Platelet and Monocyte Microvesicles as Potential Biomarkers of COVID-19 Severity: A Cross-Sectional Analysis.

Background: Coronavirus disease (COVID-19) induces inflammation, coagulopathy following platelet and monocyte activation, and fibrinolysis, resulting in elevated D-dimer levels. Activated platelets and monocytes produce microvesicles (MVs). We analyzed the differences in platelet and monocyte MV counts in mild, moderate, and severe COVID-19, as well as their correlation with D-dimer levels. Methods: In this cross-sectional study, blood specimens were collected from 90 COVID-19 patients and analyzed for D-dimers using SYSMEX CS-2500. Platelet MVs (PMVs; PMVCD42b+ and PMVCD41a+), monocyte MVs (MMVs; MMVCD14+), and phosphatidylserine-binding annexin V (PS, AnnV+) were analyzed using a BD FACSCalibur instrument. Results: PMV and MMV counts were significantly increased in COVID-19 patients. AnnV+ PMVCD42b+ and AnnV+ PMVCD41a+ cell counts were higher in patients with severe COVID-19 than in those with moderate clinical symptoms. The median (range) of AnnV+ PMVCD42b+ (MV/µL) in mild, moderate, and severe COVID-19 was 1,118.3 (328.1-1,910.5), 937.4 (311.4-2,909.5), and 1,298.8 (458.2-9,703.5), respectively (P =0.009). The median (range) for AnnV+ PMVCD41a+ (MV/µL) in mild, moderate, and severe disease was 885.5 (346.3-1,682.7), 663.5 (233.8-2,081.5), and 1,146.3 (333.3-10,296.6), respectively (P =0.007). D-dimer levels (ng/mL) weak correlated with AnnV+ PMVCD41a+ (P =0.047, r=0.258). Conclusions: PMV PMVCD42b+ and PMVCD41a+ counts were significantly increased in patients with severe clinical symptoms, and PMVCD41a+ counts correlated with D-dimer levels. Therefore, MV counts can be used as a potential biomarker of COVID-19 severity.

RUNX1/ETO regulates reactive oxygen species (ROS) levels in t(8,21) acute myeloid leukaemia via FLT3 and RAC1.

Reactive oxygen species (ROS) homeostasis is crucial for leukaemogenesisand deregulation would hamper leukaemic progression. Although the regulatory effects of RUNX1/ETO has been extensively studied, its underlying molecular mechanims in ROS production in t(8,21) AML is yet to be fully elucidated. Here, we report that RUNX1/ETO could directly control FLT3 by occupying several DNA elements on FLT3 locus. The possible hijacking mechanism by RUNX1/ETO over FLT3 mediated ROS modulation in AML t(8;21) was made apparent when suppression of RUNX1/ETO led to decrement in ROS levels and the direct oxidative marker FOXO3 but not in FLT3 and RAC1 suppressed t(8,21) AML cell line Furthermore, nuclear import of RUNX1/ETO was aberrated following RUNX1/ETO and RAC1 suppression suggesting association in ROS control. A different picture was depicted in non t(8;21) cells where suppression of RAC1 and FLT3 led to decreased levels of FOXO3a and ROS. Results alltogether indicate a possible dysregulation of ROS levels by RUNX1/ETO in t(8,21) AML.

Levels of SARS-COV-2 anti-spike protein receptor-binding domain (S-RBD) IgG in Indonesian-vaccinated healthcare workers: experimental research.

