Successful transfer of ADA gene in vitro into human peripheral blood CD34+ cells by transfecting EBV-based episomal vectors

AbstractWe report a novel non-viral system for transfecting human immature hematopoietic cells in vitro. Epstein-Barr virus (EBV)-based episomal vectors carrying human adenosine deaminase (ADA) gene cDNA were transfected by electroporation into human peripheral blood (PB) CD34+ cells. The transgene-specific mRNA were detected from 37 to 100% of CFU-c (colony forming unit in culture) colonies derived from the transfected cells. A two-fold increase in enzyme activity was also found. These results indicate the successful transfer and expression of genes in human immature hematopoietic cells using the EBV-based episomal vector system

Heterozygous B beta-chain C-terminal 12 amino acid elongation variant, B beta X462W (Kyoto VI), showed dysfibrinogenemia

A heterozygous patient with dysfibrinogenemia with slight bleeding and no thrombotic complications was diagnosed with fibrinogen Kyoto VI (K-VI). To elucidate the genetic mutation(s) and characterize the variant protein, we performed the following experiments and compared with identical and similar variants that have already been reported. The proposita's PCR-amplified DNA was analyzed by sequencing and her purified plasma fibrinogen underwent SDS-PAGE followed by immunoblotting, fibrin polymerization, and scanning electron microscopic observation of fibrin clot and fibers. Sequence analyses showed that K-VI fibrinogen substituted W (TGG) for terminal codon (TAG), resulting in 12 amino acid elongation 462-473 (WSPIRRFLLFCM) in the B beta-chain. Protein analyses indicated that the presence of some albumin-binding variant fibrinogens and a dimeric molecule of variant fibrinogens reduced fibrin polymerization, with a thinner fiber and aberrant fibrin network. These results are almost the same as for the identical variant of Magdeburg, however, different from the similar variant of Osaka VI [ 12 amino acid elongation 462-473 (KSPIRRFLLFCM) in the B beta-chain] in the presence of variant forms and clot structure. We speculate the side-chain difference at 462 residues, W in K-VI, K in Osaka VI, and/or the difference in the presence of disulfide bridged forms of variant fibrinogens, led to the notable difference in the fibrin bundle network. Although a strong evolutional and structural association between B beta-chain and gamma-chain molecules is established, the corresponding recombinant 15 residue elongation variants of the fibrinogen gamma-chain showed reduced assembly and secretion.ArticleBLOOD COAGULATION & FIBRINOLYSIS. 23(1):87-90 (2012)journal articl

Raman Fingerprints of SARS-CoV‐2 Omicron Subvariants: Molecular Roots of Virological Characteristics and Evolutionary Directions

The latest RNA genomic mutation of SARS-CoV-2 virus, termed the Omicron variant, has generated a stream of highly contagious and antibody-resistant strains, which in turn led to classifying Omicron as a variant of concern. We systematically collected Raman spectra from six Omicron subvariants available in Japan (i.e., BA.1.18, BA.2, BA.4, BA.5, XE, and BA.2.75) and applied machinelearning algorithms to decrypt their structural characteristics at the molecular scale. Unique Raman fingerprints of sulfur-containing amino acid rotamers, RNA purines and pyrimidines, tyrosine phenol ring configurations, and secondary protein structures clearly differentiated the six Omicron subvariants. These spectral characteristics, which were linked to infectiousness, transmissibility, and propensity for immune evasion, revealed evolutionary motifs to be compared with the outputs of genomic studies. The availability of a Raman “metabolomic snapshot”, which was then translated into a barcode to enable a prompt subvariant identification, opened the way to rationalize in real-time SARS-CoV-2 activity and variability. As a proof of concept, we applied the Raman barcode procedure to a nasal swab sample retrieved from a SARS-CoV-2 patient and identified its Omicron subvariant by coupling a commercially available magnetic bead technology with our newly developed Raman analyses

Tree of motility – A proposed history of motility systems in the tree of life

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility

CD5-Positive Primary Intraocular B-Cell Lymphoma Arising during Methotrexate and Tumor Necrosis Factor Inhibitor Treatment

Purpose: To report a case of CD5+ primary intraocular B-cell lymphoma arising during methotrexate (MTX) and tumor necrosis factor (TNF) inhibitor treatment in a young patient with rheumatoid arthritis and uveitis. Case Presentation: A 39-year-old woman treated with MTX and a TNF inhibitor for rheumatoid arthritis and uveitis had steroid-resistant vitreous opacity. A vitreous sample was obtained by using diagnostic vitrectomy and was categorized as class V based on cytologic examination. Flow cytometric analysis of the vitreous sample revealed that abnormal cells were CD5+, CD10-, CD19+, CD20+ and immunoglobulin light-chain kappa+, suggesting the diagnosis of CD5+ primary intraocular B-cell lymphoma. Polymerase chain reaction (PCR) detected immunoglobulin heavy-chain gene rearrangement. Epstein-Barr virus (EBV) DNA was detected in the vitreous sample by using PCR, and immunohistochemistry revealed EBV latent membrane protein-1 expression in the abnormal cells infiltrating the vitreous. Optic nerve invasion was observed on magnetic resonance imaging. Conclusion: Primary intraocular lymphoma (PIOL) may develop in patients receiving MTX and TNF inhibitor treatment. EBV infection may play an important role in the pathogenesis of PIOL arising during immunosuppressive therapy