Exosomes: Methods for Isolation and Characterization in Biological Samples.

Exosomes are small lipid bilayer-encapsulated nanosized extracellular vesicles of endosomal origin. Exosomes are secreted by almost all cell types and are a crucial player in intercellular communication. Exosomes transmit cellular information from donor to recipient cells in the form of proteins, lipids, and nucleic acids and influence several physiological and pathological responses. Due to their capacity to carry a variety of cellular cargo, low immunogenicity and cytotoxicity, biocompatibility, and ability to cross the blood-brain barrier, these nanosized vesicles are considered excellent diagnostic tools and drug-delivery vehicles. Despite their tremendous potential, the progress in therapeutic applications of exosomes is hindered by inadequate isolation techniques, poor characterization, and scarcity of specific biomarkers. The current research in the field is focused on overcoming these limitations. In this chapter, we have reviewed conventional exosome isolation and characterization methods and recent advancements, their advantages and limitations, persistent challenges in exosome research, and future directions.

Identification and development of Tetra-ARMS PCR-based screening test for a genetic variant of OLA1 (Tyr254Cys) in the human failing heart.

Obg-like ATPase 1 (OLA1) protein has GTP and ATP hydrolyzing activities and is important for cellular growth and survival. The human OLA1 gene maps to chromosome 2 (locus 2q31.1), near Titin (TTN), which is associated with familial dilated cardiomyopathy (DCM). In this study, we found that expression of OLA1 was significantly downregulated in failing human heart tissue (HF) compared to non-failing hearts (NF). Using the Sanger sequencing method, we characterized the human OLA1 gene and screened for mutations in the OLA1 gene in patients with failing and non-failing hearts. Among failing and non-failing heart patients, we found 15 different mutations in the OLA1 gene, including two transversions, one substitution, one deletion, and eleven transitions. All mutations were intronic except for a non-synonymous 5144A>G, resulting in 254Tyr>Cys in exon 8 of the OLA1 gene. Furthermore, haplotype analysis of these mutations revealed that these single nucleotide polymorphisms (SNPs) are linked to each other, resulting in disease-specific haplotypes. Additionally, to screen the 254Tyr>Cys point mutation, we developed a cost-effective, rapid genetic screening PCR test that can differentiate between homozygous (AA and GG) and heterozygous (A/G) genotypes. Our results demonstrate that this PCR test can effectively screen for OLA1 mutation-associated cardiomyopathy in human patients using easily accessible cells or tissues, such as blood cells. These findings have important implications for the diagnosis and treatment of cardiomyopathy.

Identification and development of Tetra-ARMS PCR-based screening test for a genetic variant of OLA1 (Tyr254Cys) in the human failing heart.

Obg-like ATPase 1 (OLA1) protein has GTP and ATP hydrolyzing activities and is important for cellular growth and survival. The human OLA1 gene maps on chromosome 2, at the locus 1q31, close to the Titin (TTN) gene, which is associated with familial dilated cardiomyopathy (DCM). In this study, we found that expression of OLA1 was significantly downregulated in human failing heart tissue (HF) as compared to in non-failing heart tissues (NF). Moreover, using the Sanger sequencing method, we characterized the human OLA1 gene and screened genetic mutations in patients with heart-failing and non-failing. Among failing and non-failing heart patients, we found a total of 15 mutations, including two transversions, one substitution, one indel, and eleven transition mutations in the OLA1 gene. All the mutations were intronic except for a non-synonymous mutation, 5144A>G, resulting in 254Tyr>Cys in exon 8 of the OLA1 gene. Furthermore, haplotype analysis of these mutations revealed that these single nucleotide polymorphisms (SNPs) are linked to each other, resulting in disease-specific haplotypes. Additionally, to screen for the 254Tyr>Cys point mutation, we developed a cost-effective, rapid genetic screening PCR test that can differentiate between homozygous (AA and GG) and heterozygous (A/G) genotypes. Our results show that this test can be used as a genetic screening tool for human cardiomyopathy. These findings have important implications for the diagnosis and treatment of cardiomyopathy.

A Region of UNC-89 (Obscurin) Lying between Two Protein Kinase Domains Is a Highly Elastic Spring Required for Proper Sarcomere Organization.

In Caenorhabditis elegans, unc-89 encodes a set of giant multi-domain proteins (up 8081 residues) localized to the M-lines of muscle sarcomeres and required for normal sarcomere organization and whole-animal locomotion. Multiple UNC-89 isoforms contain two protein kinase domains. There is conservation in arrangement of domains between UNC-89 and its two mammalian homologs, obscurin and SPEG: kinase, a non-domain region of 647-742 residues, Ig domain, Fn3 domain and a second kinase domain. In all three proteins, this non-domain "interkinase region" has low sequence complexity, has high proline content, and lacks predicted secondary structure. We report that a major portion of this interkinase (571 residues out of 647 residues) when examined by single molecule force spectroscopy in vitro displays the properties of a random coil and acts as an entropic spring. We used CRISPR/Cas9 to create nematodes carrying an in-frame deletion of the same 571-residue portion of the interkinase. These animals display severe disorganization of all portions of the sarcomere in body wall muscle. Super-resolution microscopy reveals extra, short-A-bands lying close to the outer muscle cell membrane and between normally spaced A-bands. Nematodes with this in-frame deletion show defective locomotion and muscle force generation. We designed our CRISPR-generatedin-frame deletion to contain an HA tag at the N terminus of the large UNC-89 isoforms. This HA tag results in normal organization of body wall muscle, but approximately half the normal levels of the giant UNC-89 isoforms, dis-organization of pharyngeal muscle, small body size, and reduced muscle force, likely due to poor nutritional uptake.