1 plays a critical role in the regulation of macrophage homeostasis by inhibiting TRAF6-mediated Akt activation.
NEDD4-like ubiquitin ligase 2 (NEDL2) is a HECT type ubiquitin ligase. NEDL2 enhances p73 transcriptional activity and degrades ATR kinase in lamin misexpressed cells. Compared with the important functions of other HECT type ubiquitin ligase, there is less study concerning the function and regulation of NEDL2. Using primary antibody immunoprecipitation and mass spectrometry, we identify a list of potential proteins that are putative NEDL2-interacting proteins. The candidate list contains many of mitotic proteins, especially including several subunits of anaphase-promoting complex/cyclosome (APC/C) and Cdh1, an activator of APC/C. Cdh1 can interact with NEDL2 in vivo and in vitro. Cdh1 recognizes one of the NEDL2 destruction boxes (R(740)GSL(743)) and targets it for degradation in an APC/C-dependent manner during mitotic exit. Overexpression of Cdh1 reduces the protein level of NEDL2, whereas knockdown of Cdh1 increases the protein level of NEDL2 but has no effect on the NEDL2 mRNA level. NEDL2 associates with mitotic spindles, and its protein level reaches a maximum in mitosis. The function of NEDL2 during mitosis is essential because NEDL2 depletion prolongs metaphase, and overexpression of NEDL2 induces chromosomal lagging. Elevated expression of NEDL2 protein and mRNA are both found in colon cancer and cervix cancer. We conclude that NEDL2 is a novel substrate of APC/C-Cdh1 as cells exit mitosis and functions as a regulator of the metaphase to anaphase transition. Its overexpression may contribute to tumorigenesis.
Casein kinase-2 interacting protein-1 (CKIP-1) has been identified to play an important role in cell morphology, differentiation and apoptosis. However, the role of CKIP-1 in other cellular processes is still unknown. Here we investigated transcriptome profiles of WT and CKIP-1-deficient mouse embryonic fibroblasts (MEFs), and found that innate immunity and cell migration related pathways were significantly correlated with CKIP-1 expression. As macrophage is a key cell type in innate immunity, we then used murine macrophage RAW264.7 cells to discover CKIP-1 interacting proteins by immunoprecipitation/mass spectrometry (IP/MS). Analysis of these proteins revealed migration related pathways were enriched. Further experiments indicated that knockdown of CKIP-1 in RAW264.7 cells resulted in impaired cell migration. Our study suggests that CKIP-1 is a novel regulator of macrophage migration.
N-methylpurine DNA glycosylase (MPG), a DNA repair enzyme, functions in the DNA base excision repair (BER) pathway. Aberrant over-expression of MPG in various cancers suggests an important role of MPG in carcinogenesis. Identification of MPG-interacting proteins will help to dissect the molecular link between MPG and cancer development. In the present study, using immunoprecipitation coupled with mass spectrometry (IP/MS), we screened ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1), an essential protein required for the maintenance of DNA methylation, as a MPG-interacting protein. Endogenous co-immunoprecipitation assay in cancer cells confirmed that UHRF1 interacted with MPG in a p53 status-independent manner. Confocal microscopy showed that endogenous MPG and UHRF1 were co-localized in the nucleoplasm. Furthermore, co-immunoprecipitation assay indicated that UHRF2, the homolog of UHRF1, could also interact with MPG. These results show that MPG and the UHRF family of proteins interact, thus providing a functional linkage between MPG and UHRF1/2.
