Role of differentially expressed LBX1 in Adolescent Idiopathic Scoliosis (AIS) paraspinal muscle phenotypes and muscle-bone crosstalk through modulating myoblasts

2021 ◽  
Author(s):  
Y Wang ◽  
Z Feng ◽  
KL Cheng ◽  
J Zhang ◽  
L Xu ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Congenital Scoliosis ◽  
Paraspinal Muscle ◽  
Muscle Fiber Types ◽  
Fiber Types ◽  
Loss Of Function ◽  
Paraspinal Muscles ◽  
Function Study ◽  
Myogenic Markers

AIS is three-dimensional spinal deformity with unclear etiopathogenesis. LBX1 is so far the only multi-centers validated AIS predisposing gene. The imbalance of posterior paraspinal muscles is an important factor in AIS etiopathogenesis. It is poorly understood how LBX1 contributes to the abnormal paraspinal muscles and onset/progression of AIS. We aimed to evaluate the expression of LBX1 in paraspinal muscles at the concave and convex side in AIS, and whether alternation of LBX1 expression could affect myoblastsactivities and potentially influence muscle-bone interaction via myokines expression. Paraspinal muscles from AIS and age- and curvature-matched congenital scoliosis (CS) patients were collected for fiber types analysis. Biopsies were also subjected to qPCR to validate expression of myogenic markers, selected myokines and LBX1. Human skeletal muscle myoblast (HSMM) was used for LBX1 loss-of-function study in vitro. Muscle fiber types analysis showed type I and type IIX/IIAX fibers proportion were significantly different between AIS concave and convex but not in two sides of CS. LBX1, myogenic markers and one myokine were significantly imbalanced in AIS but not in CS. Loss-of-function study showed knockdown of LBX1 could inhibit myogenic markers expression and myokines as well. This study provides new insight into the association between imbalanced paraspinal muscle and potential muscle-bone crosstalk in AIS patients and the biological function of predisposing gene LBX1. Further investigation with appropriate animal models is warranted to explore if asymmetric expression of LBX1 could result in distinct muscle phenotypes and bone qualities thus affect the progression of spine curvature in AIS.

Rapid characterization of candidate loss of function genes in primary organoid culture.

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 77-77
Author(s):  
Daniel James Hart ◽  
Ameen Abdulla Salahudeen ◽  
Sean de la O ◽  
Kyuho Han ◽  
David Morgens ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Suitable Model ◽  
Loss Of Function ◽  
Synonymous Mutations ◽  
Functional Control ◽  
Rapid Characterization ◽  
Air Liquid Interface ◽  
Shrna Screen ◽  
Future Work

77 Background: Genome scale sequencing efforts have identified chromosomal deletions and non-synonymous mutations as putative drivers of gastro-intestinal cancer. There is a need to rapidly validate these drivers in robust models. The culture of primary, non-transformed tissues in vitro as three-dimensional organoids that accurately recapitulate organ structure and physiology serves as an ideal model for such validation, and many other applications in biology. Methods: Mouse wild type and p53 flox/flox upper digestive tract tissue containing epithelial and mesenchymal components were cultured in an air-liquid interface and subjected to adenovirus expressing either a functional control or Cre recombinase. Resultant organoids were passaged at confluency until dysplasia was evident in histology. A lentiviral shRNA (loss-of-function) screen was then conducted, where proliferative activity was measured with barcode reads from the pooled library. Results: p53-null organoids showed histology consistent with human gastrointestinal cancer. These organoids were also able to form small tumors when injected subcutaneously into immune-deficient mice. The pooled lentiviral shRNA screen functionally validated candidate drivers. Conclusions: Gastrointestinal organoids are a suitable model for the introduction of oncogenic mutations in a tractable and replicable format. In future work we will validate loss-of function and gain-of-function screens with CRISPRi/a libraries, and replicate these screens in human gastrointestinal organoids.

scholarly journals Functional studies of twelve mutant V2 vasopressin receptors related to nephrogenic diabetes insipidus: molecular basis of a mild clinical phenotype.

1998 ◽  
Vol 9 (10) ◽  
pp. 1861-1872
Author(s):  
Y Ala ◽  
D Morin ◽  
B Mouillac ◽  
N Sabatier ◽  
R Vargas ◽  
...  
Keyword(s):  
Diabetes Insipidus ◽  
Nephrogenic Diabetes Insipidus ◽  
Three Dimensional ◽  
Urine Osmolality ◽  
Complete Loss ◽  
Loss Of Function ◽  
Hydrogen Bond Formation ◽  
Functional Studies ◽  
Three Dimensional Modeling

