Identifying transcriptomic profiles of iron-quercetin complex treated peripheral blood mononuclear cells from healthy volunteers and diabetic patients.

Peripheral blood is an alternative source of stem/progenitor cells for regenerative medicine owing to its ease of retrieval and blood bank storage. Previous in vitro studies indicated that the conditioned medium derived from peripheral blood mononuclear cells (PBMCs) treated with the iron-quercetin complex (IronQ) contains potent angiogenesis and wound-healing properties. This study aims to unveil the intricate regulatory mechanisms governing the effects of IronQ on the transcriptome profiles of human PBMCs from healthy volunteers and those with diabetes mellitus (DM) using RNA sequencing analysis. Our findings revealed 3741 and 2204 differentially expressed genes (DEGs) when treating healthy and DM PBMCs with IronQ, respectively. Functional enrichment analyses underscored the biological processes shared by the DEGs in both conditions, including inflammatory responses, cell migration, cellular stress responses, and angiogenesis. A comprehensive exploration of these molecular alterations exposed a network of 20 hub genes essential in response to stimuli, cell migration, immune processes, and the mitogen-activated protein kinase (MAPK) pathway. The activation of these pathways enabled PBMCs to potentiate angiogenesis and tissue repair. Corroborating this, quantitative real-time polymerase chain reaction (qRT-PCR) and cell phenotyping confirmed the upregulation of candidate genes associated with anti-inflammatory, pro-angiogenesis, and tissue repair processes in IronQ-treated PBMCs. In summary, combining IronQ and PBMCs brings about substantial shifts in gene expression profiles and activates pathways that are crucial for tissue repair and immune response, which is promising for the enhancement of the therapeutic potential of PBMCs, especially in diabetic wound healing.

Ultrarapid and high-resolution HLA class I typing using transposase-based nanopore sequencing applied in pharmacogenetic testing.

Nanopore sequencing has been examined as a method for rapid and high-resolution human leukocyte antigen (HLA) typing in recent years. We aimed to apply ultrarapid nanopore-based HLA typing for HLA class I alleles associated with drug hypersensitivity, including HLA-A*31:01, HLA-B*15:02, and HLA-C*08:01. Most studies have used the Oxford Nanopore Ligation Sequencing kit for HLA typing, which requires several enzymatic reactions and remains relatively expensive, even when the samples are multiplexed. Here, we used the Oxford Nanopore Rapid Barcoding kit, which is transposase-based, with library preparation taking less than 1 h of hands-on time and requiring minimal reagents. Twenty DNA samples were genotyped for HLA-A, -B, and -C; 11 samples were from individuals of different ethnicity and nine were from Thai individuals. Two primer sets, a commercial set and a published set, were used to amplify the HLA-A, -B, and -C genes. HLA-typing tools that used different algorithms were applied and compared. We found that without using several third-party reagents, the transposase-based method reduced the hands-on time from approximately 9 h to 4 h, making this a viable approach for obtaining same-day results from 2 to 24 samples. However, an imbalance in the PCR amplification of different haplotypes could affect the accuracy of typing results. This work demonstrates the ability of transposase-based sequencing to report 3-field HLA alleles and its potential for race- and population-independent testing at considerably decreased time and cost.

Iron(III)-Quercetin Complexes' Safety for MRI Cell Tracking in Cell Therapy Applications: Cytotoxic and Genotoxic Assessment.

The theranostic agent iron-quercetin complex (IronQ) provides a T1-positive magnetic resonance imaging (MRI) contrast agent. The magnetically IronQ-labeled cells can be used for cell tracking and have active biological applications in promoting cell and tissue regeneration. However, a detailed investigation of IronQ's cytotoxicity and genotoxicity is necessary. Thus, this study aimed to evaluate the possibility of IronQ inducing cytotoxicity and genotoxicity in peripheral blood mononuclear cells (PBMCs). We evaluated the vitality of cells, the production of reactive oxygen species (ROS), the level of antioxidant enzymes, and the stability of the genetic material in PBMCs treated with IronQ. The results show that IronQ had a negligible impact on toxicological parameters such as ROS production and lipid peroxidation, indicating that it is not harmful. IronQ-labeled PMBCs experienced an insignificant depletion of antioxidant enzyme levels at the highest concentration of IronQ. There is no evident genotoxicity in the magnetically IronQ-labeled PBMCs. The results show that IronQ does not potentiate the cytotoxicity and genotoxicity effects of the labeled PMBCs and might be safe for therapeutic and cell tracking purposes. These results could provide a reference guideline for the toxicological analysis of IronQ in in vivo studies.