The HECT-type ubiquitin ligase (E3) Smad ubiquitination regulatory factor 1 (Smurf1) targets various substrates, including Smad1/5, RhoA, Prickle 1, MEKK2, and JunB for degradation and thereby regulates adult bone formation and embryonic development. Here, we identify the endoplasmic reticulum (ER)-localized Wolfram syndrome protein (WFS1) as a specific degradation substrate of Smurf1. Mutations in the WFS1 gene cause Wolfram syndrome, an autosomal recessive disorder characterized by diabetes mellitus and optic atrophy. WFS1 negatively regulates the ER stress response, and WFS1 deficiency in mice increases ER stress and triggers apoptosis. We show that Smurf1 interacts with WFS1 at the ER and promotes the ubiquitination and proteasomal degradation of WFS1. A C-terminal luminal region in WFS1, including residues 667-700, is involved in this degradation. Wild-type WFS1 as well as a subset of WFS1 mutants that include this degron region are susceptible to Smurf1-mediated degradation. By contrast, pathophysiological deletion mutants of WFS1 lacking the degron, such as W648X, Y660X, and Q667X, are resistant to degradation by Smurf1. Depletion of Smurf1 by RNA interference results in increased WFS1 and decreased ATF6? levels. Furthermore, we show that ER stress induces Smurf1 degradation and WFS1 up-regulation. These findings reveal for the first time that Smurf1 targets an ER-localized protein for degradation and that Smurf1 is regulated by ER stress.
The C2-WW-HECT-type ubiquitin ligases Smurf1 and Smurf2 play a critical role in embryogenesis and adult bone homeostasis via regulation of bone morphogenetic protein, Wnt, and RhoA signaling pathways. The intramolecular interaction between C2 and HECT domains autoinhibits the ligase activity of Smurf2. However, the role of the Smurf1 C2 domain remains elusive. Here, we show that the C2-HECT autoinhibition mechanism is not observed in Smurf1, and instead its C2 domain functions in substrate selection. The Smurf1 C2 domain exerts a key role in localization to the plasma membrane and endows Smurf1 with differential activity toward RhoA versus Smad5 and Runx2. Crystal structure analysis reveals that the Smurf1 C2 domain possesses a typical anti-parallel ?-sandwich fold. Examination of the sulfate-binding site analysis reveals two key lysine residues, Lys-28 and Lys-85, within the C2 domain that are important for Smurf1 localization at the plasma membrane, regulation on cell migration, and robust ligase activity toward RhoA, which further supports a Ca(2+)-independent localization mechanism for Smurf1. These findings demonstrate a previously unidentified role of the Smurf1 C2 domain in substrate selection and cellular localization.
Krüppel-like factor 2 (KLF2) has been demonstrated to be essential for normal lung development, erythroid differentiation, T-cell differentiation, migration and homing. However, the mechanisms underlying the regulation of KLF2, in particular its responsible E3 ligase is still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets KLF2 for poly-ubiquitination and proteasomal degradation specifically in lung cancer H1299 cells. The catalytic ligase activity of Smurf1 is required for it to regulate KLF2. Consequently, Smurf1 represses the transcriptional factor activity of KLF2 and regulates the expression its downstream genes such as CD62L and Wee1. This study provided the first evidence that Smurf1 functions as an E3 ligase to promote the ubiquitination and proteasomal degradation of KLF2.
Smad ubiquitination regulatory factor 1 (Smurf1), an homologous to E6AP C-terminus (HECT)-type E3 ubiquitin ligase, performs a crucial role in the regulation of the bone morphogenetic protein (BMP) signalling pathway in both embryonic development and bone remodelling. How the stability and activity of Smurf1 are negatively regulated remains largely unclear. Here, we report that F-box and LRR domain-containing protein 15 (FBXL15), an F-box protein of the FBXL family, forms an Skp1-Cullin1-F-box protein-Roc1 (SCF)(FBXL15) ubiquitin ligase complex and targets Smurf1 for ubiquitination and proteasomal degradation. FBXL15, through its leucine-rich repeat domain, specifically recognizes the large subdomain within the N-lobe of the Smurf1 HECT domain and promotes the ubiquitination of Smurf1 on K355 and K357 within the WW-HECT linker region. In this way, FBXL15 positively regulates BMP signalling in mammalian cells. Knockdown of fbxl15 expression in zebrafish embryos by specific antisense morpholinos causes embryonic dorsalization phenocoping BMP-deficient mutants. Injection of FBXL15 siRNAs into rat bone tissues leads to a significant loss of bone mass and decrease in bone mineral density. Collectively, our results demonstrate that Smurf1 stability is suppressed by SCF(FBXL15)-mediated ubiquitination and that FBXL15 is a key regulator of BMP signalling during embryonic development and adult bone formation.