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine vasopressin V2 receptor responses due to mutations in the AVPR2 gene in Xq28. To study the cause of loss of function of mutant V2 receptors, we expressed 12 mutations (N55H, L59P, L83Q, V88M, 497CC-->GG, deltaR202, I209F, 700delC, 908insT, A294P, P322H, P322S) in COS-7 cells. Eleven of these, including P322H, were characterized by a complete loss of function, but the mutation P322S demonstrated a mild clinical and in vitro phenotype. This was characterized by a late diagnosis without any growth or developmental delay and a significant increase in urine osmolality after intravenous 1-deamino[D-Arg8]AVP administration. In vitro, the P322S mutant was able to partially activate the Gs/adenylyl cyclase system in contrast to the other V2R mutants including P322H, which were completely inactive in this regard. This showed not only that Pro 322 is important for proper V2R coupling, but also that the degree of impairment is strongly dependent on the identity of the substituting amino acid. Three-dimensional modeling of the P322H and P322S mutant receptors suggested that the complete loss of function of the P322H receptor could be due, in part, to hydrogen bond formation between the His 322 side chain and the carboxyl group of Asp 85, which does not occur in the P322S receptor.

Structure-Function Study of a Plasmodium falciparum Hsp70 Using Three Dimensional Modelling and in Vitro Analyses

2008 ◽  
Vol 15 (10) ◽  
pp. 1117-1125 ◽  
Author(s):  
Addmore Shonhai ◽  
Melissa Botha ◽  
Tjaart de Beer ◽  
Aileen Boshoff ◽  
Gregory Blatch
Keyword(s):  
Plasmodium Falciparum ◽  
Structure Function ◽  
Three Dimensional ◽  
Function Study

scholarly journals Three-dimensional in vitro models answer the right questions in ADPKD cystogenesis

2018 ◽  
Vol 315 (2) ◽  
pp. F332-F335 ◽  
Author(s):  
Eryn E. Dixon ◽  
Owen M. Woodward
Keyword(s):  
Three Dimensional ◽  
Renal Tissue ◽  
In Vitro Models ◽  
Model Systems ◽  
Membrane Composition ◽  
Loss Of Function ◽  
Novel Technologies ◽  
Autosomal Dominant Polycystic Kidney ◽  
The Right

Novel technologies, new understanding of the basement membrane composition, and better comprehension of the embryonic development of the mammalian kidney have led to explosive growth in the use of three-dimensional in vitro models to study a range of human disease pathologies (Clevers H. Cell 165: 1586–1597, 2016; Shamir ER, Ewald AJ. Nat Rev Mol Cell Biol 15: 647–664, 2014). The development of these effective model systems represents a new tool to study the progressive cystogenesis of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is a prevalent and complex monogenetic disease, characterized by the pathological formation of fluid fill cysts in renal tissue (Grantham JJ, Mulamalla S, Swenson-Fields KI. Nat Rev Nephrol 7: 556–566, 2011; Takiar V, Caplan MJ. Biochim Biophys Acta 1812: 1337–1343, 2011). ADPKD cystogenesis is attributed to loss of function mutations in either PKD1 or PKD2, which encode for two transmembrane proteins, polycystin-1 and polycystin-2, and progresses with loss of both copies of either gene through a proposed two-hit mechanism with secondary somatic mutations (Delmas P, Padilla F, Osorio N, Coste B, Raoux M, Crest M. Biochem Biophys Res Commun 322: 1374–1383, 2004; Pei Y, Watnick T, He N, Wang K, Liang Y, Parfrey P, Germino G, St George-Hyslop P. Am Soc Nephrol 10: 1524–1529, 1999; Wu G, D’Agati V, Cai Y, Markowitz G, Park JH, Reynolds DM, Maeda Y, Le TC, Hou H Jr, Kucherlapati R, Edelmann W, Somlo S. Cell 93: 177–188, 1998). The exaggerated consequences of large fluid filled cysts result in fibrosis and nephron injury, leading initially to functional compensation but ultimately to dysfunction (Grantham JJ. Am J Kidney Dis 28: 788–803, 1996; Norman J. Biochim Biophys Acta 1812: 1327–1336, 2011; Song CJ, Zimmerman KA, Henke SJ, Yoder BK. Results Probl Cell Differ 60: 323–344, 2017). The complicated disease progression has scattered focus and resources across the spectrum of ADPKD research.

