Decoding the Impact of Obesity Long Noncoding RNAs on Murine Liver Energy Homeostasis

Long noncoding RNAs (lncRNAs) have recently been recognised as multifaceted regulators of gene expression across multiple cellular and developmental contexts, yet their contribution to liver energy homeostasis remains poorly understood. Using global transcriptome profiling we demonstrate that both chronic and acute nutrient challenges elicit global anticorrelative transcriptional responses of protein-coding mRNAs and lncRNAs in liver. To address if lncRNAs functionally contribute to the control of liver metabolism, we performed in vitro characterisation of regulated hepatic lncRNAs and selected metabolically-responsive lncRNAs Gm15441 and lincIRS2 for further characterisations in vivo. Through CRISPR/Cas9-mediated genome engineering we generated deletion alleles of both selected lncRNAs and verified that expression of the respective transcripts were successfully abrogated in in vivo mouse models. Finally, we show that lincIRS2 deficiency causes hyperglycemia and impaired insulin tolerance in in vivo mouse models and provide evidence that lncIRS2 is essential for proper glucose homeostasis and insulin-evoked suppression of hepatic glucose production by impacting on AKT phosphorylation and gluconeogenic and lipogenic gene expression. Collectively, we propose the concept that nutrient-sensitive lncRNA lincIRS2 is transcriptionally coupled to alterations of systemic nutrient states and functions as molecular relay controlling liver energy homeostasis

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease

Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology

In recent years, long noncoding RNAs (lncRNAs) have emerged as multifaceted regulators of gene expression, controlling key developmental and disease pathogenesis processes. However, due to the paucity of lncRNA loss-of-function mouse models, key questions regarding the involvement of lncRNAs in organism homeostasis and (patho)-physiology remain difficult to address experimentally in vivo. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 platform provides a powerful genome-editing tool and has been successfully applied across model organisms to facilitate targeted genetic mutations, including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus. However, just a few lncRNA-deficient mouse lines have been created using CRISPR/Cas9-mediated genome engineering, presumably due to the need for lncRNA-specific gene targeting strategies considering the absence of open-reading frames in these loci. Here, we describe a step-wise procedure for the generation and validation of lncRNA loss-of-function mouse models using CRISPR/Cas9-mediated genome engineering. In a proof-of-principle approach, we generated mice deficient for the liver-enriched lncRNA Gm15441, which we found downregulated during development of metabolic disease and induced during the feeding/fasting transition. Further, we discuss guidelines for the selection of lncRNA targets and provide protocols for in vitro single guide RNA (sgRNA) validation, assessment of in vivo gene-targeting efficiency and knockout confirmation. The procedure from target selection to validation of lncRNA knockout mouse lines can be completed in 18–20 weeks, of which <10 days hands-on working time is required

Vegfr3-tdTomato, a reporter mouse for microscopic visualization of lymphatic vessel by multiple modalities

Lymphatic vessels are indispensable for tissue fluid homeostasis, transport of solutes and dietary lipids and immune cell trafficking. In contrast to blood vessels, which are easily visible by their erythrocyte cargo, lymphatic vessels are not readily detected in the tissue context. Their invisibility interferes with the analysis of the three-dimensional lymph vessel structure in large tissue volumes and hampers dynamic intravital studies on lymphatic function and pathofunction. An approach to overcome these limitations are mouse models, which express transgenic fluorescent proteins under the control of tissue-specific promotor elements. We introduce here the BAC-transgenic mouse reporter strain Vegfr3-tdTomato that expresses a membrane-tagged version of tdTomato under control of Flt4 regulatory elements. Vegfr3-tdTomato mice inherited the reporter in a mendelian fashion and showed selective and stable fluorescence in the lymphatic vessels of multiple organs tested, including lung, kidney, heart, diaphragm, intestine, mesentery, liver and dermis. In this model, tdTomato expression was sufficient for direct visualisation of lymphatic vessels by epifluorescence microscopy. Furthermore, lymph vessels were readily visualized using a number of microscopic modalities including confocal laser scanning, light sheet fluorescence and two-photon microscopy. Due to the early onset of VEGFR-3 expression in venous embryonic vessels and the short maturation time of tdTomato, this reporter offers an interesting alternative to Prox1-promoter driven lymphatic reporter mice for instance to study the developmental differentiation of venous to lymphatic endothelial cells