NuSAP is a microtubule-associated protein that plays an important role in spindle assembly. NuSAP deficiency in mice leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed in the mitotic cytoplasm. ATM is a key kinase that phosphorylates a series of substrates to mediate G1/S control. However, the role of ATM at the G2/M phase is not well understood. Here we demonstrate that ectopic expression of NuSAP lead to mitotic arrest observably dependent on the kinase activity of ATM. When endogenous ATM was depleted or its kinase activity was inhibited, NuSAP could not cause mitotic arrest. We further show ATM interacts with NuSAP and phosphorylates NuSAP on Ser124. The phosphorylation and interaction occur specifically at G2/M-phase. Collectively, our work has uncovered an ATM-dependent checkpoint pathway that prevents mitotic progression by targeting a microtubule-associated protein, NuSAP.
The KRAB-type zinc-finger protein Apak (ATM and p53 associated KZNF protein) specifically suppresses p53-mediated apoptosis. Upon DNA damage, Apak is phosphorylated and inhibited by ATM kinase, resulting in p53 activation. However, how Apak is regulated in response to oncogenic stress remains unknown. Here we show that upon oncogene activation, Apak is inhibited in the tumor suppressor ARF-dependent but ATM-independent manner. Oncogene-induced ARF protein directly interacts with Apak and competes with p53 to bind to Apak, resulting in Apak dissociation from p53. Thus, Apak is differentially regulated in the ARF and ATM-dependent manner in response to oncogenic stress and DNA damage, respectively.
The tumor suppressor p53 protein is tightly regulated by a ubiquitin-proteasomal degradation mechanism. Several E3 ubiquitin ligases, including MDM2 (mouse double minute 2), have been reported to play an essential role in the regulation of p53 stability. However, it remains unclear how the activity of these E3 ligases is regulated. Here, we show that the HECT-type E3 ligase Smurf1/2 (Smad ubiquitylation regulatory factor 1/2) promotes p53 degradation by enhancing the activity of the E3 ligase MDM2. We provide evidence that the role of Smurf1/2 on the p53 stability is not dependent on the E3 activity of Smurf1/2 but rather is dependent on the activity of MDM2. We find that Smurf1/2 stabilizes MDM2 by enhancing the heterodimerization of MDM2 with MDMX, during which Smurf1/2 interacts with MDM2 and MDMX. We finally provide evidence that Smurf1/2 regulates apoptosis through p53. To our knowledge, this is the first report to demonstrate that Smurf1/2 functions as a factor to stabilize MDM2 protein rather than as a direct E3 ligase in regulation of p53 degradation.
The ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) targets many proteins including Smad1/5 for ubiquitin-dependent proteasomal degradation. However, how Smurf1 is degraded remains unclear. Here we show that REGgamma, an activator for the 20S proteasome-mediated protein degradation, interacts with Smurf1 and mediates its degradation. We provide evidence that depletion of REGgamma stabilizes Smurf1 whereas overexpression of REGgamma promotes the degradation of Smurf1. Interestingly both Smurf2 and Smurf1 are destabilized by the REGgamma proteasome while the other members of Neural precursor cell-expressed developmentally downregulated gene 4 family were not affected. More importantly, we found that the REGgamma proteasome-mediated degradation of Smurf1 results in degradation of Smad5. These findings reveal that the REGgamma-proteasome targets a ubiquitin ligase for protein degradation.
Inhibitor of growth 2 (ING2) gene encodes a candidate tumor suppressor and is frequently reduced in many tumors. However, the mechanisms underlying the regulation of ING2, in particular its protein stability, are still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets ING2 for poly-ubiquitination and proteasomal degradation. Intriguingly, the ING2 binding domain in Smurf1 was mapped to the catalytic HECT domain. Furthermore, the C-terminal PHD domain of ING2 was required for Smurf1-mediated degradation. This study provided the first evidence that the stability of ING2 could be regulated by ubiquitin-mediated degradation.