In vitro responses of cat skeletal muscle to halothane and caffeine

1985 ◽  
Vol 58 (2) ◽  
pp. 521-527 ◽  
Author(s):  
P. A. Deuster ◽  
E. L. Bockman ◽  
S. M. Muldoon
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Soleus Muscle ◽  
Ringer Solution ◽  
Muscle Fiber Types ◽  
Type I ◽  
Fiber Types ◽  
Type Ii

Strips of soleus (100% type I) and gracilis (90% type II) muscle were obtained from anesthetized cats and mounted in organ baths filled with aerated Krebs-Ringer solution (37 degrees C). The contractile patterns in response to electrical stimulation (0.1 Hz, 25 V, 5 ms), caffeine, halothane, and caffeine in the presence of halothane were examined in the two fiber types. The ability of 25 microM dantrolene to alter the contractile patterns was also evaluated. In vitro contractile properties in response to electrical stimulation were similar to properties observed in situ, except that twitch tension in soleus muscle was significantly less in vitro than in situ. In the presence of halothane, type I soleus muscle developed a rapid contracture. The contracture was blocked by pretreatment with dantrolene and was reversed by addition of dantrolene at the peak of the response. Halothane-induced contractures were not observed at any time in type II gracilis. Type I soleus was also significantly more sensitive both to caffeine alone and to caffeine in the presence of halothane than was type II gracilis. In both fiber types, halothane increased the sensitivity of the muscles to caffeine. Dantrolene attenuated caffeine-induced contractures in both fiber types, but the attenuating effect was less in the presence of halothane. The findings of a halothane-induced contracture in the cat soleus and differential sensitivities of the two muscle fiber types to caffeine indicate that further studies in these two muscles may be useful for delineating the mechanisms inducing contracture in muscle from individuals susceptible to malignant hyperthermia.

scholarly journals A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Stefania Rosano ◽  
Davide Corà ◽  
Sushant Parab ◽  
Serena Zaffuto ◽  
Claudio Isella ◽  
...  
Keyword(s):  
Target Genes ◽  
Global Analysis ◽  
Three Dimensional ◽  
Loss Of Function ◽  
Temporal Coordination ◽  
Sprouting Angiogenesis ◽  
Network Modules ◽  
Vitro Model ◽  
Colorectal Cancer Patients

Angiogenesis requires the temporal coordination of the proliferation and the migration of endothelial cells. Here, we investigated the regulatory role of microRNAs (miRNAs) in harmonizing angiogenesis processes in a three-dimensional in vitro model. We described a microRNA network which contributes to the observed down- and upregulation of proliferative and migratory genes, respectively. Global analysis of miRNA–target gene interactions identified two sub-network modules, the first organized in upregulated miRNAs connected with downregulated target genes and the second with opposite features. miR-424–5p and miR-29a-3p were selected for the network validation. Gain- and loss-of-function approaches targeting these microRNAs impaired angiogenesis, suggesting that these modules are instrumental to the temporal coordination of endothelial migration and proliferation. Interestingly, miR-29a-3p and its targets belong to a selective biomarker that is able to identify colorectal cancer patients who are responding to anti-angiogenic treatments. Our results provide a view of higher-order interactions in angiogenesis that has potential to provide diagnostic and therapeutic insights.

Bi-allelic loss of function variants of TBX6 causes a spectrum of malformation of spine and rib including congenital scoliosis and spondylocostal dysostosis

2019 ◽  
Vol 56 (9) ◽  
pp. 622-628 ◽  
Author(s):  
Nao Otomo ◽  
Kazuki Takeda ◽  
Shunsuke Kawai ◽  
Ikuyo Kou ◽  
Long Guo ◽  
...  
Keyword(s):  
Congenital Scoliosis ◽  
Allelic Loss ◽  
Risk Haplotype ◽  
Nucleotide Polymorphisms ◽  
Loss Of Function ◽  
Missense Variants ◽  
Spondylocostal Dysostosis ◽  
Rib Anomalies ◽  
Somite Formation

BackgroundCongenital scoliosis (CS) is a common vertebral malformation. Spondylocostal dysostosis (SCD) is a rare skeletal dysplasia characterised by multiple vertebral malformations and rib anomalies. In a previous study, a compound heterozygosity for a null mutation and a risk haplotype composed by three single-nucleotide polymorphisms in TBX6 have been reported as a disease-causing model of CS. Another study identified bi-allelic missense variants in a SCD patient. The purpose of our study is to identify TBX6 variants in CS and SCD and examine their pathogenicity.MethodsWe recruited 200 patients with CS or SCD and investigated TBX6 variants. We evaluated the pathogenicity of the variants by in silico prediction and in vitro experiments.ResultsWe identified five 16p11.2 deletions, one splice-site variant and five missense variants in 10 patients. In vitro functional assays for missense variants identified in the previous and present studies demonstrated that most of the variants caused abnormal localisation of TBX6 proteins. We confirmed mislocalisation of TBX6 proteins in presomitic mesoderm cells induced from SCD patient-derived iPS cells. In induced cells, we found decreased mRNA expressions of TBX6 and its downstream genes were involved in somite formation. All CS patients with missense variants had the risk haplotype in the opposite allele, while a SCD patient with bi-allelic missense variants did not have the haplotype.ConclusionsOur study suggests that bi-allelic loss of function variants of TBX6 cause a spectrum of phenotypes including CS and SCD, depending on the severity of the loss of TBX6 function.