Studies evaluating the levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike protein receptor-binding domain (S-RBD) immunoglobulin G (IgG) antibodies in vaccinated healthcare workers in Indonesia are limited. Objectives: Evaluating time-dependent levels of anti-IgG S-RBD antibodies and monitoring the response of healthcare workers in a tertiary hospital in Indonesia after vaccination. Materials and methods: This prospective cohort observational study was conducted from January to December 2021. A total of 50 healthcare workers participated in the study. Blood samples were collected at five time points. Antibody levels were measured using a CL 1000i analyzer (Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China). Antibody levels between groups were analyzed using the Wilcoxon signed-rank test with P less than 0.05. Results: The median levels of SARS-CoV-2 anti-S-RBD IgG antibody on days 14, 28, 90, and 180 were significantly higher than the levels on day 0 (P<0.001). After the second dose, peak levels were observed on day 14; the levels decreased gradually after day 28. Despite receiving two doses of the vaccine, 10 out of 50 participants (20%) were infected with COVID-19 (coronavirus disease 2019). However, symptoms were mild, and antibody levels were significantly higher than in noninfected participants (P<0.001). Conclusion: SARS-CoV-2 anti-S-RBD IgG antibody levels increased significantly until day 14 after the second dose; the levels decreased gradually after day 28. Ten participants (20%) were infected with SARS-CoV-2, with mild symptoms.

A RUNX1/ETO-SKP2-CDKN1B axis regulates expression of telomerase in t (8;21) acute myeloid leukemia.

The fusion oncoprotein RUNX1/ETO which results from the chromosomal translocation t (8;21) in acute myeloid leukemia (AML) is an essential driver of leukemic maintenance. We have previously shown that RUNX1/ETO knockdown impairs expression of the protein component of telomerase, TERT. However, the underlying molecular mechanism of how RUNX1/ETO controls TERT expression has not been fully elucidated. Here we show that RUNX1/ETO binds to an intergenic region 18 kb upstream of the TERT transcriptional start site and to a site located in intron 6 of TERT. Loss of RUNX1/ETO binding precedes inhibition of TERT expression. Repression of TERT expression is also dependent on the destabilization of the E3 ubiquitin ligase SKP2 and the resultant accumulation of the cell cycle inhibitor CDKN1B, that are both associated with RUNX1/ETO knockdown. Increased CDKN1B protein levels ultimately diminished TERT transcription with E2F1/Rb involvement. Collectively, our results show that RUNX1/ETO controls TERT expression directly by binding to its locus and indirectly via a SKP2-CDKN1B-E2F1/Rb axis.

Mesenchymal Stromal Cells-Derived Exosome and the Roles in the Treatment of Traumatic Brain Injury.

Traumatic brain injury (TBI) could result in life-long disabilities and death. Though the mechanical insult causes primary injury, the secondary injury due to dysregulated responses following neuronal apoptosis and inflammation is often the cause for more detrimental consequences. Mesenchymal stromal cell (MSC) has been extensively investigated as the emerging therapeutic for TBI, and the functional properties are chiefly attributed to their secretome, especially the exosomes. Delivering these nanosize exosomes have shown to ameliorate post-traumatic injury and restore brain functions. Recent technology advances also allow engineering MSC-derived exosomes to carry specific biomolecules of interest to augment their therapeutic outcome. In this review, we discuss the pathophysiology of TBI and summarize the recent progress in the applications of MSCs-derived exosomes, the roles and the signalling mechanisms underlying the protective effects in the treatment of the TBI.

Cytogenetics analysis as the central point of genetic testing in acute myeloid leukemia (AML): a laboratory perspective for clinical applications.

Chromosomal abnormalities in acute myeloid leukemia (AML) have significantly contributed to scientific understanding of its molecular pathogenesis, which has aided in the development of therapeutic strategies and enhanced management of AML patients. The diagnosis, prognosis and treatment of AML have also rapidly transformed in recent years, improving initial response to treatment, remission rates, risk stratification and overall survival. Hundreds of rare chromosomal abnormalities in AML have been discovered thus far using chromosomal analysis and next-generation sequencing. As a result, the World Health Organization (WHO) has categorized AML into subgroups based on genetic, genomic and molecular characteristics, to complement the existing French-American classification which is solely based on morphology. In this review, we aim to highlight the most clinically relevant chromosomal aberrations in AML together with the technologies employed to detect these aberrations in laboratory settings.

Markers of Renal Complications in Beta Thalassemia Patients with Iron Overload Receiving Chelation Agent Therapy: A Systematic Review.

