ABSTRACT
FHL1-related myopathies are rare X-linked dominant myopathies. Though clinically classified into several subgroups, spinal and scapuloperoneal muscle involvement are common to all. In this study, we identified c.449G > A, p.C150Y mutation by clinical exome sequencing in two patients from same family (son and mother) of Indian origin who presented with multiple contractures. Muscle biopsy showed numerous intracytoplasmic aggregates intensely stained on HE and MGT. The strong reactions to M-NBT revealed aggregates to be reducing bodies and positively labeled to anti-FHL1 antibody. Ultrastructurally, Z-band streaming and granular and granulofilamentous material were seen. Further, the translational evidence of mutant peptide was confirmed using mass spectrometric analysis. To establish p.C150Y as the cause for protein aggregation, in vivo studies were carried out using transgenic Drosophila model which highlighted Z-band abnormalities and protein aggregates in indirect flight muscles with compromised physiological function. Thus, recapitulating the X-linked human disease phenotype. Additionally, the molecular dynamics simulation analysis unraveled the drastic change in α-helix of LIM2, the region immediately next to site of C150Y mutation that could be the plausible cause for protein aggregation. To the best of our knowledge, this is the first study of p.C150Y mutation in FHL1 identified in Indian patients with in vivo and in silico analysis to establish the cause for protein aggregation in muscle.
Subject(s)
Genetic Diseases, X-Linked/genetics , Intracellular Signaling Peptides and Proteins/genetics , LIM Domain Proteins/genetics , Muscle Proteins/genetics , Muscle, Skeletal/metabolism , Muscular Diseases/congenital , Mutation, Missense , Protein Multimerization , Adult , Animals , Child , Drosophila melanogaster , Female , Genes, Dominant , Genetic Diseases, X-Linked/metabolism , Genetic Diseases, X-Linked/pathology , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , LIM Domain Proteins/chemistry , LIM Domain Proteins/metabolism , Male , Muscle Proteins/chemistry , Muscle Proteins/metabolism , Muscle, Skeletal/pathology , Muscular Diseases/genetics , Muscular Diseases/metabolism , Muscular Diseases/pathology , Protein Conformation, alpha-Helical , Protein DomainsABSTRACT
Green synthesis of nanoparticles (NPs) involves the use of diverse extracts of biological origin as substrates to synthesize NPs and can overcome the hazards associated with chemical methods. Coconut inflorescence sap, which is unfermented phloem sap obtained by tapping of coconut inflorescence, is a rich source of sugars and secondary metabolites. In this study, coconut inflorescence sap was used to synthesize silver NPs (AgNPs). We have initially undertaken metabolomic profiling of coconut inflorescence sap from West Coast Tall cultivar to delineate its individual components. It was found to comprise of 64% secondary metabolites, 9% sugars, 12% lipids/fats and 9% peptides in positive mode, whereas in the negative mode, it was 33, 20, 9 and 11%, respectively. The concentration of silver nitrate, inflorescence sap and incubation temperature for the synthesis of AgNPs were optimized. Incubating the reaction mixture at 40 °C was found to enhance AgNP synthesis. The AgNPs synthesized were characterized using UV-visible (UV-Vis) spectrophotometry, X-Ray Diffraction (XRD), Fourier Transform Infrared spectroscopy (FTIR) and Transmission Electron Microscopy (TEM). The particles were crystalline in nature and the bulk of the particles were spherical with smooth (thin) shell and poly-dispersed with a diameter ranging from 10 nm to 30 nm. Antimicrobial property of AgNPs was tested in tissue culture of arecanut (Areca catechu L.) where bacterial contamination (Bacillus pumilus) was a frequent occurrence. A significant reduction in the contamination was observed when plantlets were treated with aqueous solutions of AgNPs. Notably, treatment with AgNPs did not affect the growth and development of the arecanut plantlets. Antimicrobial properties of AgNPs synthesized from inflorescence sap were also evaluated in human pathogenic bacteria viz., Escherichia coli ATCC 25922; Salmonella Typhimurium ATCC 14028 and Vibrio parahaemolyticus AQ4037. The antibacterial action was confirmed by determining the production of reactive oxygen species (ROS) and protein leakage studies. Cytotoxicity of AgNPs was quantified in HeLa cells. The viability (%) of HeLa cells declined significantly at 10 mg L-1 concentration of AgNP and complete mortality was observed at a concentration of 60 mg L-1. The study concludes that unfermented inflorescence sap, with above neutral pH, serves as an excellent reducing agent to synthesize AgNPs from Ag+.