Biallelic variants in ADAMTS15 cause a novel form of distal arthrogryposis

Purpose: We aimed to identify the underlying genetic cause for a novel form of distal arthrogryposis. Methods: Rare variant family-based genomics, exome sequencing, and disease-specific panel sequencing were used to detect ADAMTS15 variants in affected individuals. Adamts15 expression was analyzed at the single-cell level during murine embryogenesis. Expression patterns were characterized using in situ hybridization and RNAscope. Results: We identified homozygous rare variant alleles of ADAMTS15 in 5 affected individuals from 4 unrelated consanguineous families presenting with congenital flexion contractures of the interphalangeal joints and hypoplastic or absent palmar creases. Radiographic investigations showed physiological interphalangeal joint morphology. Additional features included knee, Achilles tendon, and toe contractures, spinal stiffness, scoliosis, and orthodontic abnormalities. Analysis of mouse whole-embryo single-cell sequencing data revealed a tightly regulated Adamts15 expression in the limb mesenchyme between embryonic stages E11.5 and E15.0. A perimuscular and peritendinous expression was evident in in situ hybridization in the developing mouse limb. In accordance, RNAscope analysis detected a significant coexpression with Osr1, but not with markers for skeletal muscle or joint formation. Conclusion: In aggregate, our findings provide evidence that rare biallelic recessive trait variants in ADAMTS15 cause a novel autosomal recessive connective tissue disorder, resulting in a distal arthrogryposis syndrome. (C) 2022 The Authors. Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics.US National Institute of Neurological Disorders and Stroke [R35 NS 105078]; US National Human Genome Research Institute (NHGRI); National Heart, Lung, and Blood Institute [UM1 HG006542, R01 GM106373, U01 HG011758, 512848]; Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health [P50HD103555]; International Rett Syndrome Foundation (IRSF) [3701-1]; NHGRI [K08 HG008986]; German Research Council (DFG) [KO 2891/9-1]; BIH Center for Regenerative Therapies (BCRT)We are grateful to the families for their participation in this study. We thank Aris. N. Economides and Manuel Holtgrewe for their valuable suggestions and support. J.R.L. laboratory is supported by the US National Institute of Neurological Disorders and Stroke (R35 NS 105078) and in part by the US National Human Genome Research Institute (NHGRI) and National Heart, Lung, and Blood Institute to the Baylor-Hopkins Center for Mendelian Genomics (BHCMG; UM1 HG006542), the National Institute of General Medical Sciences (NIGMS; R01 GM106373), the NHGRI Baylor College of Medicine Genomics Research Elucidates Genetics of Rare Diseases (BCM-GREGoR; U01 HG011758), the Muscular Dystrophy Association (512848), and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health under award number P50HD103555 for use of the Clinical Translation Core facilities. D.P. is supported by International Rett Syndrome Foundation (IRSF; grant #3701-1). J.E.P. was supported by NHGRI K08 HG008986. U.K. obtained funding from the German Research Council (DFG)(KO 2891/9-1) and the BIH Center for Regenerative Therapies (BCRT)(cross-field project GenoPro)

Autologous Stem Cell Transplantation in Multiple Myeloma in the Era of Novel Drug Induction: A Retrospective Single-Center Analysis.

Within this retrospective single-center study, we analyzed the survival of 320 multiple myeloma (MM) patients receiving melphalan high-dose chemotherapy (HDCT) and either single (n = 286) or tandem (n = 34) autologous stem cell transplantation (ASCT) from 1996 to 2012. Additionally, the impact of novel induction regimens was assessed. Median follow-up was 67 months, median overall survival (OS) 62 months, median progression-free survival (PFS) 33 months (95% CI 27-39), and treatment-related death (TRD) 3%. Multivariate analysis revealed age ≥60 years (p = 0.03) and stage 3 according to the International Staging System (p = 0.006) as adverse risk factors regarding PFS. Median OS was significantly better in newly diagnosed MM patients receiving induction therapy with novel agents, e.g., bortezomib, thalidomide, or lenalidomide, compared with a traditional regimen (69 vs. 58 months; p = 0.01). More patients achieved at least a very good partial remission in the period from 2005 to 2012 than from 1996 to 2004 (65 vs. 30%; p < 0.001), with a longer median OS in the later period (71 vs. 52 months, p = 0.027). In conclusion, our analysis confirms HDCT-ASCT as an effective therapeutic strategy in an unselected large myeloma patient cohort with a low TRD rate and improved prognosis due to novel induction strategies

Application of tobacco hairy roots for the removal of malachite green from aqueous solutions: Experimental design, kinetic, equilibrium, and thermodynamic studies

Tobacco hairy roots (THR) were used to evaluate its potential for the biosorption and removal of malachite green (MG) from aqueous solutions. A 32 full factorial design was applied to study the effects of pH and THR concentration on the biosorption capacity. Under the optimal conditions (pH of 7.0 and THR concentration of 1 g L−1), dye removal efficiency was around 92%. Experimental data obtained from kinetic studies demonstrated good concordance with the pseudo-second-order model. Equilibrium studies were developed and the data were evaluated by Langmuir, Freundlich, and Sips models, being the Sips model the most adequate (maximum biosorption capacity of 277.2 mg g−1). Thermodynamically, the biosorption of MG on THR proved to be endothermic, spontaneous, and favorable. Desorption was feasible under acidic conditions and the biosorbent could be reused three times. THR was tested in simulated effluent and the removal percentage was 87%, demonstrating that this material is a promising biosorbent which can be used to treat colored wastewaters.Fil: Escudero, Leticia Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales. Laboratorio de Química Analítica para Investigación y Desarrollo; ArgentinaFil: Agostini, Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Dotto, Guilherme L.. Universidade Federal de Santa Maria; Brasi