The pleckstrin homology (PH) domain-containing protein casein kinase 2 interacting protein-1 (CKIP-1) plays an important role in regulation of bone formation and muscle differentiation. How CKIP-1 localization is determined remains largely unclear. We observed that isolated CKIP-1-PH domain was predominantly localized in the nucleus and the C-terminus of CKIP-1 counteracted its nuclear localization. The net charge of basic residues and a serine-rich motif within the PH domain plays a pivotal role in the localization switch of both full-length CKIP-1 and the isolated PH domain. We propose that the N-terminal PH domain and C-terminal auto-inhibitory region of CKIP-1 coordinate to determine its subcellular localization and the nucleus-plasma membrane shuttling.
The HECT-type E3 Smad ubiquitination regulation factor 1 (Smurf1) functions in regulation of cell polarity and bone homeostasis by targeting Smads, Runx2, RhoA and MEKK2 for ubiquitination and degradation. In a yeast two-hybrid screening, we identified TNF receptor-associated factor 4 (TRAF4) as a candidate substrate and was further validated. The PY motifs of TRAF4 mediated the interaction with the second WW domain of Smurf1. Overexpression of Smurf1 reduced the protein levels of TRAF4 dependent of its E3 activity and the proteasome. Further, we showed that all six members of TRAF family could be ubiquitinated by Smurf1. Consequently, Smurf1 interfered with the functions of TRAFs in NF-kappaB signaling under stimulation or not. These results suggested a new role of Smurf1 in inflammation and immunity through controlling the degradation of TRAFs.
The tumor suppressor p53 lies at the center of a protein-signaling network that responds to many types of stress, and p53 activation leads to cell cycle arrest or apoptosis. We recently identified ATM and p53-associated KZNF protein (Apak) as a negative regulator of p53-mediated apoptosis. After treatment of cells with methyl methanesulfonate (MMS), Apak is phosphorylated by ATM kinase and dissociates from p53, resulting in p53 activation and induction of apoptosis. However, the mechanism by which Apak is regulated in response to other types of DNA damage signals remains unclear. Here, we show that four of seven types of DNA damage signals we examined (induction by etoposide, doxorubicin, camptothecin and cisplatin treatment) resulted in significant Apak phosphorylation and dissociation of Apak from p53, releasing the inhibition of p53 transcriptional activity. In contrast, Apak was not phosphorylated at Ser68 after 5-fluorouracil or alpha-lipoic acid treatment and persistently inhibited p53 activity. These findings provide evidence that the Apak-p53 interaction is regulated differentially by various DNA damage signals.
Only a few p53 regulators have been shown to participate in the selective control of p53-mediated cell cycle arrest or apoptosis. How p53-mediated apoptosis is negatively regulated remains largely unclear. Here we report that Apak (ATM and p53-associated KZNF protein), a Krüppel-associated box (KRAB)-type zinc-finger protein, binds directly to p53 in unstressed cells, specifically downregulates pro-apoptotic genes, and suppresses p53-mediated apoptosis by recruiting KRAB-box-associated protein (KAP)-1 and histone deacetylase 1 (HDAC1) to attenuate the acetylation of p53. Apak inhibits p53 activity by interacting with ATM, a previously identified p53 activator. In response to stress, Apak is phosphorylated by ATM and dissociates from p53, resulting in activation of p53 and induction of apoptosis. These findings revealed Apak to be a negative regulator of p53-mediated apoptosis and showed the dual role of ATM in p53 regulation.