Objective: The emerging renal complications in beta-thalassemia patients have raised the global exchange of views. Despite better survival due to blood transfusion and iron chelation therapy, the previously unrecognized renal complication remain a burden of disease affecting this population -the primary concern on how iron overload and chelation therapy correlated with renal impairment is still controversial. Early detection and diagnosis is crucial in preventing further kidney damage. Therefore, a systematic review was performed to identify markers of kidney complications in beta thalassemia patients with iron overload receiving chelation therapy. Methods: Searches of PubMed, Scopus, Science Direct, and Web of Science were conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) to identify studies of literature reporting renal outcome in ß-TM patients with iron overload and receiving chelation therapy. The eligible 17 studies were obtained. Results: uNGAL/NGAL, uNAG/NAG, uKIM-1 are markers that can be used as predictor of renal tubular damage in early renal complications, while Cystatin C and uß2MG showed further damage at the glomerular level. Discussion and Conclusion: The renal complication in beta-thalassemia patients with iron overload receiving chelating agent therapy may progress to kidney disease. Early detection using accurate biological markers is a substantial issue that deserves further evaluation to determine prevention and management.

Analysis of Common Beta-Thalassemia (ß-Thalassemia) Mutations in East Java, Indonesia.

Background: The frequency of the beta-thalassemia (ß-thalassemia) gene in Indonesia ranges from 3 to 10%. However, in the East Java province, there is still limited information on the prevalence of ß-thalassemia mutations in clinically diagnosed beta-thalassemia patients of East Java. Therefore, this study aimed to characterize ß-thalassemia mutations in selected patients in the East Java province of Indonesia. Methods: This is an analytical observational study. Diagnosis of ß-thalassemia was based on clinical presentation, complete blood count (CBC), and hemoglobin (Hb) electrophoresis. Blood specimens taken from each patient in three ethylenediaminetetraacetic acid (EDTA) tubes were analyzed for CBC and Hb electrophoresis and processed for DNA extraction and subsequent polymerase chain reaction (PCR). Detection of mutations in Hemoglobin Subunit Beta (HBB) gene exons 1-3 of the ß-thalassemia gene as the common mutation in Indonesia was done using PCR followed by Sanger sequencing. Results: In total, 33 (n = 33) participants were involved in this study with ages ranging from 5 to 17 years comprising 19 women and 14 men. Their ethnic origins were Javanese (n = 30) and Chinese (n = 3). CBC results showed that mean ± standard deviation (SD) for Hb, red blood cell (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW)-CV were 81.2 ± 7.0 g/L; 3.40 ± 0.39 × 109/L; 71.05 ± 5.72 fL; 24.12 ± 2.45 pg; 33.91 ± 1.47 g/dl; 24.38 ± 6.02%, respectively. Hb electrophoresis revealed that 5 out of 33 participants had beta-thalassemia and 28 out of 33 participants had hemoglobinopathy (Hb) E/beta-thalassemia. Results of Sanger sequencing showed the following genotype variations in the samples: 12 (36.4%) with ß CD26 /ß IVS-I-5; 6 (18.2%) with ß CD26 /ß CD35; 3 (9.1%) with ß CD26 /ß IVS-I-2; 2 (6.1%) with ß CD27/28 /ß CD40; 2 (6.1%) with ß IVS-I-1 /ß CAP+1; and ß CD26 /ß IVS-I-1; ß IVS-I-5 /ß CAP+1; ß IVS-I-5 /ß CD35; ß CD26 /ß CD37; ß CD26 /ß CD15; ß CD26 /ß CD40; and ß IVS-I-5 /ß CD19 in 1 (3%) sample, respectively, and 1 (3%) had no abnormality detected in sequencing even though electrophoresis showed abnormality in the migration pattern. The ß CD26 /ß IVS-I-5 mutation was found in samples that were noted to have Hb E/beta-thalassemia on Hb electrophoresis. Conclusion: The underlying genetic variations are heterogeneous in thalassemia patients in East Java, where 12 variants were found. The most common variant was ß CD26 /ß IVS-I-5, which all accounted for Hb E/beta-thalassemia on Hb electrophoresis. Furthermore, 28 out of 33 participants had hemoglobinopathy (Hb) E/beta-thalassemia.

