Browsing by Subject "EXPRESSION"

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  • Villaseñor-Altamirano, Ana B.; Watson, John D.; Prokopec, Stephenie D.; Yao, Cindy Q.; Boutros, Paul C.; Pohjanvirta, Raimo; Valdés-Flores, Jesús; Elizondo, Guillermo (2019)
    Alternative splicing is a co-transcriptional mechanism that generates protein diversity by including or excluding exons in different combinations, thereby expanding the diversity of protein isoforms of a single gene. Abnormalities in this process can result in deleterious effects to human health, and several xenobiotics are known to interfere with splicing regulation through multiple mechanisms. These changes could lead to human diseases such as cancer, neurological disorders, autoimmune diseases, and developmental disorders. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant generated as a byproduct of various industrial activities. Exposure to this dioxin has been linked to a wide range of pathologies through the alteration of multiple cellular processes. However, the effects of TCDD exposure on alternative splicing have not yet been studied. Here, we investigated whether a single po. dose of 5 μg/kg or 500 μg/kg TCDD influence hepatic alternative splicing in adult male C57BL/6Kou mouse. We identified several genes whose alternative splicing of precursor messenger RNAs was modified following TCDD exposure. In particular, we demonstrated that alternative splicing of Cyp1a1, Ahrr, and Actn1 was significantly altered after TCDD treatment. These findings show that the exposure to TCDD has an impact on alternative-splicing, and suggest a new avenue for understanding TCDD-mediated toxicity and pathogenesis.
  • Lou, Yan-Ru; Toh, Tai Chong; Tee, Yee Han; Yu, Hanry (2017)
    25-Hydroxyvitamin D-3 [25(OH)D-3] has recently been found to be an active hormone. Its biological actions are demonstrated in various cell types. 25(OH)D-3 deficiency results in failure in bone formation and skeletal deformation. Here, we investigated the effect of 25(OH)D-3 on osteogenic differentiation of human mesenchymal stem cells (hMSCs). We also studied the effect of 1 alpha, 25-dihydroxyvitamin D-3[1 alpha,25-(OH)(2)D-3], a metabolite of 25(OH)D-3. One of the vitamin D responsive genes, 25(OH)D-3-24-hydroxylase (cytochrome P450 family 24 subfamily A member 1) mRNA expression is up-regulated by 25(OH)D-3 at 250-500 nM and by 1 alpha, 25-(OH)(2)D-3 at 1-10 nM. 25(OH)D-3 and 1 alpha, 25-(OH)(2)D-3 at a time-dependent manner alter cell morphology towards osteoblast-associated characteristics. The osteogenic markers, alkaline phosphatase, secreted phosphoprotein 1 (osteopontin), and bone gamma-carboxyglutamate protein (osteocalcin) are increased by 25(OH)D-3 and 1 alpha,25-(OH)(2)D-3 in a dose-dependent manner. Finally, mineralisation is significantly increased by 25(OH)D-3 but not by 1 alpha, 25-(OH)(2)D-3. Moreover, we found that hMSCs express very low level of 25(OH)D-3-1 alpha-hydroxylase (cytochrome P450 family 27 subfamily B member 1), and there is no detectable 1 alpha, 25-(OH)(2)D-3 product. Taken together, our findings provide evidence that 25(OH)D-3 at 250-500 nM can induce osteogenic differentiation and that 25(OH)D-3 has great potential for cell-based bone tissue engineering.