Hepatitis C virus (HCV) is a major cause of liver disease. However, the detailed mechanism underlying hepatocyte infection with HCV is not yet completely understood. We previously identified a novel C-type lectin--LSECtin predominantly expressed on liver sinusoidal endothelial cells. Here we demonstrate that LSECtin can interact with two HCV receptors, DC-SIGNR and CD81, through its central ectodomain. Furthermore, cells expressing LSECtin specifically can be attached by the naturally occurring HCV in the sera of infected individuals. This binding was found to be mediated by the HCV E2 glycoprotein and could be efficiently inhibited by EGTA but not by mannan treatment. The present study suggests that LSECtin interaction with DC-SIGNR might contribute to HCV binding to liver sinusoidal endothelial cells.
CKIP-1 is an activator of the Smurf1 ubiquitin ligase acting to promote the ubiquitylation of Smad5 and MEKK2. The mechanisms involved in the recognition and degradation of these substrates by the proteasome remain unclear. Here, we show that CKIP-1, through its leucine zipper, interacts directly with the Rpt6 ATPase of the 19S regulatory particle of the proteasome. CKIP-1 mediates the Smurf1-Rpt6 interaction and delivers the ubiquitylated substrates to the proteasome. Depletion of CKIP-1 reduces the degradation of Smurf1 and its substrates by Rpt6. These findings reveal an unexpected adaptor role of CKIP-1 in coupling the ubiquitin ligase and the proteasome.
Alkylating agents induce genome-wide base damage, which is repaired mainly by N-methylpurine DNA glycosylase (MPG). An elevated expression of MPG in certain types of tumor cells confers higher sensitivity to alkylation agents because MPG-induced apurinic/apyrimidic (AP) sites trigger more strand breaks. However, the determinant of drug sensitivity or insensitivity still remains unclear. Here, we report that the p53 status coordinates with MPG to play a pivotal role in such process. MPG expression is positive in breast, lung and colon cancers (38.7%, 43.4% and 25.3%, respectively) but negative in all adjacent normal tissues. MPG directly binds to the tumor suppressor p53 and represses p53 activity in unstressed cells. The overexpression of MPG reduced, whereas depletion of MPG increased, the expression levels of pro-arrest gene downstream of p53 including p21, 14-3-3? and Gadd45 but not proapoptotic ones. The N-terminal region of MPG was specifically required for the interaction with the DNA binding domain of p53. Upon DNA alkylation stress, in p53 wild-type tumor cells, p53 dissociated from MPG and induced cell growth arrest. Then, AP sites were repaired efficiently, which led to insensitivity to alkylating agents. By contrast, in p53-mutated cells, the AP sites were repaired with low efficacy. To our knowledge, this is the first direct evidence to show that a DNA repair enzyme functions as a selective regulator of p53, and these findings provide new insights into the functional linkage between MPG and p53 in cancer therapy.
Though the mouse is the most widely used biomedical animal model, it is difficult to isolate murine mesenchymal stem cells (MSCs) from the bone marrow because of contamination by hematopoietic cells. The murine compact bone tissue of long bones is considered a novel and reliable source of MSCs with low hematopoietic cell contamination. We investigated whether the murine compact bone of the calvaria would be a promising source of MSCs due to its low bone marrow content. We isolated cells from both long bones and the calvaria using the same method. Although they shared morphological features and surface antigens similar to those of long bone-derived MSCs, the calvaria-derived cells highly expressed the osteogenic transcription factor osterix, lost their adipogenic capacity and gained a higher osteogenic capacity. These findings suggest that the cells that migrated from the calvaria were progenitor cells rather than MSCs and that the differentiation fate of mesenchymal stem/progenitor cells existing in different murine compact bone deposits is already committed.
The KRAB-type zinc-finger protein Apak was recently identified as a negative regulator of p53-mediated apoptosis. However, the mechanism of this selective regulation is not fully understood. Here, we show that Apak recognizes the TCTTN2?30TTGT consensus sequence through its zinc-fingers. This sequence is specifically found in intron 1 of the proapoptotic p53 target gene p53AIP1 and largely overlaps with the p53-binding sequence. Apak competes with p53 for binding to this site to inhibit p53AIP1 expression. Upon DNA damage, Apak dissociates from the DNA, which abolishes its inhibitory effect on p53-mediated apoptosis.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.