Elucidating miRNA Function in Cancer Biology via the Molecular Genetics' Toolbox.

Micro-RNA (miRNAs) are short non-coding RNAs of about 18-20 nucleotides in length and are implicated in many cellular processes including proliferation, development, differentiation, apoptosis and cell signaling. Furthermore, it is well known that miRNA expression is frequently dysregulated in many cancers. Therefore, this review will highlight the various mechanisms by which microRNAs are dysregulated in cancer. Further highlights include the abundance of molecular genetics tools that are currently available to study miRNA function as well as their advantages and disadvantages with a special focus on various CRISPR/Cas systems This review provides general workflows and some practical considerations when studying miRNA function thus enabling researchers to make informed decisions in regards to the appropriate molecular genetics tool to be utilized for their experiments.

Global Globin Network Consensus Paper: Classification and Stratified Roadmaps for Improved Thalassaemia Care and Prevention in 32 Countries.

The Global Globin Network (GGN) is a project-wide initiative of the Human Variome/Global Variome Project (HVP) focusing on haemoglobinopathies to build the capacity for genomic diagnosis, clinical services, and research in low- and middle-income countries. At present, there is no framework to evaluate the improvement of care, treatment, and prevention of thalassaemia and other haemoglobinopathies globally, despite thalassaemia being one of the most common monogenic diseases worldwide. Here, we propose a universally applicable system for evaluating and grouping countries based on qualitative indicators according to the quality of care, treatment, and prevention of haemoglobinopathies. We also apply this system to GGN countries as proof of principle. To this end, qualitative indicators were extracted from the IthaMaps database of the ITHANET portal, which allowed four groups of countries (A, B, C, and D) to be defined based on major qualitative indicators, supported by minor qualitative indicators for countries with limited resource settings and by the overall haemoglobinopathy carrier frequency for the target countries of immigration. The proposed rubrics and accumulative scores will help analyse the performance and improvement of care, treatment, and prevention of haemoglobinopathies in the GGN and beyond. Our proposed criteria complement future data collection from GGN countries to help monitor the quality of services for haemoglobinopathies, provide ongoing estimates for services and epidemiology in GGN countries, and note the contribution of the GGN to a local and global reduction of disease burden.

Central resources of variant discovery and annotation and its role in precision medicine.

Rapid technological advancement in high-throughput genomics, microarray, and deep sequencing technologies has accelerated the possibility of more complex precision medicine research using large amounts of heterogeneous health-related data from patients, including genomic variants. Genomic variants can be identified and annotated based on the reference human genome either within the sequence as a whole or in a putative functional genomic element. The American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) mutually created standards and guidelines for the appraisal of proof to expand consistency and straightforwardness in clinical variation interpretations. Various efforts toward precision medicine have been facilitated by many national and international public databases that classify and annotate genomic variation. In the present study, several resources are highlighted with recognition and data spreading of clinically important genetic variations.

Footprints of microRNAs in Cancer Biology.

MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional gene regulation. Over the past years, various studies have demonstrated the role of aberrant miRNA expression in the onset of cancer. The mechanisms by which miRNA exerts its cancer-promoting or inhibitory effects are apparent through the various cancer hallmarks, which include selective proliferative advantage, altered stress response, vascularization, invasion and metastasis, metabolic rewiring, the tumor microenvironment and immune modulation; therefore, this review aims to highlight the association between miRNAs and the various cancer hallmarks by dissecting the mechanisms of miRNA regulation in each hallmark separately. It is hoped that the information presented herein will provide further insights regarding the role of cancer and serve as a guideline to evaluate the potential of microRNAs to be utilized as biomarkers and therapeutic targets on a larger scale in cancer research.

Mechanism of Human Telomerase Reverse Transcriptase (hTERT) Regulation and Clinical Impacts in Leukemia.