  • Kondelin, Johanna; Tuupanen, Sari; Gylfe, Alexandra E.; Aavikko, Mervi; Renkonen-Sinisalo, Laura; Järvinen, Heikki; Bohm, Jan; Mecklin, Jukka-Pekka; Andersen, Claus L.; Vahteristo, Pia; Pitkanen, Esa; Aaltonen, Lauri A. (2015)
    Approximately 15 % of colorectal cancers exhibit instability of short nucleotide repeat regions, microsatellites. These tumors display a unique clinicopathologic profile and the microsatellite instability status is increasingly used to guide clinical management as it is known to predict better prognosis as well as resistance to certain chemotherapeutics. A panel of five repeats determined by the National Cancer Institute, the Bethesda panel, is currently the standard for determining the microsatellite instability status in colorectal cancer. Recently, a quasimonomorphic mononucleotide repeat 16T/U at the 3' untranslated region of the Ewing sarcoma breakpoint region 1 gene was reported to show perfect sensitivity and specificity in detecting mismatch repair deficient colorectal, endometrial, and gastric cancers in two independent populations. To confirm this finding, we replicated the analysis in 213 microsatellite unstable colorectal cancers from two independent populations, 148 microsatellite stable colorectal cancers, and the respective normal samples by PCR and fragment analysis. The repeat showed nearly perfect sensitivity for microsatellite unstable colorectal cancer as it was altered in 212 of the 213 microsatellite unstable (99.5 %) and none of the microsatellite stable colorectal tumors. This repeat thus represents the first potential single marker for detecting microsatellite instability.
  • Gu, Ying; Lee, Hsi-Ming; Napolitano, Nicole; Clemens, McKenzie; Zhang, Yazhou; Sorsa, Timo; Zhang, Yu; Johnson, Francis; Golub, Lorne M. (2013)
  • Buettner, Ralf; Le Xuan Truong Nguyen; Kumar, Bijender; Morales, Corey; Liu, Chao; Chen, Lisa S.; Pemovska, Tea; Synold, Timothy W.; Palmer, Joycelynne; Thompson, Ryan; Li, Ling; Dinh Hoa Hoang; Zhang, Bin; Ghoda, Lucy; Kowolik, Claudia; Kontro, Mika; Leitch, Calum; Wennerberg, Krister; Yu, Xiaochun; Chen, Ching-Cheng; Horne, David; Gandhi, Varsha; Pullarkat, Vinod; Marcucci, Guido; Rosen, Steven T. (2019)
    Nucleoside analogs represent the backbone of several distinct chemotherapy regimens for acute myeloid leukemia (AML) and combination with tyrosine kinase inhibitors has improved survival of AML patients, including those harboring the poor-risk FLT3-ITD mutation. Although these compounds are effective in killing proliferating blasts, they lack activity against quiescent leukemia stem cells (LSCs), which contributes to initial treatment refractoriness or subsequent disease relapse. The reagent 8-chloro-adenosine (8-Cl-Ado) is a ribose-containing, RNA-directed nucleoside analog that is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. In this report, we demonstrate antileukemic activities of 8-Cl-Ado in vitro and in vivo and provide mechanistic insight into the mode of action of 8-Cl-Ado in AML. 8-Cl-Ado markedly induced apoptosis in LSC, with negligible effects on normal stem cells. 8-Cl-Ado was particularly effective against AML cell lines and primary AML blast cells harboring the FLT3-ITD mutation. FLT3-ITD is associated with high expression of miR-155. Furthermore, we demonstrate that 8-Cl-Ado inhibits miR-155 expression levels accompanied by induction of DNA-damage and suppression of cell proliferation, through regulation of miR-155/ErbB3 binding protein 1(Ebp1)/p53/PCNA signaling. Finally, we determined that combined treatment of NSG mice engrafted with FLT3-ITD (+) MV4-11 AML cells with 8-Cl-Ado and the FLT3 inhibitor AC220 (quizartinib) synergistically enhanced survival, compared with that of mice treated with the individual drugs, suggesting a potentially effective approach for FLT3-ITD AML patients.