Length-tension relationship of mammalian diaphragm muscles

1983 ◽  
Vol 54 (6) ◽  
pp. 1681-1686 ◽  
Author(s):  
K. K. McCully ◽  
J. A. Faulkner
Keyword(s):  
Muscle Fiber ◽  
Relaxation Times ◽  
Isometric Tension ◽  
Diaphragm Muscle ◽  
Muscle Fiber Types ◽  
Fiber Types ◽  
Active Tension ◽  
Tension Curves ◽  
Relationship Of

We tested the hypothesis that the length-tension relationship of diaphragm muscle is different from that of other skeletal muscle. Isometric contractile properties of small bundles of diaphragm muscle from rats, cats, rhesus monkeys, dogs, and pigs were measured in vitro at 37 degrees C. For diaphragm muscles from all species, twitch contraction and relaxation times and histochemical myofibrillar ATPase indicated a mixture of fast and slow muscle fiber types. By use of tetanic stimulations of 400-ms duration, isometric tension was recorded from bundles of diaphragm muscle at lengths between 50 and 130% of the muscle fiber length at which active tension was maximal (Lo). At lengths below 60–70% of Lo, tetanic tension did not reach a plateau within 400 ms. With longer stimulation durations, active tension could be recorded between 30 and 40% of Lo. The length-tension curves for bundles of fibers from the diaphragm muscle were not different among species and were consistent with results reported for intact limb muscles.

Influence of thyroid status on skeletal muscle LPL activity and TG uptake

1980 ◽  
Vol 238 (6) ◽  
pp. E518-E523 ◽  
Author(s):  
H. Kaciuba-Uscilko ◽  
G. A. Dudley ◽  
R. L. Terjung
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fiber Types ◽  
Fiber Types ◽  
Thyroid Status ◽  
Different Types ◽  
Slow Twitch ◽  
Fast Twitch ◽  
Fractional Uptake

The influence of thyroid status on the clearance of chylomicron 14C-labeled triglycerides (14C-TG), the fractional uptake of 14C-TG, and activity of lipoprotein lipase (LPL) in the different skeletal muscle fiber types was evaluated in pentobarbital-anesthetized rats. The turnover of plasma TG was approximately fourfold greater in the hyperthyroid (HyperT) compared to either euthyroid (EUT) or hypothyroid (HypoT) animals. The uptake of 14C-TG was increased in slow-twitch red and fast-twitch red muscle sections of the HyperT group and normal in the HypoT group. The changes in LPL activity in these two fiber types were inversely related to thyroid influence, with decreases found in the HyperT group and increases found in the HypoT group. Thus, the uptake of 14C-TG in these high-oxidative fibers, relative to the LPL activity, varied directly with thyroid influence. As a result, the normal linear relationship, apparent for the different types of skeletal muscle, between LPL activity measured in vitro and TG uptake determined in situ was not maintained.

scholarly journals Identification of novel FBN1 variations implicated in congenital scoliosis

2019 ◽  
Vol 65 (3) ◽  
pp. 221-230 ◽  
Author(s):  
Mao Lin ◽  
◽  
Sen Zhao ◽  
Gang Liu ◽  
Yingzhao Huang ◽  
...  
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Missense Variant ◽  
Congenital Scoliosis ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Exact Test ◽  
Novel Variants ◽  
Burden Test

AbstractCongenital scoliosis (CS) is a form of scoliosis caused by congenital vertebral malformations. Genetic predisposition has been demonstrated in CS. We previously reported that TBX6 loss-of-function causes CS in a compound heterozygous model; however, this model can explain only 10% of CS. Many monogenic and polygenic CS genes remain to be elucidated. In this study, we analyzed exome sequencing (ES) data of 615 Chinese CS from the Deciphering Disorders Involving Scoliosis and COmorbidities (DISCO) project. Cosegregation studies for 103 familial CS identified a novel heterozygous nonsense variant, c.2649G>A (p.Trp883Ter) in FBN1. The association between FBN1 and CS was then analyzed by extracting FBN1 variants from ES data of 574 sporadic CS and 828 controls; 30 novel variants were identified and prioritized for further analyses. A mutational burden test showed that the deleterious FBN1 variants were significantly enriched in CS subjects (OR = 3.9, P = 0.03 by Fisher’s exact test). One missense variant, c.2613A>C (p.Leu871Phe) was recurrent in two unrelated CS subjects, and in vitro functional experiments for the variant suggest that FBN1 may contribute to CS by upregulating the transforming growth factor beta (TGF-β) signaling. Our study expanded the phenotypic spectrum of FBN1, and provided nove insights into the genetic etiology of CS.

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