The proliferative capacity and continuous survival of cells are highly dependent on telomerase expression and the maintenance of telomere length. For this reason, elevated expression of telomerase has been identified in virtually all cancers, including leukemias; however, it should be noted that expression of telomerase is sometimes observed later in malignant development. This time point of activation is highly dependent on the type of leukemia and its causative factors. Many recent studies in this field have contributed to the elucidation of the mechanisms by which the various forms of leukemias increase telomerase activity. These include the dysregulation of telomerase reverse transcriptase (TERT) at various levels which include transcriptional, post-transcriptional, and post-translational stages. The pathways and biological molecules involved in these processes are also being deciphered with the advent of enabling technologies such as next-generation sequencing (NGS), ribonucleic acid sequencing (RNA-Seq), liquid chromatography-mass spectrometry (LCMS/MS), and many others. It has also been established that TERT possess diagnostic value as most adult cells do not express high levels of telomerase. Indeed, studies have shown that prognosis is not favorable in patients who have leukemias expressing high levels of telomerase. Recent research has indicated that targeting of this gene is able to control the survival of malignant cells and therefore offers a potential treatment for TERT-dependent leukemias. Here we review the mechanisms of hTERT regulation and deliberate their association in malignant states of leukemic cells. Further, we also cover the clinical implications of this gene including its use in diagnostic, prognostic, and therapeutic discoveries.

Utilization of CRISPR-Mediated Tools for Studying Functional Genomics in Hematological Malignancies: An Overview on the Current Perspectives, Challenges, and Clinical Implications.

Hematological malignancies (HM) are a group of neoplastic diseases that are usually heterogenous in nature due to the complex underlying genetic aberrations in which collaborating mutations enable cells to evade checkpoints that normally safeguard it against DNA damage and other disruptions of healthy cell growth. Research regarding chromosomal structural rearrangements and alterations, gene mutations, and functionality are currently being carried out to understand the genomics of these abnormalities. It is also becoming more evident that cross talk between the functional changes in transcription and proteins gives the characteristics of the disease although specific mutations may induce unique phenotypes. Functional genomics is vital in this aspect as it measures the complete genetic change in cancerous cells and seeks to integrate the dynamic changes in these networks to elucidate various cancer phenotypes. The advent of CRISPR technology has indeed provided a superfluity of benefits to mankind, as this versatile technology enables DNA editing in the genome. The CRISPR-Cas9 system is a precise genome editing tool, and it has revolutionized methodologies in the field of hematology. Currently, there are various CRISPR systems that are used to perform robust site-specific gene editing to study HM. Furthermore, experimental approaches that are based on CRISPR technology have created promising tools for developing effective hematological therapeutics. Therefore, this review will focus on diverse applications of CRISPR-based gene-editing tools in HM and its potential future trajectory. Collectively, this review will demonstrate the key roles of different CRISPR systems that are being used in HM, and the literature will be a representation of a critical step toward further understanding the biology of HM and the development of potential therapeutic approaches.

Homozygous Southeast Asian Ovalocytosis in five live-born neonates.

Not available.

Production and differential activity of recombinant human wild-type G6PD and G6PDViangchan.

Background: Glucose-6-phosphate dehydrogenase (G6PD) is essential to produce reduced nicotinamide adenine dinucleotide phosphate, which is required to protect cells against oxidative stress. G6PD deficiency is a genetic variation that may lead to hemolysis with potential consequences, such as kidney failure, and patients often experience low quality of life. Objectives: To establish a simple, efficient, and optimized method to produce a G6PDViangchan variant and characterize the phenotypes of recombinant human wild-type G6PD and G6PDViangchan. Methods: G6PD was amplified by polymerase chain reaction (PCR) from a human cDNA plasmid, and the gene for G6PDViangchan was amplified by initiating a mutation at location 871 (G>A) through site-directed mutagenesis. Protein expression and western blotting were conducted after successful cloning. The enzymatic activity of both proteins was assessed spectrophotometrically after purification. Results: Both amplicons were successfully cloned into a pET26b(+) expression vector and transformed into Escherichia coli BL21 (DE3) cells for overexpression as C-terminally histidine-tagged recombinant proteins. Western blotting confirmed that both proteins were successfully produced at similar levels. The enzymes were purified by immobilized metal (Co) affinity chromatography. Postpurification assay of enzyme activity revealed about 2-fold differences in the levels of specific activity between the wild-type G6PD (155.88 U/mg) and G6PDViangchan (81.85 U/mg), which is consistent with earlier reports. Analysis in silico showed that the coding change in G6PDViangchan has a substantial effect on protein folding structure. Conclusions: We successfully cloned, expressed, and purified both wild-type G6PD and G6PDViangchan proteins. Such a protocol may be useful for creating a model system to study G6PD deficiency disease.