  • Radhakrishnan, Dhanya; Shanmukhan, Anju Pallipurath; Kareem, Abdul; Aiyaz, Mohammed; Varapparambathu, Vijina; Toms, Ashna; Kerstens, Merijn; Valsakumar, Devisree; Landge, Amit N.; Shaji, Anil; Mathew, Mathew K.; Sawchuk, Megan G.; Scarpella, Enrico; Krizek, Beth A.; Efroni, Idan; Mähönen, Ari Pekka; Willemsen, Viola; Scheres, Ben; Prasad, Kalika (2020)
    Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.
  • Frischknecht, Mirjam; Niehof-Oellers, Helena; Jagannathan, Vidhya; Owczarek-Lipska, Marta; Droegemueller, Cord; Dietschi, Elisabeth; Dolf, Gaudenz; Tellhelm, Bernd; Lang, Johann; Tiira, Katriina; Lohi, Hannes; Leeb, Tosso (2013)
  • Lluch, Aina; Veiga, Sonia R.; Latorre, Jèssica; Moreno-Navarrete, José M.; Bonifaci, Núria; Nguyen, Van Dien; Zhou, You; Höring, Marcus; Liebisch, Gerhard; Olkkonen, Vesa M.; Llobet-Navas, David; Thomas, George; Rodríguez-Barrueco, Ruth; Fernández-Real, José M.; Kozma, Sara C.; Ortega, Francisco J. (2022)
    The ribosomal protein S6 kinase 1 (S6K1) is a relevant effector downstream of the mammalian target of rapamycin complex 1 (mTORC1), best known for its role in the control of lipid homeostasis. Consistent with this, mice lacking the S6k1 gene have a defect in their ability to induce the commitment of fat precursor cells to the adipogenic lineage, which contributes to a significant reduction of fat mass. Here, we assess the therapeutic blockage of S6K1 in diet-induced obese mice challenged with LY2584702 tosylate, a specific oral S6K1 inhibitor initially developed for the treatment of solid tumors. We show that diminished S6K1 activity hampers fat mass expansion and ameliorates dyslipidemia and hepatic steatosis, while modifying transcriptome-wide gene expression programs relevant for adipose and liver function. Accordingly, decreased mTORC1 signaling in fat (but increased in the liver) segregated with defective epithelial-mesenchymal transition and the impaired expression of Cd36 (coding for a fatty acid translocase) and Lgals1 (Galectin 1) in both tissues. All these factors combined align with reduced adipocyte size and improved lipidomic signatures in the liver, while hepatic steatosis and hypertriglyceridemia were improved in treatments lasting either 3 months or 6 weeks.
  • Sajanti, Antti; Lyne, Sean B.; Girard, Romuald; Frantzen, Janek; Rantamaki, Tomi; Heino, Iiro; Cao, Ying; Diniz, Cassiano; Umemori, Juzoh; Li, Yan; Takala, Riikka; Posti, Jussi P.; Roine, Susanna; Koskimäki, Fredrika; Rahi, Melissa; Rinne, Jaakko; Castren, Eero; Koskimäki, Janne (2020)
    P75 neurotrophic receptor (p75NTR) is an important receptor for the role of neurotrophins in modulating brain plasticity and apoptosis. The current understanding of the role of p75NTR in cellular adaptation following pathological insults remains blurred, which makes p75NTR's related signaling networks an interesting and challenging initial point of investigation. We identified p75NTR and related genes through extensive data mining of a PubMed literature search including published works related to p75NTR from the past 20 years. Bioinformatic network and pathway analyses of identified genes (n=235) were performed using ReactomeFIViz in Cytoscape based on the highly reliable Reactome functional interaction network algorithm. This approach merges interactions extracted from human curated pathways with predicted interactions from machine learning. Genome-wide pathway analysis showed total of 16 enriched hierarchical clusters. A total of 278 enriched single pathways were also identified (p
  • Kostopoulos, Ioannis; Aalvink, Steven; Kovatcheva-Datchary, Petia; Nijsse, Bart; Backhed, Fredrik; Knol, Jan; de Vos, Willem M.; Belzer, Clara (2021)