Introduction of new alternative pipeline using multiplexed fast COLD­PCR together with sequencing approach highlighting pharmacoeconomics by detection of CYP variants.

In precision medicine, multiple factors are involved in clinical decision-making because of ethnic and racial genetic diversity, family history and other health factors. Although advanced techniques have evolved, there is still an economic obstacle to pharmacogenetic (PGx) implementation in developing countries. The aim of the present study was to provide an alternative pipeline that roughly estimate patient carrier type and prescreen out wild-type samples before sequencing or genotyping to determine genetic status. Fast co-amplification at lower denaturation temperature (COLD)-PCR was used to differentiate genetic variant non-carriers from carriers. The majority of drugs are hepatically cleared by cytochrome P450 (CYP) enzymes and genes encoding CYP enzymes are highly variable. Of all the CYPs, CYP2 family of CYP2C9, CYP2C19, and CYP2D6 isoforms have clinically significant impact on drugs of PGx testing. Therefore, five variants associated with these CYPs were selected for preliminary testing with this novel pipeline. For fast COLD-PCR, the optimal annealing temperature and critical denaturation temperature were determined and evaluated via Sanger sequencing of 27 randomly collected samples. According to precise Tc, to perform in a single-reaction is difficult. However, in this study, this issue was resolved by combination of precise Tc using 10+10+20 cycles. The results showed 100% sensitivity and specificity, with perfect agreement (κ=1.0) compared with Sanger sequencing. The present study provides a prescreening platform by introducing multiplex fast COLD-PCR as a pharmacoeconomic implementation. Our study just present in five variants which are not enough to describe patient metabolic status. Therefore, other actional genetic variants are still needed to cover the actual patient's genotypes. Nevertheless, the proposed method can well-present its efficiency and reliability for serving as a PGx budget platform in the future.

Two novel alleles on Fucosyltransferase 2 from northern Thai para-Bombay family and computational prediction on mutation effect.

BACKGROUND: Major characteristics of the para-Bombay phenotype are the absence of ABH antigens on red blood cells due to fucosyltransferase 1 (FUT1) gene mutation and the presence of these antigens in body secretions due to the active fucosyltransferase 2 (FUT2) gene. An ABO blood group discrepancy can be identified via serological testing, and additional tests can be performed for confirmation. This study aimed to resolve the ABO discrepancy and report two novel alleles on the FUT2 gene in northern Thai para-Bombay families. STUDY DESIGN AND METHODS: Twelve blood samples were collected from five suspected para-Bombay donors and their families. Nucleotide sequences of ABO, FUT1, and FUT2 were analyzed by polymerase chain reaction-sequence-based typing. Bioinformatics tools were used to predict the effect of suspected novel FUT2 alleles. RESULTS: All samples exhibited normal ABO alleles, concordant with serological test results. FUT1 exhibited three known variants (c.328G>A, c.424C>T, and c.658C>T). Although FUT2 exhibited two known variants (c.357C>T and c.385A>T), two novel alleles were observed. One allele consisted of c.98A>G, c.101T>G, and c.357C>T with predicted normal transferase activity, whereas the other consisted of c.357C>T and c.617T>C with predicted abnormal enzyme activity. DISCUSSION: Two novel alleles in FUT2 were reported among the affected para-Bombay individuals of northern Thai families. The c.617T>C variant caused an amino acid change from valine to alanine at position 206, predicted to be an inactive FUT2 enzyme. Inheritance of this variant with the recessive FUT1 allele may lead to inheritance of the rare Bombay blood group in the progeny.