The BRCA1 and BRCA2 Genes in Early-Onset Breast Cancer Patients.

Approximately 5-10% of breast cancers are attributable to genetic susceptibility. Mutations in the BRCA1 and BRCA2 genes are the best known genetic factors to date. The goal of this study was to determine the structure and distribution of haplotypes of the BRCA1 and BRCA2 genes in early-onset breast cancer patients. We enrolled 70 patients diagnosed with early-onset breast cancer. A total of 21 SNPs (11 on BRCA1 and 10 on BRCA2) and 1 dinucleotide deletion on BRCA1 were genotyped using nested allele-specific PCR methods. Linkage disequilibrium (LD) analysis was conducted, and haplotypes were deduced from the genotype data. Two tightly linked LD blocks were observed on each of the BRCA1 and BRCA2 genes. Variant-free haplotypes (TAT-AG for BRCA1 and ATA-AAT for BRCA2) were observed at a frequency of more than 50% on each gene along with variable frequencies of derived haplotypes. The variant 3'-subhaplotype CGC displayed strong LD with 5'-subhaplotypes GA, AA, and GG on BRCA1 gene. Haplotypes ATA-AGT, ATC-AAT, and ATA-AAC were the variant haplotypes frequent on BRCA2 gene. Although the clinical significance of these derived haplotypes has not yet been established, it is expected that some of these haplotypes, especially the less frequent subhaplotypes, eventually will be shown to be indicative of a predisposition to early-onset breast cancer.

Conditioned Medium of Human Menstrual Blood-Derived Endometrial Stem Cells Protects Against MPP+-Induced Cytotoxicity in vitro.

Mesenchymal stem cells (MSCs) showed the potential to treat Parkinson's disease (PD). However, it is unknown whether the conditioned medium of human menstrual blood-derived endometrial stem cells (MenSCs-CM) has the function to alleviate syndromes of PD. In this study, human neuroblastoma SH-SY5Y cells were exposed to neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) for inducing a range of response characteristics of PD. After culturing this cell model with 24 h/48 h collected MenSCs-CM for different days, cell viability, pro-inflammation cytokines, mitochondrial membrane potential (ΔΨm), oxidative stress, and cell apoptosis were detected. Finally, protein assay was performed to detect 12 kinds of neurotrophic factors inside MenSCs-CM. Our results showed that MPP+ caused SH-SY5Y cell viability reduction as an increasing dose and time dependent manner. MPP+ treatment resulted in inflammation, mitochondrial dysfunction, reactive oxygen species (ROS) production accumulation, and apoptosis of SH-SY5Y at its IC50 concentration. Forty-eight hours-collected MenSCs-CM and culturing with the MPP+-treated SH-SY5Y for 2 days are the optimized condition to increase cell viability. Besides, MenSCs-CM was efficacious against MPP+ induced inflammation, ΔΨm loss, ROS generation, and it could significantly decrease cells numbers in late apoptosis stage. What's more, protein assay showed that MenSCs-CM contained various neuroprotective factors. Our study provided the first evidence that MenSCs-CM has a protective effect on MPP+-induced cytotoxicity in various aspects, and firstly showed that MenSCs can release at least 12 kinds of neurotrophic factors to medium, which may contribute to the protective function of MenSCs-CM to treat PD. This research enlightening that MenSCs-CM is beneficial in the therapy for PD and probably also for other neurodegenerative diseases.