    The human gastrointestinal tract is colonized by a diverse microbial community, which plays a crucial role in human health. In the gut, a protective mucus layer that consists of glycan structures separates the bacteria from the host epithelial cells. These host-derived glycans are utilized by bacteria that have adapted to this specific compound in the gastrointestinal tract. Our study investigated the close interaction between two distinct gut microbiota members known to use mucus glycans, the generalist Bacteroides thetaiotaomicron and the specialist Akkermansia muciniphila in vitro and in vivo. The in vitro study, in which mucin was the only nutrient source, indicated that B. thetaiotaomicron significantly upregulated genes coding for Glycoside Hydrolases (GHs) and mucin degradation activity when cultured in the presence of A. muciniphila. Furthermore, B. thetaiotaomicron significantly upregulated the expression of a gene encoding for membrane attack complex/perforin (MACPF) domain in co-culture. The transcriptome analysis also indicated that A. muciniphila was less affected by the environmental changes and was able to sustain its abundance in the presence of B. thetaiotaomicron while increasing the expression of LPS core biosynthesis activity encoding genes (O-antigen ligase, Lipid A and Glycosyl transferases) as well as ABC transporters. Using germ-free mice colonized with B. thetaiotaomicron and/or A. muciniphila, we observed a more general glycan degrading profile in B. thetaiotaomicron while the expression profile of A. muciniphila was not significantly affected when colonizing together, indicating that two different nutritional niches were established in mice gut. Thus, our results indicate that a mucin degrading generalist adapts to its changing environment, depending on available carbohydrates while a mucin degrading specialist adapts by coping with competing microorganism through upregulation of defense related genes.
  • Rotgers, E.; Cisneros-Montalvo, S.; Jahnukainen, K.; Sandholm, J.; Toppari, J.; Nurmio, M. (2015)
    Accurate analysis and quantification of different testicular cell populations are of central importance in studies of male reproductive biology. The traditional histomorphometric and immunohistochemical methods remain the gold standard in studying the complex dynamics of the testicular tissue. Through past years advances have been made in the application of flow cytometry for the rapid analysis of testicular cell populations. Detection of DNA content and of surface antigens and fluorescent reporters have been widely used to analyze and sort cells. Detection of intracellular antigens can broaden the possibilities of applying flow cytometry in studies of male reproduction. Here, we report a detailed protocol for the preparation of rat testicular tissue for detection of intracellular antigens by flow cytometry, and a pipeline for subsequent data analysis and troubleshooting. Rat testicular ontogenesis was chosen as the experimental model to validate the performance of the assay using vimentin and gamma H2AX as intracellular markers for the somatic and spermatogenic cells, respectively. The results show that the assay is reproducible and recapitulates the rat testis ontogenesis.
  • Comai, Glenda; Heude, Eglantine; Mella, Sebastian; Paisant, Sylvain; Pala, Francesca; Gallardo, Mirialys; Langa, Francina; Kardon, Gabrielle; Gopalakrishnan, Swetha; Tajbakhsh, Shahragim (2019)
    In most vertebrates, the upper digestive tract is composed of muscularized jaws linked to the esophagus that permits food ingestion and swallowing. Masticatory and esophagus striated muscles (ESM) share a common cardiopharyngeal mesoderm (CPM) origin, however ESM are unusual among striated muscles as they are established in the absence of a primary skeletal muscle scaffold. Using mouse chimeras, we show that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to MET/HGF signaling for antero-posterior migration of esophagus muscle progenitors, where Hgf ligand is expressed in adjacent smooth muscle cells. These observations highlight the functional relevance of a smooth and striated muscle progenitor dialogue for ESM patterning. Our findings establish a Tbx1-Isl1-Met genetic hierarchy that uniquely regulates esophagus myogenesis and identify distinct genetic signatures that can be used as framework to interpret pathologies arising within CPM derivatives.
  • Mollerup, Filip; Aumala, Ville; Parikka, Kirsti Maria; Mathieu, Yan; Brumer, Harry; Tenkanen, Tiina Maija; Master, Emma (2019)
    Copper radical alcohol oxidases belonging to auxiliary activity family 5, subfamily 2 (AA5_2) catalyze the oxidation of galactose and galactosides, as well as aliphatic alcohols. Despite their broad applied potential, so far very few AA5_2 members have been biochemically characterized. We report the recombinant production and biochemical characterization of an AA5_2 oxidase from Penicillium rubens Wisconsin 54-1255 (PruAA5_2A), which groups within an unmapped clade phylogenetically distant from those comprising AA5_2 members characterized to date. PruAA5_2 preferentially oxidized raffinose over galactose; however, its catalytic efficiency was 6.5 times higher on glycolaldehyde dimer compared to raffinose. Deep sequence analysis of characterized AA5_2 members highlighted amino acid pairs correlated to substrate range and conserved within the family. Moreover, PruAA5_2 activity spans substrate preferences previously reported for AA5 subfamily 1 and 2 members, identifying possible functional overlap across the AA5 family.
  • Vanhatalo, Jarno; Li, Zitong; Sillanpää, Mikko J. (2019)
    Motivation: Recent advances in high dimensional phenotyping bring time as an extra dimension into the phenotypes. This promotes the quantitative trait locus (QTL) studies of function-valued traits such as those related to growth and development. Existing approaches for analyzing functional traits utilize either parametric methods or semi-parametric approaches based on splines and wavelets. However, very limited choices of software tools are currently available for practical implementation of functional QTL mapping and variable selection. Results: We propose a Bayesian Gaussian process (GP) approach for functional QTL mapping. We use GPs to model the continuously varying coefficients which describe how the effects of molecular markers on the quantitative trait are changing over time. We use an efficient gradient based algorithm to estimate the tuning parameters of GPs. Notably, the GP approach is directly applicable to the incomplete datasets having even larger than 50% missing data rate (among phenotypes). We further develop a stepwise algorithm to search through the model space in terms of genetic variants, and use a minimal increase of Bayesian posterior probability as a stopping rule to focus on only a small set of putative QTL. We also discuss the connection between GP and penalized B-splines and wavelets. On two simulated and three real datasets, our GP approach demonstrates great flexibility for modeling different types of phenotypic trajectories with low computational cost. The proposed model selection approach finds the most likely QTL reliably in tested datasets.
  • Jylhava, Juulia; Lyytikainen, Leo-Pekka; Kahonen, Mika; Hutri-Kahonen, Nina; Kettunen, Johannes; Viikari, Jorma; Raitakari, Olli T.; Lehtimaki, Terho; Hurme, Mikko (2012)
  • Porcu, Eleonora; Medici, Marco; Pistis, Giorgio; Volpato, Claudia B.; Wilson, Scott G.; Cappola, Anne R.; Bos, Steffan D.; Deelen, Joris; den Heijer, Martin; Freathy, Rachel M.; Lahti, Jari; Liu, Chunyu; Lopez, Lorna M.; Nolte, Ilja M.; O'Connell, Jeffrey R.; Tanaka, Toshiko; Trompet, Stella; Arnold, Alice; Bandinelli, Stefania; Beekman, Marian; Bohringer, Stefan; Brown, Suzanne J.; Buckley, Brendan M.; Camaschella, Clara; de Craen, Anton J. M.; Davies, Gail; de Visser, Marieke C. H.; Ford, Ian; Forsen, Tom Johan; Frayling, Timothy M.; Fugazzola, Laura; Goegele, Martin; Hattersley, Andrew T.; Hermus, Ad R.; Hofman, Albert; Houwing-Duistermaat, Jeanine J.; Jensen, Richard A.; Kajantie, Eero; Kloppenburg, Margreet; Lim, Ee M.; Masciullo, Corrado; Mariotti, Stefano; Minelli, Cosetta; Mitchell, Braxton D.; Nagaraja, Ramaiah; Netea-Maier, Romana T.; Palotie, Aarno; Persani, Luca; Piras, Maria G.; Psaty, Bruce M.; Räikkönen, Katri; Richards, J. Brent; Rivadeneira, Fernando; Sala, Cinzia; Sabra, Mona M.; Sattar, Naveed; Shields, Beverley M.; Soranzo, Nicole; Starr, John M.; Stott, David J.; Sweep, Fred C. G. J.; Usala, Gianluca; van der Klauw, Melanie M.; van Heemst, Diana; van Mullem, Alies; Vermeulen, Sita H.; Visser, W. Edward; Walsh, John P.; Westendorp, Rudi G. J.; Widen, Elisabeth; Zhai, Guangju; Cucca, Francesco; Deary, Ian J.; Eriksson, Johan G.; Ferrucci, Luigi; Fox, Caroline S.; Jukema, J. Wouter; Kiemeney, Lambertus A.; Pramstaller, Peter P.; Schlessinger, David; Shuldiner, Alan R.; Slagboom, Eline P.; Uitterlinden, Andre G.; Vaidya, Bijay; Visser, Theo J.; Wolffenbuttel, Bruce H. R.; Meulenbelt, Ingrid; Rotter, Jerome I.; Spector, Tim D.; Hicks, Andrew A.; Toniolo, Daniela; Sanna, Serena; Peeters, Robin P.; Naitza, Silvia (2013)
  • Bhutta, Mahmood F.; Lambie, Jane; Hobson, Lindsey; Goel, Anuj; Hafren, Lena; Einarsdottir, Elisabet; Mattila, Petri S.; Farrall, Martin; Brown, Steve; Burton, Martin J. (2017)
    Chronic otitis media with effusion (COME) is the most common cause of hearing loss in children, and known to have high heritability. Mutant mouse models have identified Fbxo11, Evi1, Tgif1, and Nisch as potential risk loci. We recruited children aged 10 and under undergoing surgical treatment for COME from 35 hospitals in the UK, and their nuclear family. We performed association testing with the loci FBXO11, EVI1, TGIF1 and NISCH and sought to replicate significant results in a case-control cohort from Finland. We tested 1296 families (3828 individuals), and found strength of association with the T allele at rs881835 (p = 0.006, OR 1.39) and the G allele at rs1962914 (p = 0.007, OR 1.58) at TGIF1, and the A allele at rs10490302 (p = 0.016, OR 1.17) and the G allele at rs2537742 (p = 0.038, OR 1.16) at FBXO11. Results were not replicated. This study supports smaller studies that have also suggested association of otitis media with polymorphism at FBX011, but this is the first study to report association with the locus TGIF1. Both FBX011 and TGIF1 are involved in TGF-beta signalling, suggesting this pathway may be important in the transition from acute to chronic middle ear inflammation, and a potential molecular target.
  • Strauss, Philipp; Rivedal, Mariell; Scherer, Andreas; Eikrem, Oystein; Nakken, Sigrid; Beisland, Christian; Bostad, Leif; Flatberg, Arnar; Skandalou, Eleni; Beisvag, Vidar; Furriol, Jessica; Marti, Hans-Peter (2022)
    Clear cell renal cell carcinoma (ccRCC) is the most common renal cancer. Identification of ccRCC likely to progress, despite an apparent low risk at the time of surgery, represents a key clinical issue. From a cohort of adult ccRCC patients (n = 443), we selected low-risk tumors progressing within a 5-years average follow-up (progressors: P, n = 8) and non-progressing (NP) tumors (n = 16). Transcriptome sequencing, miRNA sequencing and proteomics were performed on tissues obtained at surgery. We identified 151 proteins, 1167 mRNAs and 63 miRNAs differentially expressed in P compared to NP low-risk tumors. Pathway analysis demonstrated overrepresentation of proteins related to “LXR/ RXR and FXR/RXR Activation”, “Acute Phase Response Signaling” in NP compared to P samples. Integrating mRNA, miRNA and proteomic data, we developed a 10-component classifier including two proteins, three genes and five miRNAs, effectively differentiating P and NP ccRCC and capturing underlying biological differences, potentially useful to identify “low-risk” patients requiring closer surveillance and treatment adjustments. Key results were validated by immunohistochemistry, qPCR and data from publicly available databases. Our work suggests that LXR, FXR and macrophage activation pathways could be critically involved in the inhibition of the progression of low-risk ccRCC. Furthermore, a 10-component classifier could support an early identification of apparently low-risk ccRCC patients.
  • Berger, Claudia; Heyne, Henrike O.; Heiland, Tina; Dommel, Sebastian; Hoefling, Corinna; Guiu-Jurado, Esther; Lorenz, Jana; Rossner, Steffen; Dannemann, Michael; Kelso, Janet; Kovacs, Peter; Blueher, Matthias; Kloeting, Nora (2021)
    The leptin receptor (Lepr) pathway is important for food intake regulation, energy expen-diture, and body weight. Mutations in leptin and the Lepr have been shown to cause early-onset severe obesity in mice and humans. In studies with C57BL/ 6NCrl mice, we found a mouse with extreme obesity. To identify a putative spontaneous new form of monogenic obesity, we performed backcross studies with this mouse followed by a quantitative trait locus (QTL) analysis and sequencing of the selected chro-mosomal QTL region. We thereby identified a novel Lepr mutation (C57BL/6N-Lepr(L536Hfs*6-1NKB)), which is located at chromosome 4, exon 11 within the CRH2-leptin-binding site. Compared with C57BL/6N mice, Lepr(L536Hfs*6) develop early onset obesity and their body weight exceeds that of Leprdb/db mice at an age of 30 weeks. Similar to Leprdb/db mice, the Lepr(L536Hfs*6) model is characterized by hyperphagia, obesity, lower energy expenditure and activity, hyperglycemia, and hyperinsulinemia compared with C57BL/6N mice. Crossing Leprdb/wt with Lepr(L536Hfs*6/wt) mice results in compound heterozygous Lepr(L536Hfs*6/db) mice, which develop even higher body weight and fat mass than both homozygous Lepr(db/db) and Lepr(L536Hfs*6) mice. Compound heterozygous Lepr deficiency affecting functionally different regions of the Lepr causes more severe obesity than the parental homozygous mutations.
  • Leigh, Robert S.; Ruskoaho, Heikki J.; Kaynak, Bogac L. (2020)
    Reliable in vitro models to assess developmental toxicity of drugs and chemicals would lead to improvement in fetal safety and a reduced cost of drug development. The validated embryonic stem cell test (EST) uses cardiac differentiation of mouse embryonic stem cells (mESCs) to predict in vivo developmental toxicity, but does not take into account the stage-specific patterning of progenitor populations into anterior (ventricular) and posterior (atrial) compartments. In this study, we generated a novel dual reporter mESC line with fluorescent reporters under the control of anterior and posterior cardiac promoters. Reporter expression was observed in nascent compartments in transgenic mouse embryos, and mESCs were used to develop differentiation assays in which chemical modulators of Wnt (XAV939: 3, 10 mu M), retinoic acid (all-trans retinoic acid: 0.1, 1, 10 mu M; 9-cis retinoic acid: 0.1, 1, 10 mu M; bexarotene 0.1, 1, 10 mu M), and Tgf-beta (SB431542: 3, 10 mu M) pathways were tested for stage- and dose-dependent effects on in vitro anterior-posterior patterning. Our results suggest that with further development, the inclusion of anterior-posterior reporter expression could be part of a battery of high-throughput tests used to identify and characterize teratogens.